e new england journal o medicine

The new england journal of medicine

n engl j med 382;16 nejm.org April 16, 2020 1497

established in 1812 April 16, 2020 vol. 382 no. 16

From Sunovion Pharmaceuticals, Marl-borough, MA (K.S.K., J.K., S.C.H., H.C., R.G., A.L.); and the Department of Psy-chiatry, Yale University, the Department of Neuroscience, Yale University School of Medicine, and Behavioral Health Ser-vices, Yale New Haven Hospital, New Haven (J.H.K.), and the Clinical Neuro-sciences Division, Veterans Affairs Na-tional Center for PTSD, Veterans Affairs Connecticut Healthcare System, West Haven (J.H.K.) — all in Connecticut. Ad-dress reprint requests to Dr. Koblan at Sunovion Pharmaceuticals, 84 Water-ford Dr., Marlborough, MA 01752, or at kenneth . koblan@ sunovion . com.

N Engl J Med 2020;382:1497-506.DOI: 10.1056/NEJMoa1911772Copyright © 2020 Massachusetts Medical Society.

BACKGROUNDAn oral compound, SEP-363856, that does not act on dopamine D2 receptors but has agonist activity at trace amine–associated receptor 1 (TAAR1) and 5-hydroxy-tryptamine type 1A (5-HT1A) receptors, may represent a new class of psychotropic agent for the treatment of psychosis in schizophrenia.

METHODSWe performed a randomized, controlled trial to evaluate the efficacy and safety of SEP-363856 in adults with an acute exacerbation of schizophrenia. The patients were randomly assigned in a 1:1 ratio to receive once-daily treatment with SEP-363856 (50 mg or 75 mg) or placebo for 4 weeks. The primary end point was the change from baseline in the total score on the Positive and Negative Symptom Scale (PANSS; range, 30 to 210; higher scores indicate more severe psychotic symptoms) at week 4. There were eight secondary end points, including the changes from baseline in the scores on the Clinical Global Impressions Severity (CGI-S) scale and the Brief Negative Symptom Scale (BNSS).

RESULTSA total of 120 patients were assigned to the SEP-363856 group and 125 to the placebo group. The mean total score on the PANSS at baseline was 101.4 in the SEP-363856 group and 99.7 in the placebo group, and the mean change at week 4 was −17.2 points and −9.7 points, respectively (least-squares mean difference, −7.5 points; 95% confidence interval, −11.9 to −3.0; P = 0.001). The reductions in the CGI-S and BNSS scores at week 4 were generally in the same direction as those for the primary outcome, but the results were not adjusted for multiple comparisons. Adverse events with SEP-363856 included somnolence and gastrointestinal symp-toms; one sudden cardiac death occurred in the SEP-363856 group. The incidence of extrapyramidal symptoms and changes in the levels of lipids, glycated hemo-globin, and prolactin were similar in the trial groups.

CONCLUSIONSIn this 4-week trial involving patients with an acute exacerbation of schizophrenia, SEP-363856, a non–D2-receptor-binding antipsychotic drug, resulted in a greater reduction from baseline in the PANSS total score than placebo. Longer and larger trials are necessary to confirm the effects and side effects of SEP-363856, as well as its efficacy relative to existing drug treatments for patients with schizophrenia. (Funded by Sunovion Pharmaceuticals; ClinicalTrials.gov number, NCT02969382.)

a bs tr ac t

A Non–D2-Receptor-Binding Drug for the Treatment of Schizophrenia

Kenneth S. Koblan, Ph.D., Justine Kent, M.D., Seth C. Hopkins, Ph.D., John H. Krystal, M.D., Hailong Cheng, Ph.D., Robert Goldman, Ph.D., and Antony Loebel, M.D.

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T h e n e w e ngl a nd j o u r na l o f m e dic i n e

A Quick Take is available at

NEJM.org

Antipsychotic drugs have produced clinical benefit in patients with psychosis presumably through antagonist or partial

agonist effects at postsynaptic dopamine D2 re-ceptors.1 The efficacy of D2-receptor–binding antipsychotics is often limited in the treatment of negative symptoms (e.g., blunted affect and anhedonia) and cognitive impairment.2-5 Atypical antipsychotics that have antagonist activity at both D2 and serotonin 5-hydroxytryptamine type 2A (5-HT2A) receptors were shown to have fewer side effects related to movement disorders than first-generation antipsychotics, but they did not improve efficacy and were associated with adverse metabolic effects and weight gain.6-10

SEP-363856 was developed by researchers at Sunovion Pharmaceuticals in collaboration with PsychoGenics. A high-throughput phenotypic screening platform (SmartCube) involving a mouse behavioral model, in combination with in vitro antitarget screening, was used to identify candidate non–D2-receptor-binding compounds that exhibited antipsychotic-like activity. Methods used in medicinal chemistry research were im-plemented to screen selected compounds and ultimately led to obtaining SEP-363856.11,12

The mechanism of action of SEP-363856 has not been fully elucidated. Preclinical data sug-gest that agonism at trace amine–associated re-ceptor 1 (TAAR1) and serotonin 5-HT type 1A (5-HT1A) receptors contributes to its efficacy.11 The drug inhibits firing of a subset of neurons in the ventral tegmental area of the midbrain, partly through TAAR1 agonism.11 This inhibitory effect is consistent with a report of inhibition of dopaminergic neurons through activation of TAAR1.13 Several studies have suggested that the G-protein–coupled TAAR1 receptor has a role in modulating dopaminergic circuitry and has po-tential as a therapeutic target in patients with schizophrenia.13-16 In addition to its inhibitory effect on neurons in the ventral tegmental area, SEP-363856 attenuates ketamine-induced increas-es in striatal dopamine synthesis in mice,17 a finding that suggests that it could modulate presynaptic dopamine dysfunction, a possible contributor to the pathophysiology of schizo-phrenia.18 The drug also has functional activity at the 5-HT1A receptor, which in rodent models inhibits dorsal raphe neuron firing and partially attenuates phencyclidine-induced hyperactivity

through activation of 5-HT1A.11 Preclinical and

clinical studies have implicated 5-HT1A receptor agonists as also having a potential role in the treatment of schizophrenia.19-21

These lines of evidence suggest that SEP-363856 may represent a new class of psychotropic agent with a non–D2-receptor-binding mechanism of action for the treatment of psychosis in schizo-phrenia. We performed a randomized, placebo-controlled trial to evaluate the efficacy and safety of SEP-363856 in patients with an acute exacerbation of schizophrenia.

Me thods

Trial Oversight

The sponsor, Sunovion Pharmaceuticals, designed the trial and provided the trial drug and placebo. All the patients provided written informed con-sent before trial entry. The trial protocol, avail-able with the full text of this article at NEJM.org, was approved by independent ethics committees or institutional review boards at each participat-ing institution, and the trial was conducted in accordance with the International Conference on Harmonisation Good Clinical Practice guidelines and the ethical principles of the Declaration of Helsinki. The informed consent form and the statistical analysis plan are available with the protocol, and a list of the members of the inde-pendent ethics committees and institutional review boards is provided in Table S1 in the Supplemen-tary Appendix, available at NEJM.org. A contract research organization (IQVIA, formerly Quintiles) paid by the sponsor supervised the conduct of the trial under the direction of the sponsor. The analysis and interpretation of the data were performed by the sponsor. A medical writer paid by the sponsor wrote the first draft of the manu-script. All the authors had full access to the trial data, reviewed and approved the manuscript before submission, and vouch for the adherence of the trial to the protocol, the completeness and accuracy of the data and analyses, and the re-porting of adverse events. There are confidenti-ality agreements in place between the authors and the sponsor.

Patient Population

Patients were eligible for inclusion if they were 18 to 40 years of age, met the criteria for schizo-




A Non–D2-Receptor-Binding Drug for Schizophrenia

phrenia in the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-5),22 for at least 6 months, and had (at the time of screen-ing) an acute exacerbation of psychotic symp-toms with a duration of 2 months or less. The Structured Clinical Interview for DSM-5, Clinical Trials Version (SCID-5-CT), was conducted by a trained clinician to confirm the DSM-5 diagno-sis.23 Eligible patients had a score of at least 4 on the Clinical Global Impression of Severity (CGI-S) scale (scores range from 1 to 7, with higher scores indicating higher global severity of schizo-phrenic illness) and a total score on the Positive and Negative Syndrome Scale (PANSS) of at least 80 (scores range from 30 to 210, with higher scores indicating more severe psychotic symp-toms). Patients were excluded if they had three or more prior hospitalizations for treatment of an acute exacerbation of schizophrenia (full in-clusion and exclusion criteria are provided in the protocol). Patients who were receiving treatment with depot neuroleptic agents were excluded un-less the neuroleptic drug had been discontinued at least 30 days before the screening visit.

Trial Design and Procedures

The trial was conducted at 34 clinical sites in five countries in Eastern Europe and North America (Hungary, Romania, Russia, Ukraine, and the United States) from December 2016 through July 2018. The trial consisted of a screening and washout period of up to 14 days, during which all psychotropic medications were discontinued; a double-blind treatment period, during which hospitalized patients who continued to meet entry criteria were randomly assigned in a 1:1 ratio to receive once-daily SEP-363856 (at flexible doses of 50 mg or 75 mg) or placebo for 4 weeks; and a follow-up period, during which patients who decided not to continue in an op-tional 26-week, open-label extension study were assessed 7 days after discontinuation of SEP-363856 or placebo (patients who discontinued the trial drug or placebo before week 4 also completed an assessment at day 7).

Randomization was performed centrally by a biostatistician (not otherwise involved in the trial) with the use of a computer-generated ran-dom-number sequence.24 Capsules containing either the 50-mg or 75-mg doses of SEP-363856 or placebo were provided in blister packs that

were identical in packaging, labeling, weight, appearance, and schedule of administration. The randomized assignment sequence was concealed from the patients and all the trial personnel and investigators. In addition, the sponsor, clinical team members of the contract research organi-zation involved in the trial, data analysts, and personnel at the central laboratories were un-aware of the trial-group assignments.

SEP-363856 or placebo was administered orally once daily at bedtime. Patients received a dose of 50 mg per day of SEP-363856 or matching pla-cebo on day 1 through day 3. On the basis of the investigator’s judgment of the efficacy and safe-ty in individual patients, adjustment of the dose to a maximum of 75 mg per day of SEP-363856 or equivalent placebo was permitted (but not required) on day 4 and at subsequent regularly scheduled weekly trial visits. A dose reduction (from 75 mg to 50 mg per day of SEP-363856 or equivalent placebo) because of unacceptable side effects was permitted at any time.

Treatment with anticholinergic agents or pro-pranolol was permitted for akathisia and move-ment disorders. Lorazepam, temazepam, and eszopiclone (or their equivalents) were permitted as needed for anxiety or insomnia, but not within 8 hours before any trial assessment.

At the completion of the trial, patients were given the option to enroll in an outpatient study in which they received open-label treatment with SEP-363856 in flexible doses of 25 mg, 50 mg, or 75 mg per day for 26 weeks. The patients provided written informed consent before par-ticipation in the extension study. To maintain the double-blind nature of the placebo-controlled trial, the patients enrolled in the extension study received a starting dose of SEP-363856 of 50 mg per day for 3 days, regardless of their initial trial-group assignment. The patients were assessed at weekly intervals for the first 4 weeks and then every 4 weeks thereafter up to week 24; a final assessment was performed at week 26.

End Points

Efficacy was assessed with the use of the PANSS, the CGI-S scale,25 the Brief Negative Symptom Scale (BNSS; total scores range from 0 to 78, with higher scores indicating greater severity of negative symptoms),26 and the Montgomery–





Åsberg Depression Rating Scale (MADRS; total scores range from 0 to 60, with higher scores indicating greater severity of symptoms of de-pression).27 Training and certification of investi-gators for all efficacy assessments were provided before trial enrollment.

The primary efficacy end point was the change from baseline in the total score on the PANSS at week 4. Prespecified secondary effi-cacy end points were the least-squares mean changes from baseline in the CGI-S scale score, the PANSS subscale (positive, negative, and gen-eral psychopathology) scores, and the total scores on the BNSS and MADRS; a PANSS response (defined as an improvement of ≥20% in the total score on the PANSS); and the change from base-line in the uncorrelated PANSS score matrix (UPSM)-transformed PANSS factor severity scores.28 The UPSM-transformed PANSS factors measure drug effects on clinical symptom domains of schizophrenia with greater specificity by cor-recting for correlated improvements among the individual PANSS items.

Safety

Safety assessments included monitoring for ad-verse events and serious adverse events, evalua-tion of vital signs and weight, laboratory tests (including fasting lipid and glucose levels), and 12-lead electrocardiography. Extrapyramidal symp-toms were assessed with the use of the Simpson–Angus Rating Scale (SAS; scores range from 0 to 40, with higher scores indicating more extrapy-ramidal signs in 10 domains, each scored from 0 to 4),29 the Barnes Rating Scale for Drug- Induced Akathisia (BARS; global clinical assess-ment scores range from 0 to 5, with higher scores indicating greater akathisia),30 and the Abnormal Involuntary Movement Scale (AIMS; scores range from 0 to 44, with higher scores indicating more frequent and severe abnormal involuntary movements).31 Suicidality was assessed with the use of the Columbia–Suicide Severity Rating Scale (C-SSRS).32 Sleep quality was as-sessed with the use of the Pittsburgh Sleep Quality Index (PSQI; global scores range from 0 to 21, with higher scores indicating worse sleep quality).33

Statistical Analysis

Analyses of efficacy were performed in the modi-fied intention-to-treat population, which included

all patients who underwent randomization, re-ceived at least one dose of SEP-363856 or place-bo, and had a baseline measurement and at least one postbaseline measurement of efficacy based on the PANSS or CGI-S scale. However, all the patients fulfilled these criteria, and the modified intention-to-treat population was the same as the intention-to-treat population. The safety popula-tion included all patients who underwent ran-domization and received at least one dose of SEP-363856 or placebo. No interim analyses or unblinded data monitoring were performed in this trial.

The primary efficacy end point was evaluated with the use of a mixed model for repeated mea-sures; effect sizes were calculated as the absolute value of the difference between the SEP-363856 group and the placebo group in the change in score from baseline at week 4, divided by the pooled standard deviation of the between-group difference in the change in score. In order to assess the robustness of the mixed model for repeated-measures analysis of the primary end point and the potential effect of missing data due to early withdrawals, a tipping-point analy-sis and pattern-mixture modeling with placebo-based multiple imputation were performed as sensitivity analyses.

The secondary efficacy end points were also evaluated with the use of a mixed model for re-peated measures, with the exception of the UPSM-transformed PANSS factors, which were evaluated with the use of a prespecified analysis-of-covariance model to assess domain-specific change in patients who completed the 4-week trial. Because no adjustment for multiple com-parisons was performed, no inferences can be drawn regarding the secondary efficacy end points; these results are presented as point estimates with unadjusted 95% confidence intervals only. PANSS response was evaluated with the use of a logistic-regression model with total score on the PANSS at baseline as a covariate.

Descriptive statistics were used to analyze safety data, including adverse events, laboratory values, findings from clinical evaluations, and C-SSRS scores. Changes from baseline in scores on the SAS, BARS, and AIMS were evaluated with the use of a mixed model for repeated measures. Only one postbaseline PSQI assessment was per-formed, and thus these data were evaluated with the use of analysis of covariance.





R esult s

Patients

A total of 295 patients were screened, of whom 245 underwent randomization, received at least one dose of SEP-363856 or placebo, and had at least one postbaseline efficacy evaluation (Fig. 1). The 4-week trial was completed by 78.3% of the patients in the SEP-363856 group and by 79.2% of those in the placebo group; the reasons for discontinuation of SEP-363856 or placebo are summarized in Figure 1. The percentages of patients in the SEP-363856 group who were re-ceiving the 75-mg-per-day dose were 67.2% at week 1, 70.0% at week 2, and 72.5% at week 3; the SEP-363856 dose was reduced from 75 mg to 50 mg per day in 4 patients. The mean (±SD) duration of exposure to SEP-363856 or placebo was 24.3±7.6 days in the SEP-363856 group and 25.4±6.4 days in the placebo group. The use of concomitant medications included anticholiner-gic agents (1 patient in the placebo group), anti-psychotic agents (1 patient in the placebo group), anxiolytics (32 patients in the SEP-363856 group and 30 in the placebo group), and hypnotics and

sedative agents (10 patients in the SEP-363856 group and 15 in the placebo group).

Clinical and demographic characteristics among the patients at baseline were similar in the two trial groups (Table 1); the mean age was 30.3 years, 81.6% were white, 63.7% were male, and the mean time since the onset of schizo-phrenia was 5 years. The mean total scores on the PANSS at baseline were 101.4 in the SEP-363856 group and 99.7 in the placebo group.

Efficacy

The least-squares mean change from baseline in the total score on the PANSS at week 4 was −17.2 points in the SEP-363856 group and −9.7 points in the placebo group (least-squares mean differ-ence, −7.5 points; 95% confidence interval [CI], −11.9 to −3.0; P = 0.001) (Table 2 and Fig. 2). The between-group least-squares mean differences in the changes from baseline in the CGI-S scale score and the PANSS positive, negative, and gen-eral psychopathology subscale scores at week 4 were −0.5 points (95% CI, −0.7 to −0.2), −1.7 points (95% CI, −3.1 to −0.3), −1.5 points (95% CI, −2.6 to −0.4), and −4.3 points (95% CI, −6.6

Figure 1. Assessment, Randomization, and Analysis.

245 Underwent randomization

295 Patients were assessed for eligibility

50 Were excluded39 Did not meet inclusion criteria8 Declined to participate3 Had other reasons

26 Discontinued treatment4 Had lack of efficacy8 Had adverse event

14 Withdrew consent

26 Discontinued treatment5 Had lack of efficacy

10 Had adverse event9 Withdrew consent2 Had other reasons

94 Completed the double-blind trial78 Continued into the extension study

120 Were included in the analysis

99 Completed the double-blind trial 79 Continued into the extension study

125 Were included in the analysis

120 Were assigned to and received SEP-363856

125 Were assigned to and receivedplacebo





to −2.0), respectively (Table 2). Effect sizes with respect to the changes in the uncorrelated UPSM-transformed PANSS factor severity scores are shown in Figure S1 in the Supplementary Ap-pendix. The between-group least-squares mean differences in the changes from baseline in the

total scores on the BNSS and MADRS at week 4 were −4.3 points (95% CI, −6.8 to −1.8) and −1.8 points (95% CI, −3.2 to −0.3), respectively (Ta-ble 2). A PANSS response at week 4 was observed in 64.6% of the patients in the SEP-363856 group and in 44.0% of those in the placebo group (odds ratio, 2.6; 95% CI, 1.4 to 4.9). Because of the lack of a prespecified plan for adjustment for multiple comparisons of secondary end points, the confidence intervals are not adjusted for multiple comparisons and no inferences can be drawn from any secondary end-point data. The results of planned sensitivity analyses were in the same direction as the results of the primary efficacy analysis (Table S2). A trial-group-by-country analysis showed no significant interac-tion effect at week 4 (Fig. S2).

Safety

Adverse events are summarized in Table 3. Se-vere adverse events occurred in seven patients (5.8%) in the SEP-363856 group and in two pa-tients (1.6%) in the placebo group. The incidence of insomnia was 3.3% in the SEP-363856 group and 10.4% in the placebo group, and concomitant hypnotics were used by 8.3% and 12.0% of the patients, respectively. Treatment with SEP-363856, as compared with placebo, was associated with an improvement in sleep quality, with a least-squares mean (±SE) change from baseline in the PSQI global score at week 4 of −2.5±0.4 points in the SEP-363856 group and −1.7±0.4 points in the placebo group.

The incidence of extrapyramidal symptoms (akathisia, restlessness, musculoskeletal or joint stiffness, tremor, and nuchal rigidity) was 3.3% in the SEP-363856 group and 3.2% in the placebo group. At week 4, the least-squares mean changes from baseline in the scores on the SAS, the BARS, and the AIMS, which were used to deter-mine the effects on movement disorders, were −0.01±0.01 points, 0.0±0.06 points, and 0.0±0.01 points, respectively, in the SEP-363856 group and 0.01±0.01 points, 0.1±0.05 points, and 0.0±0.01 points, respectively, in the placebo group. A con-comitant medication to treat extrapyramidal symptoms was prescribed to one patient in the SEP-363856 group (lorazepam for restlessness) and one patient in the placebo group (trihexy-phenidyl for hand tremor and restlessness).

The two serious adverse events that occurred in the SEP-363856 group were worsening of

Table 1. Characteristics of the Patients at Baseline (Intention-to-Treat Population).*

CharacteristicSEP-363856

(N = 120)Placebo (N = 125)

Age — yr 30.0±5.8 30.6±6.1

Male sex — no. (%) 77 (64.2) 79 (63.2)

Race — no. (%)†

White 96 (80.0) 104 (83.2)

Black 19 (15.8) 20 (16.0)

Other 5 (4.2) 1 (0.8)

Hispanic ethnic group — no. (%)† 5 (4.2) 6 (4.8)

Body-mass index‡ 25.0±4.3 24.7±3.7

Years since initial onset of schizophrenia 5.3±4.8 5.5±4.8

No. of prior psychiatric hospitalizations 1.3±0.7 1.2±0.7

PANSS score§

Total 101.4±8.4 99.7±7.8

Positive subscale 25.8±3.3 25.4±3.1

Negative subscale 24.7±3.9 24.9±4.0

CGI-S scale score¶ 5.0±0.4 4.9±0.5

BNSS total score‖ 37.2±11.5 37.4±12.0

MADRS total score** 13.1±7.2 12.6±7.1

Country — no. (%)

United States 27 (22.5) 25 (20.0)

Hungary 6 (5.0) 6 (4.8)

Romania 5 (4.2) 5 (4.0)

Russia 46 (38.3) 52 (41.6)

Ukraine 36 (30.0) 37 (29.6)

* Plus–minus values are means ±SD. The intention-to-treat population com-prised all patients who underwent randomization.

† Race and Hispanic ethnic group were reported by the patients.‡ The body-mass index is the weight in kilograms divided by the square of the

height in meters.§ The total score on the Positive and Negative Syndrome Scale (PANSS)

ranges from 30 to 210, and the scores on the PANSS positive and negative subscales range from 7 to 49; higher scores indicate greater severity of psy-chotic symptoms.

¶ The score on the Clinical Global Impression of Severity (CGI-S) scale ranges from 1 to 7, with higher scores indicating greater global illness severity.

‖ The total score on the Brief Negative Symptom Scale (BNSS) ranges from 0 to 78, with higher scores indicating greater severity of negative symptoms.

** The total score on the Montgomery–Åsberg Depression Rating Scale (MADRS) ranges from 0 to 60, with higher scores indicating greater severity of symp-toms of depression.





schizophrenia and acute cardiovascular insuf-ficiency in a 37-year-old woman that resulted in sudden death 7 days after she had taken the first 50-mg dose of SEP-363856. The patient had a history of essential hypertension and was found on autopsy to have coronary artery disease and pulmonary embolism. There were four serious events in the placebo group (three patients had worsening of schizophrenia and one patient at-tempted suicide). According to the scores on the C-SSRS, no suicidal ideation or behavior was present among the patients in the SEP-363856 group, and there were two instances of suicidal-ity among those in the placebo group.

Differences in vital signs (including ortho-static hypotension or tachycardia), weight, body-mass index, and laboratory values (including prolactin and fasting metabolic measures) be-tween the SEP-363856 group and the placebo group are shown in Table 3. There were no clinically significant electrocardiographic abnor-malities after baseline, and no patient in the SEP-363856 group or the placebo group had a prolongation of the corrected QT interval, calcu-

lated with the use of Fridericia’s formula (QTcF), of more than 450 msec or an increase in the QTcF interval of more than 60 msec.

Extension Study

A total of 156 patients (80.8% of those who com-pleted the 4-week trial) were enrolled and re-ceived treatment with SEP-363856 in the 26-week, open-label extension study. Among 77 patients who had initially been randomly assigned to receive SEP-363856 in the double-blind trial and then continued to receive treatment in the open-label extension, the mean (±SD) change from the extension-study baseline in the PANSS total score at week 26 was −17.1±12.4 points (Fig. S3). Among 79 patients who had initially been ran-domly assigned to receive placebo and then switched to open-label SEP-363856, the mean change from the extension-study baseline in the PANSS total score at week 26 was −27.9±16.4 points. The adverse events related to extrapyra-midal symptoms that occurred among the 156 patients included parkinsonism (2 patients), dys-kinesia (1 patient), tremor (1 patient), and rest-

Table 2. Changes from Baseline in Efficacy Measures at Week 4.*

Efficacy MeasureLeast-Squares Mean Change from Baseline

at Week 4Least-Squares Mean Difference (95% CI)

SEP-363856, 50 mg or 75 mg Placebo

Primary end point

PANSS total score −17.2±1.7 −9.7±1.6 −7.5 (−11.9 to −3.0)†

Secondary end points‡

CGI-S score −1.0±0.1 −0.5±0.1 −0.5 (−0.7 to −0.2)

PANSS positive subscale score −5.5±0.5 −3.9±0.5 −1.7 (−3.1 to −0.3)

PANSS negative subscale score −3.1±0.4 −1.6±0.4 −1.5 (−2.6 to −0.4)

PANSS general psychopathology subscale score −9.0±0.9 −4.7±0.8 −4.3 (−6.6 to −2.0)

BNSS total score −7.1±1.0 −2.7±0.9 −4.3 (−6.8 to −1.8)

MADRS total score −3.3±0.6 −1.6±0.6 −1.8 (−3.2 to −0.3)

* Plus–minus values are means ±SE. Changes in efficacy measures were evaluated with the use of a mixed model for repeated measures. The model included trial group, visit (day 4 and weeks 1, 2, 3, and 4 [categorical variables]), clinical site (pooled by country), and trial-group-by-visit interaction as factors and included the baseline PANSS total score as a covariate. At baseline, the PANSS total score and positive, negative, and general psychopathology subscale scores, the CGI-S score, and the MADRS total score were evaluated in 120 patients in the SEP-363856 group and in 125 patients in the placebo group, and at week 4, these scores were evaluated in 96 and 100 patients, respectively. At baseline, the BNSS total score was evaluated in 113 patients in the SEP-363856 group and in 119 patients in the placebo group, and at week 4, the BNSS score was evaluated in 89 and 96 patients, respectively.

† The estimated effect size was 0.45 (P = 0.001). Effect size was calculated as the absolute value of the difference between the SEP-363856 group and the placebo group in the change in score from baseline at week 4, divided by the pooled standard deviation of the between-group difference in the change in score.

‡ No inferences can be made from the results for the secondary end points because there was no plan for adjustment for multiple comparisons.





lessness (1 patient). Additional safety data are summarized in Table S3.

Discussion

In this 4-week, flexible-dose trial involving pa-tients with an acute exacerbation of schizophre-nia, SEP-363856, a drug that does not bind to the dopamine D2 receptor, resulted in a greater re-duction from baseline in the PANSS total score at week 4 (the primary efficacy end point) than placebo. Treatment with SEP-363856 was associ-ated with changes in the severity scores on the secondary efficacy measures (including the CGI-S scale and the PANSS positive, negative, and gen-eral psychopathology subscales) at week 4 that were in the same direction as the scores in the primary efficacy analysis. Treatment with SEP-363856 was also associated with changes in se-verity scores of negative symptoms of schizo-phrenia at week 4 (as measured by both the BNSS total score and the UPSM-transformed PANSS negative symptom factor scores) that were in the same direction as the scores in the primary efficacy analysis. Changes in the UPSM-transformed PANSS negative symptom factor scores (apathy or avolition and deficit of expres-

sion) were shown previously to have minimal correlations with change in the UPSM-trans-formed PANSS positive symptom factor score,29 a finding that suggests that specific effects on negative symptoms are measured by the UPSM negative symptom factors. However, because there was no plan for adjustment for multiple comparisons with respect to secondary efficacy outcomes, no inferences can be drawn from any of these secondary outcome data. A reduction in the total score on the PANSS was observed dur-ing the additional 26-week extension study of open-label treatment with SEP-363856 (Fig. S2); however, because no control group was available for comparison, no conclusions can be drawn from this reduction in PANSS score.

The percentage of patients who discontinued SEP-363856 or placebo was similar in the two groups (21.7% vs. 20.8%) and was similar to or lower than the percentage of patients who had dropped out of previous short-term trials of first- and second-generation antipsychotics; how-ever, the design of the previous trials does not allow direct comparison with our trial.34 The incidence of adverse events was generally similar in the SEP-363856 group and the placebo group, with a difference of 2.5% or less for each event. The SEP-363856 and placebo groups were similar with respect to the percentage of patients who reported extrapyramidal symptoms (3.3% vs. 3.2%), the percentage who used medications to treat extrapyramidal symptoms, and the findings on movement disorder scales. In addition, minimal effects on prolactin were observed in the SEP-363856 group. These findings are consistent with the absence of D2-receptor binding for SEP-363856. Short-term treatment with SEP-363856 was asso-ciated with a mean increase in body weight of 0.3 kg, reductions in total and LDL cholesterol levels, and no change in other metabolic labora-tory values. No clinically significant electrocar-diographic abnormalities, including prolongation in the QTcF interval, were present after baseline.

A limitation of the trial should be noted. The generalizability of the findings from this trial is limited by its short duration and by enrollment criteria that excluded persons older than 40 years of age and those who had more than two prior hospitalizations for exacerbation of schizophrenia.

In conclusion, in this 4-week trial involving patients with an acute exacerbation of schizophre-nia, SEP-363856, a drug with a non–D2-receptor-

Figure 2. Least-Squares Mean Change from Baseline in the Total Score on the PANSS.

Shown is the mixed model for repeated-measures analysis performed in the modified intent-to-treat population, which included all patients who under-went randomization, received at least one dose of SEP-363856 or placebo, and had a baseline and at least one postbaseline measurement of efficacy based on the Positive and Negative Symptom Scale (PANSS) or the Clinical Global Impression of Severity scale. I bars indicate the standard error.

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−6

−8

−12

−14

−10

−16

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Day4

Baseline 1 2 3 4

Week

P=0.001

No. of PatientsPlaceboSEP-363856

125120

125120

122115

117109

113102

10096

SEP-363856

Placebo





binding mechanism of action but with agonist activity at TAAR1 and 5-HT1A receptors, led to a greater reduction in the total score on the PANSS than placebo. Longer and larger trials are necessary to determine the effects and safety of SEP-363856, as well as its efficacy relative to existing drug treatments for schizophrenia.

Supported by Sunovion Pharmaceuticals.Disclosure forms provided by the authors are available with

the full text of this article at NEJM.org.A data sharing statement provided by the authors is available

with the full text of this article at NEJM.org.We thank all the patients, investigators, and site staff for par-

ticipating in this trial and Edward Schweizer, M.D., for provid-ing writing and editorial assistance (funded by Sunovion Phar-maceuticals) with earlier versions of the manuscript.

Table 3. Adverse Events and Changes from Baseline in Body Weight, Body-Mass Index, Fasting Metabolic Laboratory Values, and Prolactin Level at Week 4 (Safety Population).*

VariableSEP-363856,

50 mg or 75 mg Placebo

Adverse events — no./total no. (%)†

Any adverse event 55/120 (45.8) 63/125 (50.4)

Somnolence 8/120 (6.7) 6/125 (4.8)

Agitation 6/120 (5.0) 6/125 (4.8)

Nausea 6/120 (5.0) 4/125 (3.2)

Diarrhea 3/120 (2.5) 1/125 (0.8)

Dyspepsia 3/120 (2.5) 0/125

Serious adverse events — no./total no. (%)‡

Worsening of schizophrenia 1/120 (0.8) 3/125 (2.4)

Sudden cardiac death 1/120 (0.8)§ 0/125

Suicide attempt 0/120 1/125 (0.8)

Changes from baseline in body weight and body-mass index at week 4¶

Body weight — kg 0.3±1.9 −0.1±2.3

Body-mass index 0.1±0.6 0.0±0.8

Median changes from baseline in fasting metabolic laboratory values at week 4‖

Total cholesterol — mmol/liter −0.2 0.0

LDL cholesterol — mmol/liter −0.1 0.0

Triglycerides — mmol/liter 0.0 −0.1

Glucose — mmol/liter 0.0 0.1

Glycated hemoglobin — % 0.0 0.0

Median change from baseline in prolactin level at week 4, male/female — nmol/liter

−0.037/−0.175 −0.036/−0.101

* Plus–minus values are means ±SD. The safety population included all patients who underwent randomization and re-ceived at least one dose of SEP-363856 or placebo.

† Shown are the adverse events that occurred during the 4-week treatment and 7-day follow-up periods, had a reported frequency of at least 2%, and were more common in the SEP-363856 group than in the placebo group.

‡ Shown are serious adverse events that occurred during the 4-week treatment and 7-day follow-up periods.§ The patient had a history of essential hypertension and was found on autopsy to have coronary artery disease and pul-

monary embolism.¶ The changes in body weight and body-mass index at week 4 were assessed in 120 patients in the SEP-363856 group

and 125 patients in the placebo group.‖ The changes from baseline in total cholesterol and triglyceride levels were assessed in 104 patients in the SEP-363856

group and 107 patients in the placebo group; the change in low-density lipoprotein (LDL) cholesterol level, in 104 and 106 patients, respectively; the change in glucose level, in 103 and 107 patients, respectively; the change in glycated hemoglobin level, in 113 and 111 patients, respectively; and the change in prolactin level, in 74 male and 40 female pa-tients and 71 male and 42 female patients, respectively. To convert the values for cholesterol to milligrams per deciliter, divide by 0.02586. To convert the values for triglycerides to milligrams per deciliter, divide by 0.01129. To convert the values for glucose to milligrams per deciliter, divide by 0.05551.





References1. Kaar SJ, Natesan S, McCutcheon R, Howes OD. Antipsychotics: mechanisms underlying clinical response and side-effects and novel treatment approaches based on pathophysiology. Neuropharma-cology 2019 July 9 (Epub ahead of print).2. Leucht S, Leucht C, Huhn M, et al. Sixty years of placebo-controlled antipsy-chotic drug trials in acute schizophrenia: systematic review, Bayesian meta-analy-sis, and meta-regression of efficacy pre-dictors. Am J Psychiatry 2017; 174: 927-42.3. Keefe RS, Bilder RM, Davis SM, et al. Neurocognitive effects of antipsychotic medications in patients with chronic schizophrenia in the CATIE Trial. Arch Gen Psychiatry 2007; 64: 633-47.4. Stahl SM, Buckley PF. Negative symp-toms of schizophrenia: a problem that will not go away. Acta Psychiatr Scand 2007; 115: 4-11.5. Krause M, Zhu Y, Huhn M, et al. Anti-psychotic drugs for patients with schizo-phrenia and predominant or prominent negative symptoms: a systematic review and meta-analysis. Eur Arch Psychiatry Clin Neurosci 2018; 268: 625-39.6. Lieberman JA, Stroup TS, McEvoy JP, et al. Effectiveness of antipsychotic drugs in patients with chronic schizophrenia. N Engl J Med 2005; 353: 1209-23.7. Leucht S, Cipriani A, Spineli L, et al. Comparative efficacy and tolerability of 15 antipsychotic drugs in schizophrenia: a multiple-treatments meta-analysis. Lan-cet 2013; 382: 951-62.8. Laursen TM, Nordentoft M, Mortensen PB. Excess early mortality in schizophre-nia. Annu Rev Clin Psychol 2014; 10: 425-48.9. Cooper SJ, Reynolds GP, Barnes T, et al. BAP guidelines on the management of weight gain, metabolic disturbances and cardiovascular risk associated with psychosis and antipsychotic drug treat-ment. J Psychopharmacol 2016; 30: 717-48.10. Taipale H, Mittendorfer-Rutz E, Alex-anderson K, et al. Antipsychotics and mortality in a nationwide cohort of 29,823 patients with schizophrenia. Schizophr Res 2018; 197: 274-80.11. Dedic N, Jones PG, Hopkins SC, et al. SEP-363856, a novel psychotropic agent with a unique, non-D2 receptor mecha-nism of action. J Pharmacol Exp Ther 2019; 371: 1-14.

12. Shao L, Campbell UC, Fang QK, et al. In vivo phenotypic drug discovery: apply-ing a behavioral assay to the discovery and optimization of novel antipsychotic agents. MedChemComm 2016; 7: 1093-101.13. Revel FG, Moreau J-L, Gainetdinov RR, et al. TAAR1 activation modulates monoaminergic neurotransmission, pre-venting hyperdopaminergic and hypo-glutamatergic activity. Proc Natl Acad Sci U S A 2011; 108: 8485-90.14. Lindemann L, Meyer CA, Jeanneau K, et al. Trace amine-associated receptor 1 modulates dopaminergic activity. J Phar-macol Exp Ther 2008; 324: 948-56.15. Rutigliano G, Accorroni A, Zucchi R. The case for TAAR1 as a modulator of central nervous system function. Front Pharmacol 2018; 8: 987.16. Schwartz MD, Canales JJ, Zucchi R, Espinoza S, Sukhanov I, Gainetdinov RR. Trace amine-associated receptor 1: a mul-timodal therapeutic target for neuropsy-chiatric diseases. Expert Opin Ther Tar-gets 2018; 22: 513-26.17. Kokkinou M, Irvine EE, Bonsall DR, et al. Mesocorticolimbic circuit mecha-nisms underlying the effects of ketamine on dopamine: a translational imaging study. bioRxiv (in press) (https://www .biorxiv .org/ content/ 10 .1101/ 748665v1).18. McCutcheon R, Beck K, Jauhar S, Howes OD. Defining the locus of dopa-minergic dysfunction in schizophrenia: a meta-analysis and test of the mesolim-bic hypothesis. Schizophr Bull 2018; 44: 1301-11.19. Miyamoto S, Duncan GE, Marx CE, Lieberman JA. Treatments for schizophre-nia: a critical review of pharmacology and mechanisms of action of antipsychotic drugs. Mol Psychiatry 2005; 10: 79-104.20. Celada P, Bortolozzi A, Artigas F. Sero-tonin 5-HT1A receptors as targets for agents to treat psychiatric disorders: ra-tionale and current status of research. CNS Drugs 2013; 27: 703-16.21. Newman-Tancredi A. The importance of 5-HT1A receptor agonism in antipsy-chotic drug action: rationale and perspec-tives. Curr Opin Investig Drugs 2010; 11: 802-12.22. Diagnostic and statistical manual of mental disorders, 5th ed.: DSM V. Wash-

ington, DC: American Psychiatric Associ-ation, 2013.23. First MB, Williams JBW, Karg RS, Spitzer RL. Structured clinical interview for DSM-5 disorders: clinical trials ver-sion (SCID-5-CT). Washington, DC: Amer-ican Psychiatric Association, 2015.24. Ruikar V. Interactive voice/Web re-sponse system in clinical research. Per-spect Clin Res 2016; 7: 15-20.25. Guy W, ed. ECDEU assessment manual for psychopharmacology. Rockville, MD: Department of Health, Education, and Welfare, 1976.26. Kirkpatrick B, Strauss GP, Nguyen L, et al. The Brief Negative Symptom Scale: psychometric properties. Schizophr Bull 2011; 37: 300-5.27. Montgomery SA, Asberg M. A new de-pression scale designed to be sensitive to change. Br J Psychiatry 1979; 134: 382-9.28. Hopkins SC, Ogirala A, Loebel A, Ko-blan KS. Transformed PANSS factors in-tended to reduce pseudospecificity among symptom domains and enhance under-standing of symptom change in antipsy-chotic-treated patients with schizophre-nia. Schizophr Bull 2018; 44: 593-602.29. Simpson GM, Angus JWS. A rating scale for extrapyramidal side effects. Acta Psychiatr Scand Suppl 1970; 212: 11-9.30. Barnes TR. A rating scale for drug-induced akathisia. Br J Psychiatry 1989; 154: 672-6.31. Rush AJ Jr, ed. Handbook of psychiat-ric measures. Washington, DC: American Psychiatric Association, 2000: 166-8.32. Posner K, Brown GK, Stanley B, et al. The Columbia-Suicide Severity Rating Scale: initial validity and internal consis-tency findings from three multisite stud-ies with adolescents and adults. Am J Psy-chiatry 2011; 168: 1266-77.33. Buysse DJ, Reynolds CF III, Monk TH, Berman SR, Kupfer DJ. The Pittsburgh Sleep Quality Index: a new instrument for psychiatric practice and research. Psychi-atry Res 1989; 28: 193-213.34. Kemmler G, Hummer M, Widschwend-ter C, Fleischhacker WW. Dropout rates in placebo-controlled and active-control clin-ical trials of antipsychotic drugs: a meta-analysis. Arch Gen Psychiatry 2005; 62: 1305-12.Copyright © 2020 Massachusetts Medical Society.



JPET#260281

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TITLE: SEP-363856, A NOVEL PSYCHOTROPIC AGENT WITH A UNIQUE, NON-D2

RECEPTOR MECHANISM OF ACTION

NINA DEDIC1*, PHILIP G. JONES1*, SETH C. HOPKINS1, ROBERT LEW1, LIMING

SHAO1, JOHN E. CAMPBELL1, KERRY L. SPEAR1, THOMAS H. LARGE1, UNA C.

CAMPBELL1, TALEEN HANANIA2, EMER LEAHY2, KENNETH S. KOBLAN1

*both authors contributed equally to the work

1 Sunovion Pharmaceuticals, 84 Waterford Drive, Marlborough, MA 01752

2 Psychogenics Inc., 215 College Road, Paramus, NJ 07652

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on August 1, 2019 as DOI: 10.1124/jpet.119.260281

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Running title: SEP-363856, a novel, non-D2 psychotropic agent

Corresponding author:

Kenneth S. Koblan, PhD

Chief Scientific Officer

Sunovion Pharmaceuticals

84 Waterford Drive, Marlborough, MA 01752

Email: [email protected]

Word Count (including references):

Abstract: 249 words

Introduction: 609

Discussion: 1932

Non-standard abbreviations:

MOA – Mechanism of action

LMA – locomotor activity

Tb – body temperature

NREM – non-rapid eye movement sleep

PPI – prepulse inhibition

VTA – ventral tegmental area

DRN – dorsal raphe nucleus

s.e.m. – standard error of the mean

s.d. – standard deviation


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ABSTRACT

For the past 50 years, the clinical efficacy of antipsychotic medications has relied on

blockade of dopamine D2 receptors. Drug development of non-D2 compounds, seeking

to avoid the limiting side effects of dopamine receptor blockade, has failed to date to

yield new medicines for patients. Here we report the discovery of SEP-363856 (SEP-

856), a novel psychotropic agent with a unique mechanism of action. SEP-856 was

discovered in a medicinal chemistry effort utilizing a high throughput, high content,

mouse-behavior phenotyping platform, in combination with in vitro screening, aimed at

developing non-D2 (anti-target) compounds that could nevertheless retain efficacy

across multiple animal models sensitive to D2-based pharmacological mechanisms.

SEP-856 demonstrated broad efficacy in putative rodent models relating to aspects of

schizophrenia, including phencyclidine (PCP)-induced hyperactivity, prepulse inhibition

and PCP-induced deficits in social interaction. In addition to its favorable

pharmacokinetic properties, lack of D2 receptor occupancy and the absence of

catalepsy, SEP-856’s broad profile was further highlighted by its robust suppression of

rapid eye movement sleep in rats. Although the mechanism of action has not been fully

elucidated, in vitro and in vivo pharmacology data as well as slice and in vivo

electrophysiology recordings suggest that agonism at both trace amine-associated

receptor 1 (TAAR1) and 5-HT1A receptors are integral to its efficacy. Based on the

preclinical data and its unique mechanism of action, SEP-856 is a promising new agent

for the treatment of schizophrenia and represents a new pharmacological class

expected to lack the side effects stemming from blockade of D2 signaling.


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SIGNIFICANCE STATEMENT (120 words max)

Since the discovery of chlorpromazine in the 1950’s, the clinical efficacy of antipsychotic

medications has relied on blockade of dopamine D2 receptors, which is associated with

substantial side effects and little to no efficacy in treating the negative and cognitive

symptoms of schizophrenia. Here we describe the discovery and pharmacology of SEP-

363856, a novel psychotropic agent that does not exerts its antipsychotic-like effects

through direct interaction with D2 receptors. Although the mechanism of action has not

been fully elucidated, our data suggest that agonism at both TAAR1 and 5-HT1A

receptors are integral to its efficacy. Based on its unique profile in preclinical species,

SEP-363856 represents a promising candidate for the treatment of schizophrenia and

potentially other neuropsychiatric disorders.


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INTRODUCTION

Schizophrenia is a chronic and disabling psychiatric disorder that affects approximately

1% of the global population. It is characterized by positive symptoms (eg. hallucinations,

delusions and thought disorders), negative symptoms (eg. flat affect, anhedonia, alogia

and avolition) and cognitive deficits (eg. impaired memory, attention and executive

functioning). Despite advances in our understanding of the pathophysiology,

schizophrenia remains one of the most challenging diseases to treat due to the diversity

of clinical symptoms, the heterogeneity of clinical response, the side effects of current

treatments and its association with high morbidity and mortality (Insel, 2010; Meyer-

Lindenberg, 2010; Girgis et al., 2018).

Antipsychotics have been the standard of care for schizophrenia since the discovery of

chlorpromazine in the 1950s (Charpentier et al., 1952; Laborit et al., 1952; Lehmann

and Ban, 1997). Since then, numerous “new” antipsychotics have been launched but

they have essentially the same mechanism of action, mediating their efficacy against

the positive symptoms through antagonism of dopamine D2 and/or serotonin 5-HT2A

receptors. While improvements in drug safety have been made, a focus on the same

molecular targets has not led to improved efficacy (Lieberman et al., 2005; Girgis et al.,

2018). In fact, the negative and cognitive symptoms remain largely untreated by

currently available antipsychotics. Furthermore, approximately 30% of patients have

treatment-resistant schizophrenia (Samara et al., 2016). The urgency for new

treatments is therefore apparent.

More recently, drug development efforts have focused on molecular targets other than

D2 and 5-HT2A receptors including GlyT1, D1, D4, D3, NMDA, mGluR2/3, AMPA, 5-HT2C,


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nicotinic α7, muscarinic M1/M4, H3, NK-3, and sigma receptors (Miyamoto et al., 2000;

Karam et al., 2010; Girgis et al., 2018). However, despite promising efficacy in

preclinical models for many of these targets, most novel, non-D2/5HT2A mechanisms

have shown limited or no success in clinical trials (Girgis et al., 2018). Thus, it is crucial

to pursue alternative strategies for novel drug development for schizophrenia.

Traditional drug discovery efforts have been focused on designing compounds with high

selectivity and potency for a target protein of interest. Unfortunately, in psychiatry there

are few validated drug targets, in part due to the complexity of the disorders, rendering

this approach largely unsuccessful. Phenotypic drug discovery (PDD) does not require

any knowledge of a molecular target(s) associated with a disease and has been

associated with the discovery of “first-in-class” medications (Swinney and Anthony,

2011; Moffat et al., 2017). Examples include most anticonvulsants as well as antiviral

drugs such as daclatasvir, which was discovered using a cell-based phenotypic screen

(Belema and Meanwell, 2014). An in vivo PDD approach could be particularly valuable

for the discovery of new therapeutics for psychiatric indications that have a complex

underlying pathophysiology and where polypharmacology is common and likely

necessary for clinical efficacy. However, the selection of an appropriate target

combination and the design of a safe and efficacious polypharmacological molecule is

extremely difficult. We therefore took a target-agnostic in vivo approach by utilizing a

mouse behavioral platform (Roberds et al., 2011; Alexandrov et al., 2015; Shao et al.,

2016) together with anti-target in vitro screening to identify antipsychotic-like

compounds that don’t exert their effects through direct modulation of D2 or 5-HT2A

receptors.


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Here we report the discovery of SEP-363856 (SEP-856), a novel psychotropic agent

with a unique, non-D2/5-HT2A mechanism of action. SEP-856 exhibits antipsychotic-like

efficacy in vivo and demonstrates the potential for treating the positive and negative

symptoms of schizophrenia. Although the mechanism of action has not been fully

elucidated, in vitro and in vivo pharmacology data suggest that agonism at both 5-HT1A

receptors and TAAR1 are integral to its efficacy. The data presented here suggest that

SEP-856 may have broad therapeutic efficacy in schizophrenia and potentially other

psychiatric disorders.


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MATERIALS AND METHODS

Animals

For behavioral screening, PCP-induced hyperactivity, catalepsy and prepulse inhibition

(PPI) studies as well as patch-clamp recordings, adult male C57BL/6J mice were used.

The forced swim test (FST) was performed in adult male BalbC/J mice. For the

electroencephalogram (EEG) recordings, microdialysis, in vivo cellular recordings,

autoradiography and PCP-induced deficits in social interaction, adult male Sprague

Dawley rats were used. In vivo assessment of D2 occupancy of SEP-856 was performed

in non-human primate female baboons (Papio Anubis). In vivo pharmacokinetic studies

were performed in male adult ICR mice, adult male Sprague Dawley rats and male

Rhesus macaques (Macaca mulatta). Animals were maintained on a 12 /12 light/dark

cycle. The room temperature was maintained between 20 and 23C with a relative

humidity maintained between 30% and 70%. Chow and water were provided ad libitum

for the duration of the study unless otherwise stated. Animals were randomly assigned

across treatment groups and studies conducted with experimenters blinded to the drug

treatment. Further details (eg. Vendor, age and/or weight range) are provided in the

Supplemental Information.

All animal studies were conducted in accordance with the institutional animal care

protocols complying with Federal regulations and were approved by the respective

Institutional Animal Care and Use Committees.


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Test compounds

Haloperidol, Risperidone, Quetiapine, Clozapine, Sertraline, PCP, 8-OH-DPAT and

WAY-100635 were purchased from Sigma Aldrich. SEP-856 ((S)-1-(4,7-dihydro-5H-

thieno[2,3-c]pyran-7-yl)-N-methylmethanamine hydrochloride) and its enantiomer SEP-

855 were synthesized by Sunovion Pharmaceuticals and all doses corrected for salt

content. Further information regarding formulation for each study are provided in the


Behavior-based, mouse phenotypic screening

The central nervous system (CNS) properties of SEP-856 were evaluated using the

SmartCube® system, a high-throughput, automated mouse behavioral platform

(Roberds et al., 2011; Alexandrov et al., 2015; Shao et al., 2016). Further experimental

details are provided in the Supplemental Information.

Behavioral phenotyping

Details on PPI, PCP-induced hyperactivity, catalepsy, FST and PCP-induced deficits in

social Interaction are provided in the Supplemental Information.

EEG recordings

EEG recordings were performed in 7 adult male Sprague Dawley rats using a cross-

over design. Animals were implanted with chronic recording devices for continuous

recordings of electroencephalograph (EEG), electromyograph (EMG), core body

temperature (Tb), and locomotor activity (LMA) via telemetry (DQ ART 4.1 software;

Data Sciences Inc., St Paul, MN). Following completion of the data collection, expert


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scorers determined states of sleep and wakefulness in 10 second (s) epochs by

examining the recordings visually using NeuroScore software (Data Sciences Inc., St

Paul, MN). All doses of SEP-856, caffeine, and vehicle were administered by oral

gavage. A minimum of 3 days elapsed between doses. In order to evaluate the effects

of SEP-856 on sleep/wake parameters during the inactive period, dosing occurred

during the middle of the rats’ normal inactive period. The first 6 hours of the recording

were scored and analyzed. For additional details, please refer to the Supplemental

Information.

In vivo Microdialysis

Extracellular dopamine and serotonin levels were assessed in the prefrontal cortex and

dorsal striatum using in vivo microdialysis in freely moving Sprague Dawley rats. For

detailed methods, please refer to the Supplemental Information.

In vivo Pharmacokinetics studies

Details on in vivo pharmacokinetic measurements are provided in the Supplemental

Information.

In vitro and in vivo 5-HT1A and D2 receptor occupancy studies

In vitro autoradiography was used to determine the effects of SEP-856 on [3H]-8-OH-

DPAT binding to 5-HT1A receptors in rat brain sections. In vivo occupancy of SEP-856

at D2 receptors was measured with [3H]-raclopride in Sprague Dawley rats and with

[18F]-Fallypride-PET in non-human primates (Papio Anubis). For details, please refer to



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Patch-Clamp recordings in the DRN and VTA

In vitro whole-cell patch-clamp recording techniques were used in isolated slice

preparations (male C57BL/6J mice, 4-16 weeks) of the dorsal raphe nucleus (DRN) and

ventral tegmental area (VTA) to investigate the effects of SEP-856 on neuronal activity.

The experiments examined the effects of SEP-856 (1-30 μM) on the activity of DRN and

VTA neurons that were characterized by their electrophysiological properties and their

sensitivity to application of the 5-HT1A receptor agonist 8-OH-DPAT (DPAT; 10 μM).

Subsequently, effects mediated via the trace amine-associated receptor 1 (TAAR1)

and/or via the 5-HT1A receptor were investigated using the selective antagonist EPPTB

(0.05-1 μM) and the selective antagonist WAY-100635 (WAY-635; 10 μM) respectively.

All compounds were dissolved in either DMSO or ddH2O and diluted with aCSF to final

concentration from a minimum 1000-fold higher stock concentration (maximum slice

DMSO concentration 0.1%). Whole-cell patch-clamp recordings were performed at

room temperature using the ‘blind’ version of the patch-clamp technique with either

Axopatch 1D or Multiclamp 700B amplifiers. For detailed methods, please refer to the


In vivo extracellular single-unit recordings in the DRN

In vivo extracellular single-unit recordings were used to characterize the effects of SEP-

856 on firing of serotonergic DRN neurons in anesthetized, male Sprague Dawley rats.

Following surgery and insertion of the recording electrode, baseline firing activity of the

neuron was recorded for at least 10 minutes prior to the compound administration. SEP-

856 was tested at 1, 2, and 5 mg/kg by i.v. injection. After clear inhibitory effects were

observed (3 – 5 minutes after compound administration), WAY-100635 (80 µg/kg, i.v.)


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was given to determine whether it could antagonize the inhibitory effect of SEP-856.

Blood samples were taken 30 minutes following compound administration. For

additional details, please refer to the Supplemental Information.

In Vitro Pharmacology

The in vitro pharmacology of SEP-856 at known receptors and enzymes was assessed

in broad panel screens (Eurofins CEREP SA, Celle-Lévescault, France, Ricerca, Taipei

Taiwan). For those targets at which SEP-856 (10M) demonstrated greater than 50%

inhibition, dose response curves were generated and inhibitory constant (Ki) values

were determined. Equilibrium radioligand binding was carried out performed using the

following incubation conditions: Incubation conditions and additional details are listed in

the Supplemental Information.

The functional (both agonist and antagonist) effects were also determined. Assays used

to study the functional effects were as follows: Intracellular cAMP levels were

determined for 5-HT1A, 5-HT7, TAAR1 and D2, using either the DiscoveRx HitHunter

cAMP XS+ assay or the Cisbio HTRF® cAMP assay. 5-HT1A was also studied using

GTPS binding. Impedance was used for 5-HT1B, 5-HT1D, and 2A. Intracellular Ca2+

release was used for 5-HT2A and 5-HT2C. IP1 accumulation was used for 5-HT2B. D2 was

also studied using the DiscoveRx PathHunter® -arrestin recruitment assay.

Statistical Analyses

Statistical analyses were performed using the commercially available software

GraphPad Prism v6.0, unless otherwise noted. Results are either presented as mean ±

s.e.m. or mean ± s.d. (for PK analyses). Simple comparisons were evaluated with two-


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tailed, Student’s t-test. Multiple groups comparisons were assessed with One-way

ANOVA, followed by appropriate post-hoc analyses. Time-dependent measures were

assessed with repeated measures ANOVA followed appropriate post-hoc analyses.

Statistical significance was defined as p < 0.05.


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RESULTS

SEP-856 EXHIBITS ANTIPSYCHOTIC-LIKE ACTIVTY IN THE SMARTCUBE®

SYSTEM

SEP-856 (Fig. 1A) was identified during a medicinal chemistry program designed to

develop structurally and mechanistically novel antipsychotics using in vivo mouse

phenotypic screening in combination with comprehensive in vitro and in vivo molecular

profiling. Screening was conducted during a 45-minute automated test session, in which

mice were exposed to multiple challenges, and different behavioral domains were

captured and analyzed using proprietary computer vision software and machine

learning algorithms (Roberds et al., 2011; Alexandrov et al., 2015; Shao et al., 2016).

The behavioral platform was established and validated with marketed CNS drugs,

producing a library of drug-class signatures. Each class is represented by a different

color, for example, purple and yellow indicate antipsychotic– and anxiolytic-like activity

respectively (Fig. 1E), and the behavioral activity is shown as a scale of 0 to 100%. The

data in Figure 1C demonstrated that SEP-856 was behaviorally active at three out of

four doses tested (0.3, 1 and 10 mg/kg, i.p.). At 0.3 mg/kg, SEP-856 was classified as

an anxiolytic (represented by the primarily yellow color of the column) but showed a

dose-dependent increase in an antipsychotic classification (purple), such that the

signatures at 1 and 10 mg/kg were predominantly antipsychotic. As a comparison, the

signatures of marketed antipsychotic drugs are shown in Supplemental Figure 1. In

addition, SEP-856 showed a modest antidepressant-like signal, illustrated by the green

signal at 0.3, 1 and 10 mg/kg. Overall, the results indicate that SEP-856 is CNS active

and exhibits a behavioral signature similar to known antipsychotic drugs. Interestingly,


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its enantiomer, SEP-363855, showed little behavioral activity at 10 mg/kg (i.p.), a dose

at which SEP-856 was fully efficacious (Fig. 1B and D).

IN VITRO PHARMACOLOGY

To investigate the molecular targets mediating the response to SEP-856, the compound

was run against several panels of known molecular targets (ion channels, GPCRs and

enzymes; Supplemental Tables 1-4). At 10 µM, SEP-856 showed >50% inhibition of

specific binding at α2A, α2B, D2, 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT2C, and 5-

HT7 receptors. Ki values are shown in Table 1 ranging from 0.031 to 21 µM. No

significant activity of SEP-856 was observed at any of the enzymes studied (up to a

concentration of 100 µM).

Receptor panel screening and follow up functional testing showed that SEP-856

exhibited a range of activities at several receptors (Table 2). The most notable activity

ofSEP-856 was agonism at the human TAAR1 receptor (EC50 of 0.14 ± 0.062 μM, Emax

= 101.3 ± 1.3%) and the 5-HT1A receptor (EC50 = 2.3 μM with values ranging from 0.1- 3

μM, Emax = 74.7 ± 19.6%; Figure 2B). Interestingly, activity at the human TAAR1

receptor demonstrated stereoselectivity in that SEP-855 (an enantiomer of SEP-856),

which was inactive in the behavioral screening platform at 10 mg/kg (i.p.), had an EC50

of 1.7 μM (Fig. 2A). In D2 receptor functional assays, SEP-856 exhibited weak partial

agonism with EC50 values of 10.44 ± 4 µM (cAMP, Emax = 23.9 ± 7.6 %; Fig. 2C) and 8

µM (ß-arrestin recruitment, Emax = 27.1%). At 100 µM, 34 ± 1.16 % inhibition was seen

in the cAMP assay and no antagonism was seen at concentrations up to 100 μM in the

β-arrestin recruitment assay. Low potency partial agonist activities were also observed


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at 5-HT1B (EC50 = 15.6 ± 11.6 μM, Emax = 22.4 ± 10.9 %), 5-HT1D (EC50 = 0.262 ± 0.09

μM and Emax = 57.1 ± 5.5 %) and 5-HT7 receptors (EC50 = 6.9 ± 1.32 μM and Emax =

41.0 ± 9.5 %). In a functional assay of 5-HT2B activity, SEP-856 showed no agonism up

to a concentration of 100 µM while norfenfluramine, the positive control, was a full

agonist with an EC50 value of 0.140 µM. Little to no activity was detected at the 5-HT2A

receptor, with 29.3% agonism seen only at the highest tested concentration of 10 M.

SEP-856 EXHIBITS HIGH BRAIN PENETRANCE AND GOOD SYSTEMIC

BIOAVAILIABILITY FOLLOWING ORAL ADMINISTRATION

Behavioral phenotypic screening demonstrated that SEP-856 is CNS active in mice

following 0.3, 1 and 10 mg/kg intraperitoneal administration. Consequently, the

pharmacokinetics of SEP-856 in plasma was characterized in ICR mice, Sprague

Dawley rats and Rhesus macaques following oral and/or i.v. dosing. Biological samples

were collected over 8 or 24 hours post-dose. Brain exposure was also assessed in mice

and rats following p.o. administration. SEP-856 was rapidly absorbed with maximum

plasma and brain concentrations reached within 0.25 to 0.5 hours in mice and rats and

maximum plasma concentrations reached within 6 ± 2.83 hours in monkeys

(Supplemental Table 5). SEP-856 penetrated mouse and rat brains after oral

administration (10 mg/kg), with average brain-to-plasma AUC ratios of ~3 respectively

(Supplemental Figure 2). In addition, SEP-856 plasma and brain levels were still

detectable at 8 hours post dose with fairly consistent brain/plasma ratios over time.

SEP-856’s brain penetration and elimination pharmacokinetics as indicated by tmax and

t1/2 were similar to the plasma pharmacokinetics (Supplemental Table 5).


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Oral bioavailability of SEP-856, determined by plasma AUC ratio after cross-over oral

and intravenous administrations, was high in rat and monkey with 58-120% and ~71 %

respectively. Total plasma clearance of SEP-856 was relatively high in rat (5 mg/kg,

i.v.) and monkey (5 mg/kg, i.v.) with 1.54 and 0.797 L/h/kg, respectively, and elimination

half-lives of 1.2 and 3.1 hours, respectively.

SEP-856 DEMONSTRATES ANTIPSCHOTIC-LIKE EFFICACY IN RODENTS

In order to demonstrate the antipsychotic-like profile of SEP-856, we performed a series

of additional pharmacological studies that assess endophenotypes of schizophrenia and

antidepressant efficacy in rodents.

Acute treatment with phencyclidine (PCP), which induces robust hyperactivity in rodents

and psychosis-like symptoms in humans, is considered a valuable assay in preclinical

research and is widely used to screen novel compounds for antipsychotic efficacy

(Ratajczak et al., 2013; Steeds et al., 2015; Moffat et al., 2017). Single oral

administration of SEP-856 (0.3, 1 and 3 mg/kg; 30 min pretreatment time) resulted in a

dose-dependent inhibition of PCP-induced hyperactivity responses in C57Bl/6J mice (1-

way ANOVA F(5, 59) = 18.96, p < 0.0001; Tukey’s post-hoc test, p < 0.05) with a 50%

effective dose (ED50) of approximately 0.3 mg/kg (Fig. 3A). The positive control,

clozapine, also significantly reduced PCP-induced hyperactivity. Small but significant

decreases in baseline activity were observed with SEP-856 at the highest dose of 3

mg/kg (1-way ANOVA F(5, 59) = 5.5, p < 0.001; Tukey’s post-hoc test, p < 0.05;

Supplemental Figure 3A).


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Another behavioral assay that is routinely used to identify novel antipsychotic agents is

prepulse inhibition (PPI) of the acoustic startle response (Geyer et al., 2001). PPI

occurs when a startle-eliciting stimulus (i.e., the pulse) is preceded by a stimulus of

lower intensity (i.e., the prepulse) and the amplitude of the startle response is reduced.

Single oral administration of SEP-856 (0.3, 1, 3, 10, and 30 mg/kg; 30 minute

pretreatment time) in C57BL/6J mice resulted in a dose-dependent increase in PPI

compared to the respective vehicle treatment (Fig. 3B), with significant increases

observed at 3, 10, and 30 mg/kg (2-way ANOVA: treatment effect F(3, 102) = 19.9, p <

0.0001; db effect F(2, 102) = 30.97, p <0.0001; Dunnett’s post hoc test, p < 0.05). Unlike

the positive control Haloperidol, SEP-856 improved PPI at dose levels that had no

confounding effects on baseline startle responses (Supplemental Figure 3B).

In contrast to positive symptoms, negative symptoms of schizophrenia, including

anhedonia, blunted affect, and social withdrawal, are more difficult to model in animals

(Jones et al., 2011; Wilson et al., 2015). However, subchronic treatment with PCP

reliably produces social interaction deficits in rodents that may mimic certain aspects of

negative symptoms such as social withdrawal (Steeds et al., 2015). Sprague Dawley

rats were subcutaneously injected with PCP (2 mg/kg) or vehicle twice daily for five

consecutive days followed by behavioral assessment on day six. The PCP-induced

deficit in social interaction was significantly attenuated by single oral administration of

SEP-856 at 1, 3, and 10 mg/kg (1-way ANOVA F(5, 56) = 8.33, p < 0.0001; Tukey’s post-

hoc test, p < 0.05). The positive control, clozapine (2.5 mg/kg, i.p.), also showed

efficacy that was comparable to SEP-856 (Fig. 3C). This suggests that SEP-856 may


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have potential benefits against some of the negative symptoms of schizophrenia such

as social withdrawal.

The effects of SEP-856 (0.3, 1, 3, and 10 mg/kg) were additionally evaluated in the

mouse FST, a behavioral assay that is acutely sensitive to all major classes of marketed

antidepressants drugs (Porsolt et al., 1977). Single oral administration of SEP-856

significantly reduced immobility time at 1, 3, and 10 mg/kg compared to vehicle (1-way

ANOVA F(5, 44) = 13.24, p < 0.0001; Tukey’s post-hoc test, p < 0.05; Fig. 3D), suggesting

that SEP-856 may also exhibit antidepressant-like activity. However, no clear dose-

dependent effect was observed, and the magnitude of response was smaller than that

produced by the positive control sertraline (20 mg/kg, i.p.).

One of the issues associated with antipsychotic drugs is the potential to develop

extrapyramidal symptoms (EPS), which can be assessed in mice by measuring the

induction of catalepsy using the bar test. Oral administration of SEP-856 (100 mg/kg)

produced no effects in the bar test while haloperidol (1 mg/kg, i.p.) significantly

increased the amount of time mice spent holding the bar at 30 and 90 min post dosing

(2-way ANOVA: treatment effect F(2, 54) = 107.7, p < 0.0001; time effect F(1, 54) = 9.4, p

<0.005; Dunnet’s post hoc test, p < 0.05). Importantly, these data indicate that SEP-856

was not associated with cataleptic effects at doses at least 30-fold higher than

efficacious dose levels in mice (Fig. 3E).

The plasma and brain exposures to SEP-856 in the mouse PCP-induced hyperactivity

and PPI tests as well as the rat subchronic PCP-induced social interaction test are

shown in Supplemental Table 6. Taken together, our results indicate a clear


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antipsychotic-like profile of SEP-856 in rodents and corroborate the initial findings

obtained in the behavioral screening platform.

SEP-856 DECREASES RAPID-EYE-MOVEMENT (REM) SLEEP

5-HT1A and TAAR1 activation have been shown to promote a generalized increase in

wakefulness, increased latency to sleep, and suppression of REM sleep (F G Revel et

al., 2012; Black et al., 2017; Schwartz et al., 2017). Since SEP-856 exhibits full agonism

at 5-HT1A and TAAR1, we investigated whether it affects sleep architecture, as

determined by telemetric recordings of the EEG, electromyogram, body temperature

(Tb) and locomotor activity in Sprague-Dawley rats. SEP-856, and the positive control

caffeine, were administered during the middle of the light (inactive) period followed by 6

hours of recordings. Oral SEP-856 administration (1, 3 and 10 mg/kg) produced a dose-

dependent decrease in REM sleep, increase in latency to REM sleep and increase in

cumulative wake (W) time (Fig. 4A, B and Supplemental Figure 4). SEP-856 had no

effect on the cumulative non-REM (NREM) time and latency to NREM (Supplemental

Figure 4). The differential effect on REM was further evident by a dose-dependent

decrease in the REM:NR ratio (Fig. 4C). Caffeine promoted wakefulness as expected

with additional characteristic increases in locomotion and Tb that SEP-856 did not

produce (Supplemental Figure 4). Collectively, these results suggest that SEP-856

promotes vigilance when given during the light (inactive) phase.

SEP-856 INTERACTS WITH CENTRAL 5-HT1A BUT NOT D2 RECEPTORS

In addition to TAAR1, in vitro testing revealed that SEP-856 exhibits full and partial

agonist activity at 5-HT1A and D2 receptors, respectively. Consequently, we conducted a


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series of experiments to determine whether SEP-856 interacts with these two targets in

vivo.

First, receptor autoradiography was used to assess SEP-856 occupancy at D2 receptors

in the rat brain using the radioligand [3H]-raclopride. SEP-856 (10 mg/kg, i.p.) or vehicle

was administered to Sprague Dawley rats followed by administration of [3H]-raclopride

(60 µCi/kg, i.v.) 30 minutes later. D2 receptor occupancy was assessed at 60 minutes

post SEP-856 (or vehicle) administration in coronal sections of the striatum (region of

interest) and cerebellum (reference region). Despite high plasma (~1300 ng/ml), brain

(~7900 ng/g), and CSF (~1800 ng/ml) exposures, SEP-856 resulted in 12.6 ± 6.4%

receptor occupancy at D2 (Supplemental Table 7), which was not statistically different

from vehicle controls (two-tailed t-test, p = 0.27). For comparison, activity in the rat

PCP-induced social interaction deficit assay (1 - 10 mg/kg) and mouse PCP-induced

hyperactivity test (0.3 mg/kg – 3 mg/kg) was seen at much lower exposures

(Supplemental Table 6). Thus, acute administration of SEP-856 did not produce

significant occupancy at D2 receptors in the rat brain at concentrations up to 200-fold

greater than those that were behaviorally efficacious.

We additionally conducted in vivo Positron Emission Tomography (PET) imaging to

determine whether SEP-856 also fails to occupy D2 receptors in the non-human primate

brain. Imaging was conducted in 2 anesthetized baboons using the radiotracer [18F]-

Fallypride. The percent occupancy was evaluated using a blockade protocol comparing

[18F]-Fallypride regional binding potential at baseline and following SEP-856

administration (~7.25 mg/kg, i.v. 30 minutes prior to [18F]-Fallypride injection). Venous

blood samples were taken before and at various time points after SEP-856


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administration. SEP-856 showed D2 receptor occupancy levels of 9.1 ± 3.3 %, 6.2 ± 6.1

%, and 9.6 ± 8.8 % in the caudate, putamen, and globus pallidus, respectively

(Supplemental Table 8 and Fig. 5). Similar to the observations in rats, SEP-856 did not

produce significant occupancy at D2 receptors despite achieving high plasma

concentrations (i.e., 2850 ± 250 ng/ml at 60 minutes and 1765 ± 125 ng/ml at 180

minutes after SEP-856 administration). Importantly, this finding demonstrates that the

antipsychotic-like behavioral profile of SEP-856 is independent of direct D2 receptor

modulation.

Next, we utilized in vitro autoradiography to evaluate the effects of SEP-856 on [3H]-8-

OH-DPAT (a 5-HT1A agonist radioligand) binding to 5-HT1A receptors in the rat brain.

Slide-mounted rat brain sections were incubated with 2 nM [3H]-8-OH-DPAT in the

presence and absence of SEP-856 (0.1, 1 and 10 µM). Non-specific binding was

defined by 10 µM 5-HT. SEP-856 produced a concentration-dependent displacement of

[3H]-8-OH-DPAT binding in all regions evaluated (Supplemental Figure 5). Given the

similar degree of [3H]-8-OH-DPAT in all brain areas, IC50 values for SEP-856

displacement of [3H]-8-OH-DPAT binding were only determined for the septum and

motor/somatosensory cortex (referred to as cortex). SEP-856 displaced [3H]-8-OH-

DPAT in a concentration-dependent manner, with a mean IC50 of 619 nM and 791 nM in

the cortex and septum, respectively (Fig. 6). These results demonstrate that SEP-856

can bind to central 5-HT1A receptor sites in vitro.


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SEP-856 INHIBITS NEURONAL FIRING IN THE DORSAL RAPHE NEUCLEUS AND

VENTRAL TEGMENTAL AREA IN VITRO

To investigate the effect of SEP-856 on neuronal activity and obtain further insights into

its mechanism of action, we performed whole-cell patch-clamp recordings in isolated

slice preparations of the DRN and the VTA of C57BL/6J mice. The DRN and VTA

express a high abundance of 5-HT1A and TAAR1 receptors, respectively (Lindemann et

al., 2008; Celada et al., 2013; Christian and Berry, 2018). After the determination of

repeatable responses, administration of SEP-856 was repeated in the presence of the

selective TAAR1 antagonist EPPTB and/or the selective 5-HT1A receptor antagonist

WAY-100635.

Whole-cell current-clamp recordings were made from 29 neurons within the DRN with a

mean resting membrane potential of -46.1 ± 1.0 mV and a mean input resistance of 849

± 61 MΩ. Based on changes in membrane potential and firing rates, 16 of the DRN

neurons were characterized as being sensitive to the 5-HT1A agonist 8-OH-DPAT

(DPAT), and 13 were classified as DPAT-insensitive (Supplemental Figure 6). Based on

their initial threshold activity, 44% (7/16) of DPAT-sensitive neurons were characterized

as spontaneously active (discharging action potentials) and 56% (9/16) were quiescent.

SEP-856 (10 µM) induced significant membrane hyperpolarization in 8 out of 9

quiescent neurons (45.5 ± 1.9 to -49.0 ± 2.2 mV, post-SEP-856 washout to 46.1 ± 1.9

mV; paired two-tailed t-test, t7 = 4.3, p = 0.004; Fig. 7A). In contrast, one single neuron

was depolarized by 2.1 mV. SEP-856 also significantly reduced the activity of 5 out of 7

spontaneously active DRN neurons (0.31 ± 0.10 Hz to 0.11 ± 0.04, post-SEP-856

washout to 0.20 ± 0.06 Hz; 69 ± 9.7% reduction; paired two-tailed t-test, t4 = 3.2, p =


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0.03; Figure 7A), whilst the remaining two neurons were largely unaffected. Collectively,

administration of 10 μM SEP-856 induced an inhibitory response, determined by either

a membrane hyperpolarization or a reduction in spontaneous firing rate, in 81% (13 out

of 16) of DPAT-sensitive neurons. In contrast, SEP-856 only induced an inhibitory

response in 15% (2 out of 13) of DPAT-insensitive neurons (Supplemental Figure 6).

Next, the effects of 10 µM SEP-856 were examined in the presence of the 5-HT1A

antagonist WAY-100635 (1 μM) in five DPAT-sensitive DRN neurons, two of which were

spontaneously active and three of which were quiescent. In three of the five neurons, a

membrane hyperpolarization of 1.9 ± 0.4 mV induced by SEP-856 was markedly

reduced to 0.4 ± 0.1 mV when SEP-856 was reapplied in the presence of WAY-100635

(79% reduction, Fig. 7B and Supplemental Figure 6). Similarly, the inhibitory effect of

SEP-856 was almost completely blocked in a further neuron in the presence of WAY-

100635, with the spontaneous firing rate being reduced by 47% (0.21 to 0.11 Hz) during

control SEP-856 administration compared to just a 2% reduction (0.83 to 0.82 Hz) when

SEP-856 was applied in the presence of WAY-100635 (Fig. 7B). In the remaining

neuron, a 2.4 mV hyperpolarization induced by SEP-856 was slightly increased to 2.6

mV in the presence of WAY-635. Thus, the inhibitory response induced by SEP-856

was reduced in the presence of the 5-HT1A antagonist WAY-635, in the majority (4/5,

80%) of DRN neurons analyzed.

In addition, the effects of SEP-856 were examined in the presence of EPPTB in six

DPAT-sensitive DRN neurons, three of which were spontaneously firing action

potentials and three of which were quiescent. Overall, the inhibitory response induced


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by SEP-856 was unaffected in the presence of the TAAR1 antagonist EPPTB, in the

majority (5/6, 83%) of DRN neurons tested (Fig. 7C).

Following the assessment of SEP-856 effects on DRN neuronal firing, whole-cell patch-

clamp recordings were also made from 23 neurons within the VTA (mean resting

membrane potential of -40.7 ± 0.7 mV and a mean input resistance of 935 ± 104 MΩ)

(Supplemental Figure 7). All of these neurons were spontaneously active once whole-

cell configuration had been established with a mean firing rate of 1.97 ± 0.60 Hz,

although activity did not persist in all cases. SEP-856 induced an inhibitory response in

approximately half of the recorded VTA neurons (Fig. 7D). This response was

characterized by a significant hyperpolarization of 2.8 ± 0.5 mV in 42% (8/19) of the

neurons (-42.6 ± 1.3 mV to -45.3 ± 1.4 mV; paired two-tailed t-test, t7 = 5.8, p = 0.0007)

and a significant reduction in spontaneous firing rate in 55% (11/20) of the neurons

(from 0.6 ± 0.18 to 0.18 ± 0.07 Hz; 71.4 ± 7% reduction; paired two-tailed t-test, t10 =

3.3, p < 0.008, Fig. 7D).

Interestingly, recordings from two neurons showed that the inhibitory response of SEP-

856 (1 μM) was reduced in the presence of 1 μM EPPTB (Fig. 7F, Supplemental Figure

7), but persisted in the presence of the 5-HT1A antagonist WAY-100635 (Fig. 7E).

However, observations from additional neurons are necessary to further validate the

ability of EPPTB to attenuate the inhibitory effect of SEP-856 in the VTA.

Overall, SEP-856 induced significant inhibitory responses in DPAT-sensitive neurons of

the DRN and a subset of VTA neurons. In contrast to the inhibitory effects of SEP-856

in the DRN, which were primarily mediated via activation of 5-HT1A receptors, the


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response in the VTA appeared to be at least partially mediated via the activation of

TAAR1.

SEP-856 INHIBITS DRN NEURONAL FIRING IN VIVO

The observation that SEP-856’s inhibitory effects in the DRN were mediated by 5-HT1A

led us to additionally investigate DRN neuron firing in vivo by recording extracellular

single-unit activities in anesthetized Sprague-Dawley rats. In accordance with previous

work (Martin et al., 1999), application of 8-OH-DPAT inhibited DRN firing, which was

subsequently reversed by 0.08 mg/kg WAY-100635 (Supplemental Figure 8). SEP-856

(2 mg/kg, i.v.) significantly decreased DRN neuron discharges to 87% of the baseline

rate (from 9.83 ± 1.4 to 1.4 ± 0.33 spikes/10 sec; two-tailed t-test, t2 = 5.9, p < 0.03)

during the initial 10 minutes post dosing (Fig. 8A,B). 30 minutes after SEP-856

administration the inhibitory effect was no longer detectable and firing-rates were

restored to baseline levels. Plasma exposures determined 30 minutes post dosing were

422 ± 22.5 ng/ml. When SEP-856 was tested at a higher dose (5 mg/kg, i.v.), firing

activity was completely suppressed and the inhibition was fully reversed by intravenous

administration of 0.08 mg/kg WAY-100635 (Fig. 8C,D). Similarly, the inhibitory effect of

2 mg/kg SEP-856 was also reversed by WAY-100635 (Supplemental Figure 8). These

results support the earlier in vitro finding in mice, and further suggest that part of SEP-

856’s mechanism of action is characterized by suppression of serotonergic neuronal

firing via activation of 5-HT1A autoreceptors.


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SEP-856 DOES NOT ALTER DOPAMINE AND SEROTONIN RELEASE IN THE

STRIATUM OR PREFRONTAL CORTEX

In order to determine whether the inhibitory action of SEP-856 in DRN neurons

translates into changes in synaptic serotonin levels, we measured 5-HT release in vivo

using microdialysis in freely-moving rats. Although SEP-856 demonstrated no

occupancy at D2 receptors in vivo, its’ antipsychotic-like behavioral profile, TAAR1

activity and ability to inhibit VTA neurons suggest potential modulation of dopaminergic

circuits independent of direct dopamine receptor regulation. We therefore also assessed

dopamine release. Changes in monoamine levels were monitored over a 240-minute

period following oral administration of vehicle, 3, 10, or 30 mg/kg SEP-856.

Interestingly, no significant changes in extracellular levels of dopamine or 5-HT were

observed in striatum or prefrontal cortex following SEP-856 administration

(Supplemental Figure 9).

ANTIPSYCHOTIC-LIKE EFFECTS OF SEP-856 ARE PARTIALLY MEDIATED BY 5-

HT1A RECEPTORS

Although the microdialysis findings revealed no changes in SEP-856-mediated 5-HT

release, the in vitro pharmacology results as well as the slice and in vivo

electrophysiology data suggest that agonism at 5-HT1A receptors is integral to SEP-

856’s mechanism of action in both mice and rats. Consequently, we evaluated whether

the 5-HT1A receptor antagonist WAY-100635 would attenuates the inhibitory behavioral

effects of SEP-856 in the mouse PCP-induced hyperactivity test. C57BL/6J mice were

injected with WAY-100635 (1 mg/kg, i.p.) or saline (i.p.) 10 minutes prior to SEP-856


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dosing (3 mg/kg, oral) followed by PCP administration (5 mg/kg, i.p.) 30 minutes later.

Consistent with our previous findings, single oral administration of SEP-856 significantly

inhibited PCP-induced hyperactivity responses (Fig. 8E). Pretreatment with WAY-

100635 partially attenuated the inhibitory effects of SEP-856 on the total distance

traveled (1-way ANOVA F(5, 51) = 22.11, p < 0.0001; Tukey’s post-hoc test, p < 0.05).

Taken together, the results indicate that the antipsychotic-like effects of SEP-856 in the

PCP-induced hyperactivity test may be partially mediated through 5-HT1A receptors.


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DISCUSSION

Numerous advances have been made in understanding the potential role of receptors

other than D2 in contributing to drug efficacy and specific side effects in schizophrenia.

Recent antipsychotic drug development has focused on non-D2 targets including D1, D4,

D3, NMDA, 5-HT2A, 5-HT2C, M1, M4, H3, NK3, and sigma receptors (Miyamoto et al.,

2005; Karam et al., 2010; Girgis et al., 2018). However, thus far, no compound lacking

D2 receptor blockade has proven effective for any symptom dimension of schizophrenia.

While several non-D2 mechanisms, including mGlu2/3 receptor agonism and GlyT1

inhibition, have demonstrated efficacy in non-clinical and clinical proof-of-concept

studies, positive results in Phase III clinical trials are still lacking (Marsman et al., 2013;

Kinon et al., 2015; Girgis et al., 2018). Here we utilized an in vivo mouse phenotypic

screening platform in combination with comprehensive in vitro and in vivo profiling to

identify compounds that exhibited behavioral similarity to known antipsychotics but do

not act through D2 or 5-HT2A mechanisms. This led to the discovery of SEP-856, a

potent compound that showed antipsychotic-like properties without inducing catalepsy,

demonstrated potential antidepressant-like effects, suppressed REM sleep and

modulated firing in a subset of VTA and DRN neurons.

In order to decipher the molecular MOA of SEP-856, its effects were initially studied in

panel screens of large numbers of receptors, ion channels and enzymes. Subsequent

studies identified activity at TAAR1, 5-HT1A, 5-HT1D, α2A and D2 receptors. Weak partial

agonism of D2 receptors (EC50 = 8-10 M, Emax 24-27%), and some very low potency

antagonism (34% inhibition at 100 M), was seen in in vitro assays. It should be noted

that this antagonism was assay dependent as, for instance, no antagonist response was


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seen in the -arrestin recruitment assay. Despite this in vitro activity, additional

experiments indicated that the MOA of SEP-856 does not include in vivo blockade at D2

given the lack of receptor occupancy in rat and monkey seen at doses/exposures that

are efficacious in rodent behavioral assays. Furthermore, the demonstrated central

occupancy of 5-HT1A receptors, as well as the partial reversal of PCP-induced

hyperactivity in the presence of WAY-100635, indicate that 5-HT1A agonism contributes

to the MOA of SEP-856. This was further supported by SEP-856’s ability to induce

significant inhibitory responses via 5-HT1A receptors in putative serotonergic neurons in

the DRN in vitro and in vivo. Notably, the inhibitory response induced in a subset of VTA

neurons was mediated, at least in part, via activation of TAAR1, suggesting that SEP-

856 modulates firing of putative serotonergic and dopaminergic neurons through distinct

mechanisms.

TAAR1 is a GPCR activated by trace amines and expressed in multiple regions of the

mammalian brain. The highest expression is reported in the DRN and VTA, whereas

lowers levels are observed in the limbic system and several other brain areas

(Borowsky et al., 2001; Burchett and Hicks, 2006; Lindemann et al., 2008; Rutigliano et

al., 2018). Recent studies have extensively characterized TAAR1 functions, elucidating

its important role in modulating dopaminergic circuitry and its potential implications in

neuropsychiatric disorders (Borowsky et al., 2001; Leo et al., 2014; Gainetdinov et al.,

2018; Schwartz et al., 2018). Revel et al. reported the first selective TAAR1 agonist

(RO5166017) and demonstrated its inhibitory effects on the firing of dopaminergic and

serotonergic neurons (Revel et al., 2011). In addition, the antipsychotic-like profile of full

and partial TAAR1-selective agonists was shown by their ability to inhibit cocaine and


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PCP-induced hyperlocomotion in rodents despite their lack of affinity for D2 receptors

(Revel et al., 2011; F G Revel et al., 2012; Florent G. Revel, Moreau, et al., 2012).

Similarly, SEP-856 was able to block PCP-induced hyperactivity without significantly

occupying D2 receptors. Unfortunately, the lack of suitable TAAR1 antagonists

prevented direct assessment off TAAR1’s contribution to the antipsychotic-like activity of

SEP-856 in vivo.

TAAR1’s ability to regulate presynaptic dopaminergic neurotransmission makes it an

interesting target for a number of psychiatric disorders (Lindemann et al., 2008; Revel et

al., 2011; Leo et al., 2014). Along these lines, TAAR1 agonists have demonstrated

efficacy in genetic mouse models of hyperdopaminergia, including DAT knockout (KO)

mice and rats (Revel et al., 2011; Florent G. Revel, Meyer, et al., 2012; Florent G.

Revel, Moreau, et al., 2012). The observed effects on dopaminergic signaling

presumably occur via functional physical interaction of TAAR1 with D2 receptors and

potentially also with the dopamine transporter (Espinoza et al., 2011; Leo et al., 2014;

Harmeier et al., 2015; Leo and Espinoza, 2016). Cell culture studies have shown that

TAAR1 is normally located intracellularly but can translocate to the plasma membrane

when co-expressed with D2 receptors (Espinoza et al., 2011; Harmeier et al., 2015).

The ability of TAAR1 to modulate dopaminergic tone was also demonstrated in TAAR1

KO mice. Under baseline conditions, striatal dopamine release was not altered in KO

animals compared to wild-type controls but was augmented following an amphetamine

challenge (Wolinsky et al., 2007; Lindemann et al., 2008). Follow up work by Leo and

colleagues, using microdialysis and fast-scan cyclic voltammetry, revealed increased

levels of dopamine specifically in the nucleus accumbens but not in the dorsal striatum


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of TAAR1 KO mice (Lindemann et al., 2008; Leo et al., 2014). In accordance, TAAR1

agonists have been reported to suppress VTA neuronal firing (Revel et al., 2011) and

inhibit electrically-evoked dopamine release (Leo et al., 2014). SEP-856 exerted

inhibitory effects in VTA neurons, which were likely mediated via activation of TAAR1.

Interestingly, the inhibitory response was only observed in half of the recorded cells,

pointing to a selective suppression of a subset of VTA neurons by SEP-856. The lack of

D2 occupancy, in addition to the inhibitory effects on VTA neuronal firing, potentially

suggest modulation of presynaptic dopamine dysfunction by SEP-856. Abnormalities in

dopamine synthesis capacity, baseline synaptic dopamine levels, and dopamine release

have been reported in schizophrenia patients, and are not targeted by current

antipsychotic treatments (Howes et al., 2012; Jauhar et al., 2017; Kim et al., 2017;

McCutcheon et al., 2018). Although SEP-856 demonstrated clear inhibitory effects on

DRN and VTA neurons, it did not alter dopamine or 5-HT release in the striatum or

prefrontal cortex. This could be due to a number of reasons including the difficulty in

detecting potential decreases in neurotransmitter release under baseline conditions

using conventional microdialysis. Differential effects of SEP-856 on dopamine and/or

serotonin release might primarily be detectable under stimulated conditions (e.g.

following electrical, pharmacological or behavioral challenges) or in a disease context

(e.g. hyperdopaminergic state). On the other hand, changes in dopaminergic and

serotonergic cell firing do not necessarily translate into changes in dopamine/5-HT

release (Berke, 2018). For example, dopamine release can be locally controlled by the

presynaptic terminals themselves and thus show spatiotemporal patterns independent

of cell body spiking (Floresco et al., 1998; Jones et al., 2010; Berke, 2018). In addition,


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SEP-856 might affect tonic versus phasic dopaminergic firing differentially, which could

ultimately result in marked behavioral effects without necessarily altering overall

dopamine levels. Additional experiments, applying both microdialysis and fast-scan

cyclic voltammetry will help to further elucidate the effect of SEP-856 on

neurotransmitter release and firing kinetics under baseline and stimulated conditions.

In addition to TAAR1-mediated activity, 5-HT1A agonism was identified as an integral

part of the MOA of SEP-856. The 5-HT1A receptor is highly expressed in the DRN,

cortex and limbic forebrain areas (e.g. hippocampus and amygdala), with lower

densities detected in the basal ganglia, thalamus, substantia nigra and VTA (Kia et al.,

1996; Hiroshi Ito, 1999; De Almeida and Mengod, 2008). In the DRN, 5-HT1A receptors

are primarily somatodendritic autoreceptors which function to inhibit neuronal firing. In

contrast, they are present as postsynaptic receptors in the hippocampus and amygdala

(Celada et al., 2013). Thus, it will be interesting to test whether SEP-856 also acts in an

inhibitory fashion in forebrain regions such as the hippocampus or prefrontal cortex.

The serotonergic circuitry has repeatedly been implicated in the pathophysiology of

schizophrenia, which is in part due to the antipsychotic properties of 5-HT2A antagonists

(although primarily in combination with D2 blockers). In addition, 5-HT2C antagonists and

compounds that target both receptors 5-HT2A/5-HT2C (e.g.ritanserin, vabicaserin,

mianserin, SR46349B, etc.) have demonstrated some efficacy in schizophrenia (Girgis

et al., 2018). Although there is multiple evidence for the therapeutic efficacy of 5-HT1A

agonists in depression and anxiety, less is known about the potential contribution of

these receptors to schizophrenia. Postmortem (Burnet et al., 1996, 1997; Simpson et

al., 1996; Sumiyoshi et al., 1996) and neuroimaging studies (Kasper et al., 2002;


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Tauscher et al., 2002) have revealed alterations in 5-HT1A-receptor density in the cortex

and amygdala of schizophrenic patients (Yasuno et al., 2004). Evidence in rodent

models indicates that the activation of 5-HT1A receptors prevents EPS induced by D2

receptor blockade, modulates dopaminergic neurotransmission in the frontal cortex,

positively influences mood, and attenuates NMDA receptor antagonist-induced cognitive

and social interaction deficits (Newman-Tancredi, 2010; Celada et al., 2013). In

addition, a number of compounds that combine partial agonism at 5-HT1A receptors with

antagonism (or partial agonism) at D2 receptors (e.g. aripiprazole, perospirone,

lurasidone, cariprazine, PF-217830, F-97013-GD, F-15063 and bifeprunox) appear to

provide therapeutic benefits against a broader range of schizophrenia symptoms

(Newman-Tancredi, 2010; Celada et al., 2013).

Interestingly, earlier work demonstrated that modulation of TAAR1 activity via the

selective agonist RO5166017 increases the potency of 5-HT1A partial agonists and

alters the desensitization rate at the 5-HT1A autoreceptors in the DRN (Revel et al.,

2011). This can occur by direct interaction of TAAR1 and 5-HT1A, or by favoring

interactions with the GPCR desensitization machinery. Consequently, Revel and

colleagues proposed that co-treatment with a TAAR1 agonist might improve therapeutic

efficacy of classical antidepressants, providing further support for compounds with dual

5-HT1A/TAAR1 activity in the treatment of psychosis and mood. Notably, TAAR1

agonists and many antidepressants (especially those with 5-HT1A activity) exert REM

sleep suppression, which is not observed for most D2-based antipsychotics (Tribl et al.,

2013; Wichniak et al., 2017; Goonawardena et al., 2019). Thus, the robust REM

suppression observed with SEP-856 could result from synergistic action on TAAR1 and


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5-HT1A receptors. However, agonism at TAAR1 and 5-HT1A receptors might only

partially contributes to SEP-856’s MOA, with other (possibly not yet identified) targets

likely playing key roles as well.

Although this work made use of multiple pharmacological rodent models and assays, it

is important to mention that no animal model is, or ever will be, truly reflective of the

underlying disease etiology of schizophrenia. Nevertheless, certain models serve as

important investigational tools and are useful in the development of novel treatments,

especially when the goals for a specific model and/or approach are clearly defined

(Nestler and Hyman, 2010). Our goal was to identify a novel MOA for the treatment of

schizophrenia by focusing on a desired clinical outcome – the combination of

antipsychotic efficacy and lack of D2-mediated side effects (e.g EPS and endocrine

effects). Ultimately, proof-of-concept studies in humans are required to determine

whether preclinical findings will translate into therapeutic efficacy. SEP-856 is currently

being evaluated for the treatment of schizophrenia in randomized controlled clinical

trials.

In summary, the approach utilized here represents an alternative to target-driven drug

discovery as it relied on in vivo phenotypic and in in vitro (anti-target) screening followed

by subsequent verification in putative animal models of schizophrenia. Accordingly, the

MOA of SEP-856 has not been fully elucidated. However, the selective agonism of

TAAR1 and 5-HT1A, in addition to the lack of D2/5-HT2A mediated efficacy, has the

potential to translate into a significantly improved safety profile compared to available

therapies, while still maintaining antipsychotic efficacy across a broad array of

symptoms. Based on its unique MOA and preclinical profile in animals, SEP-856


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represents a promising candidate for the treatment of schizophrenia and potentially

other neuropsychiatric disorders.


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ACKNOWLEDGEMENTS

This manuscript is dedicated to the memory of a beloved colleague, Dr. Una C.

Campbell.


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AUTHORSHIP CONTRIBUTIONS

Participated in Research Design: Philip G. Jones, Seth C. Hopkins, Robert Lew,

Liming Shao, John E. Campbell, Kerry L. Spear, Thomas H. Large, Una C. Campbell,

Taleen Hanania, Emer Leahy, and Kenneth S. Koblan.

Conducted Experiments: Philip G. Jones, Robert Lew, Una C. Campbell, John E.

Campbell and Taleen Hanania.

Performed data analyses: Nina Dedic, Philip G. Jones, Seth C. Hopkins, Robert Lew,

John E. Campbell, Thomas H. Large, Una C. Campbell and Taleen Hanania.

Wrote or contributed to the writing of the manuscript: Nina Dedic, Philip G. Jones,

Seth C. Hopkins, Robert Lew, Thomas H. Large, Taleen Hanania and Kenneth S.

Koblan.


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REFERENCES

Aghajanian GK, Wang RY, and Baraban JM (1978) Serotonergic and non-serotonergic neurons

of the dorsal raphe: reciprocal changes in firing induced by peripheral nerve stimulation.

Brain Res 153:169–175.

Alexandrov V, Brunner D, Hanania T, and Leahy E (2015) High-throughput analysis of behavior

for drug discovery. Eur J Pharmacol 750:82–89, Elsevier.

Belema M, and Meanwell NA (2014) Discovery of daclatasvir, a pan-genotypic hepatitis C virus

NS5A replication complex inhibitor with potent clinical effect. J Med Chem 57:5057–5071.

Berke JD (2018) What does dopamine mean? Nat Neurosci 21:787–793, Springer US.

Black SW, Schwartz MD, Chen TM, Hoener MC, and Kilduff TS (2017) Trace Amine-Associated

Receptor 1 Agonists as Narcolepsy Therapeutics. Biol Psychiatry 82:623–633.

Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, Durkin MM,

Lakhlani PP, Bonini JA, Pathirana S, Boyle N, Pu X, Kouranova E, Lichtblau H, Ochoa FY,

Branchek TA, and Gerald C (2001) Trace amines: Identification of a family of mammalian

G protein-coupled receptors. Proc Natl Acad Sci 98:8966–8971.

Burchett SA, and Hicks TP (2006) The mysterious trace amines: Protean neuromodulators of

synaptic transmission in mammalian brain. Prog Neurobiol 79:223–246.

Burnet PW, Eastwood SL, and Harrison PJ (1996) 5HT1A and 5HT2A R mRNA and Binding

Sites are Differentially Altered in Schizophrenia. Neurop 15:442-55

Burnet PWJ, Eastwood SL, and Harrison PJ (1997) [3H]WAY-100635 for 5-HT(1A) receptor

autoradiography in human brain: A comparison with [3H]8-OH-DPAT and demonstration of

increased binding in the frontal cortex in schizophrenia. Neurochem Int 30:565–574.


at ASPE



Dow

nloaded from


JPET#260281

40

Celada P, Bortolozzi A, and Artigas F (2013) Serotonin 5-HT1A receptors as targets for agents

to treat psychiatric disorders: Rationale and current status of research. CNS Drugs 27:703–

716.

Charpentier P, Gailliot P, and Jacob R (1952) Recherches sur les diméthylaminopropyl-N

phénothiazines substituées. Comptes rendus l’Académie des Sci 235:59–60.

Christian SL, and Berry MD (2018) Trace amine-associated receptors as novel therapeutic

targets for immunomodulatory disorders. Front Pharmacol 9:1–14.

Clifford EM, Gartside SE, Umbers V, Cowen PJ, Hajos M, and Sharp T (1998)

Electrophysiological and neurochemical evidence that pindolol has agonist properties at

the 5-HT1A autoreceptor in vivo. Br J Pharmacol 124:206–212.

De Almeida J, and Mengod G (2008) Serotonin 1A receptors in human and monkey prefrontal

cortex are mainly expressed in pyramidal neurons and in a GABAergic interneuron

subpopulation: Implications for schizophrenia and its treatment. J Neurochem 107:488–

496.

Espinoza S, Salahpour A, Masri B, Sotnikova TD, Messa M, Barak LS, Caron MG, and

Gainetdinov RR (2011) Functional Interaction between Trace Amine-Associated Receptor

1 and Dopamine D2 Receptor. Mol Pharmacol 80:416–425.

Floresco SB, Yang CR, Phillips AG, and Blaha CD (1998) Basolateral amygdala stimulation

evokes glutamate receptor- dependent dopamine efflux in the nucleus accumbens of the

anaesthetized rat. Eur J Neurosci 10:1241–1251.

Gainetdinov RR, Hoener MC, and Berry MD (2018) Trace Amines and Their Receptors.

Pharmacol Rev 70:549–620.


at ASPE



Dow

nloaded from


JPET#260281

41

Geyer MA, Krebs-Thomson K, Braff DL, and Swerdlow NR (2001) Pharmacological studies of

prepulse inhibition models of sensorimotor gating deficits in schizophrenia: A decade in

review. Psychopharmacology (Berl) 156:117–154.

Girgis RR, Zoghbi AW, Javitt DC, and Lieberman JA (2018) The past and future of novel, non-

dopamine-2 receptor therapeutics for schizophrenia: A critical and comprehensive review.

J Psychiatr Res, 108:57-83.

Goonawardena A V., Morairty SR, Dell R, Orellana GA, Hoener MC, Wallace TL, and Kilduff TS

(2019) Trace amine-associated receptor 1 agonism promotes wakefulness without

impairment of cognition in Cynomolgus macaques. Neuropsychopharmacology, 44:1485-

1493.

Hajós M, Sharp T, Kocsis B, Allers KA, Jennings K, Charette G, and Sík A (2007)

Neurochemical identification of stereotypic burst-firing neurons in the rat dorsal raphe

nucleus using juxtacellular labelling methods. Eur J Neurosci 25:119–126.

Harmeier A, Obermueller S, Meyer CA, Revel FG, Buchy D, Chaboz S, Dernick G, Wettstein

JG, Iglesias A, Rolink A, Bettler B, and Hoener MC (2015) Trace amine-associated

receptor 1 activation silences GSK3β signaling of TAAR1 and D2R heteromers. Eur

Neuropsychopharmacol 25:2049–2061, Elsevier.

Hiroshi Ito CH and LF (1999) Localization of 5-HT1AReceptors in the Living Human Brain Using

[Carbonyl-nC]WAY-100635: PET with Anatomic Standardization Technique. J Nucl Med

40:9.

Howes OD, Kambeitz J, Kim E, Stahl D, Slifstein M, Abi-Dargham A, and Kapur S (2012) The

nature of dopamine dysfunction in schizophrenia and what this means for treatment: Meta-

analysisof imaging studies. Arch Gen Psychiatry 69:776–786.


at ASPE



Dow

nloaded from


JPET#260281

42

Insel TR (2010) Rethinking schizophrenia. Nature 468:187–93.

Jauhar S, Veronese M, Rogdaki M, Bloomfield M, Natesan S, Turkheimer F, Kapur S, and

Howes OD (2017) Regulation of dopaminergic function: An [18F]-DOPA PET apomorphine

challenge study in humans. Transl Psychiatry 7: e1027. doi: 10.1038/tp.2016.270.

Jones C a, Watson DJG, and Fone KCF (2011) Animal models of schizophrenia. Br J

Pharmacol 164:1162–94.

Jones JL, Day JJ, Aragona BJ, Wheeler RA, Wightman RM, and Carelli RM (2010) Basolateral

Amygdala Modulates Terminal Dopamine Release in the Nucleus Accumbens and

Conditioned Responding. Biol Psychiatry 67:737–744.

Karam CS, Ballon JS, Bivens NM, Freyberg Z, Girgis RR, Lizardi-Ortiz JE, Markx S, Lieberman

JA, and Javitch JA (2010) Signaling pathways in schizophrenia: Emerging targets and

therapeutic strategies. Trends Pharmacol Sci 31:381–390.

Kasper S, Tauscher J, Willeit M, Stamenkovic M, Neumeister A, Kufferle B, Barnas C, Stastny

J, Praschak-Rieder N, Pezawas L, de Zwaan M, Quiner S, Pirker W, Asenbaum S,

Podreka I, and Bruecke T (2002) Receptor and transporter imaging studies in

schizophrenia, depression, bulimia and Tourette’s disorder--implications for

psychopharmacology. World J Biol Psychiatry 3:133–46.

Kia HK, Miquel M, Brisorgueil M, Daval G, Riad M, Mestikawy SEL, Hamon M, Verge D,

Neurobiologie D De, Neurosciences I, Ura C, Pierre U, France HKK, Neurobiologie L De,

and France MR (1996) Immunocytochemical Localization of Serotonin 1A Receptors in the

Rat Central Nervous System. J Comp Neurol 365:289–305.

Kim E, Howes OD, Veronese M, Beck K, Seo S, Park JW, Lee JS, Lee YS, and Kwon JS (2017)

Presynaptic Dopamine Capacity in Patients with Treatment-Resistant Schizophrenia


at ASPE



Dow

nloaded from


JPET#260281

43

Taking Clozapine: An [18F]DOPA PET Study. Neuropsychopharmacology 42:941–950.

Kinon BJ, Millen BA, Zhang L, and McKinzie DL (2015) Exploratory Analysis for a Targeted

Patient Population Responsive to the Metabotropic Glutamate 2/3 Receptor Agonist

Pomaglumetad Methionil in Schizophrenia. Biol Psychiatry 78:754–762.

Laborit H, Huguenard P, and Alluaume R (1952) Un noveau stabilisateur végétatif (le 4560 RP).

Presse Med 60:206–208.

Lehmann HE, and Ban TD (1997) The History of the Psychopharmacology of Schizophrenia.

Can J Psychiatry 42:152–162.

Leo D, and Espinoza S (2016) Trace Amine-Associated Receptor 1 Modulation of Dopamine

System, Elsevier Inc.

Leo D, Mus L, Espinoza S, Hoener MC, Sotnikova TD, and Gainetdinov RR (2014) Taar1-

mediated modulation of presynaptic dopaminergic neurotransmission: Role of D2

dopamine autoreceptors. Neuropharmacology 81:283–291.

Lieberman JA, Stroup ST, McEvoy JP, Swartz MS, Rosenheck RA, Perkins DO, Keefe RS,

Davis SM, Davis CE, Lebowitz BD, Severe J, and Hsiao JK (2005) Effectiveness of

Antipsychotic Drugs in Patients with Chronic Schizophrenia. N Engl J Med 353:1209–1223.

Lindemann L, Meyer CA, Jeanneau K, Bradaia A, Ozmen L, Bluethmann H, Bettler B, Wettstein

JG, Borroni E, Moreau J-L, and Hoener M (2008) Trace Amine-Associated Receptor 1

Modulates Dopaminergic Activity. J Pharmacol Exp Ther 324:948–956.

Marsman A, Van Den Heuvel MP, Klomp DWJ, Kahn RS, Luijten PR, and Hulshoff Pol HE

(2013) Glutamate in schizophrenia: A focused review and meta-analysis of 1H-MRS

studies. Schizophr Bull 39:120–129.


at ASPE



Dow

nloaded from


JPET#260281

44

Martin LP, Jackson DM, Wallsten C, and Waszczak BL (1999) Electrophysiological comparison

of 5-Hydroxytryptamine1A receptor antagonists on dorsal raphe cell firing. J Pharmacol

Exp Ther 288:820–6.

McCutcheon R, Beck K, Jauhar S, and Howes OD (2018) Defining the Locus of Dopaminergic

Dysfunction in Schizophrenia: A Meta-analysis and Test of the Mesolimbic Hypothesis.

Schizophr Bull 44:1301–1311.

Meyer-Lindenberg A (2010) From maps to mechanisms through neuroimaging of schizophrenia.

Nature 468:194–202.

Miyamoto S, Duncan GE, Marx CE, and Lieberman JA (2005) Treatments for schizophrenia: A

critical review of pharmacology and mechanisms of action of antipsychotic drugs. Mol

Psychiatry 10:79–104.

Miyamoto S, Leipzig JN, Lieberman JA, and Duncan GE (2000) Effects of ketamine, MK-801,

and amphetamine on regional brain 2-deoxyglucose uptake in freely moving mice.

Neuropsychopharmacology 22:400–412.

Moffat JG, Vincent F, Lee JA, Eder J, and Prunotto M (2017) Opportunities and challenges in

phenotypic drug discovery: an industry perspective John. Nat Rev Drug Discov 7:43–54.

Nestler EJ, and Hyman SE (2010) Animal models of neuropsychiatric disorders. Nat Neurosci

13:1161–9.

Newman-Tancredi A (2010) The importance of 5-HT1A receptor agonism in antipsychotic drug

action: rationale and perspectives. Curr Opin Investig Drugs 7:802–12.

Porsolt RD, Le Pichon M, and Jalfre M (1977) Depression: a new animal model sensitive to

antidepressant treatments. Nature 267:730–732.


at ASPE



Dow

nloaded from


JPET#260281

45

Ratajczak P, Wozniak A, and Nowakowska E (2013) Animal models of schizophrenia:

Developmental preparation in rats. Acta Neurobiol Exp (Wars) 73:472–84.

Revel Florent G., Meyer CA, Bradaia A, Jeanneau K, Calcagno E, André CB, Haenggi M, Miss

MT, Galley G, Norcross RD, Invernizzi RW, Wettstein JG, Moreau JL, and Hoener MC

(2012) Brain-specific overexpression of trace amine-associated receptor 1 alters

monoaminergic neurotransmission and decreases sensitivity to amphetamine.

Neuropsychopharmacology 37:2580–2592.

Revel FG, Moreau J-L, Gainetdinov RR, Bradaia A, Sotnikova TD, Mory R, Durkin S, Zbinden

KG, Norcross R, Meyer CA, Metzler V, Chaboz S, Ozmen L, Trube G, Pouzet B, Bettler B,

Caron MG, Wettstein JG, and Hoener MC (2011) TAAR1 activation modulates

monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic

activity. Proc Natl Acad Sci 108:8485–8490.

Revel F G, Moreau J, Pouzet B, Mory R, Bradaia A, Buchy D, Metzler V, Chaboz S, Groebke

Zbinden K, Galley G, Norcross RD, Tuerck D, Bruns A, Morairty SR, Kilduff TS, Wallace

TL, Risterucci C, Wettstein JG, and Hoener MC (2013) A new perspective for

schizophrenia: TAAR1 agonists reveal antipsychotic- and antidepressant-like activity,

improve cognition and control body weight. Mol Psychiatry 18:543-56. Epub 2012 May 29.

Revel Florent G., Moreau JL, Gainetdinov RR, Ferragud A, Velázquez-Sánchez C, Sotnikova

TD, Morairty SR, Harmeier A, Groebke Zbinden K, Norcross RD, Bradaia A, Kilduff TS,

Biemans B, Pouzet B, Caron MG, Canales JJ, Wallace TL, Wettstein JG, and Hoener MC

(2012) Trace amine-associated receptor 1 partial agonism reveals novel paradigm for

neuropsychiatric therapeutics. Biol Psychiatry 72:934–942.

Roberds SL, Filippov I, Alexandrov V, Hanania T, and Brunner D (2011) Rapid, computer vision-

enabled murine screening system identifies neuropharmacological potential of two new


at ASPE



Dow

nloaded from


JPET#260281

46

mechanisms. Front Neurosci 5:103. doi: 10.3389/fnins.2011.00103.

Rutigliano G, Accorroni A, and Zucchi R (2018) The case for TAAR1 as a modulator of central

nervous system function. Front Pharmacol 8: 987. doi: 10.3389/fphar.2017.00987.

Samara MT, Dold M, Gianatsi M, Nikolakopoulou A, Helfer B, Salanti G, and Leucht S (2016)

Efficacy, acceptability, and tolerability of antipsychotics in treatment-resistant

schizophrenia: A network meta-analysis. JAMA Psychiatry 73:199–210.

Schwartz MD, Black SW, Fisher SP, Palmerston JB, Morairty SR, Hoener MC, and Kilduff TS

(2017) Trace Amine-Associated Receptor 1 Regulates Wakefulness and EEG Spectral

Composition. Neuropsychopharmacology 42:1305–1314.

Schwartz MD, Canales JJ, Zucchi R, Espinoza S, Sukhanov I, and Gainetdinov RR (2018)

Trace amine-associated receptor 1: a multimodal therapeutic target for neuropsychiatric

diseases. Expert Opin Ther Targets 22:513–526.

Shao L, Campbell UC, Fang QK, Powell NA, Campbell JE, Jones PG, Hanania T, Alexandrov V,

Morganstern I, Sabath E, Zhong HM, Large TH, and Spear KL (2016) In vivo phenotypic

drug discovery: Applying a behavioral assay to the discovery and optimization of novel

antipsychotic agents. Medchemcomm 7:1093–1101, Royal Society of Chemistry.

Simpson MDC, Lubman DI, Slater P, and Deakin JFW (1996) Autoradiography with [3H]8-OH-

DPAT reveals increases in 5-HT1Areceptors in ventral prefrontal cortex in schizophrenia.

Biol Psychiatry 39:919–928.

Steeds H, Carhart-Harris RL, and Stone JM (2015) Drug models of schizophrenia. Ther Adv

Psychopharmacol 5:43–58.

Sumiyoshi T, Stockmeier CA, Overholser JC, Dilley GE, and Meltzer HY (1996) Serotonin1A


at ASPE



Dow

nloaded from


JPET#260281

47

receptors are increased in postmortem prefrontal cortex in schizophrenia. Brain Res

708:209–214.

Swinney DC, and Anthony J (2011) How were new medicines discovered? Nat Rev Drug Discov

10:507–519.

Tauscher J, Kapur S, Verhoeff P, Hussey D, Daskalakis Z, Tauscher-Wisniewski S, Wilson A,

Houle S, Kasper S, and Zipursky R (2002) Brain Serotonin 5-HT1A Receptor Binding in

Schizophrenia Measured by Positron Emission Tomography and [11 C]WAY-100635. Arch

Gen Psychitary 59:514–520.

Tribl GG, Wetter TC, and Schredl M (2013) Dreaming under antidepressants: A systematic

review on evidence in depressive patients and healthy volunteers. Sleep Med Rev 17:133–

142.

Wichniak A, Wierzbicka A, Walęcka M, and Jernajczyk W (2017) Effects of Antidepressants on

Sleep. Curr Psychiatry Rep 19: 63. doi: 10.1007/s11920-017-0816-4.

Wilson CA, Koenig JI, and Psychiatric M (2015) Social interaction and social withdrawal in

rodents as readouts for investigating the negative symptoms of schizophrenia Christina.

24:759–773.

Wolinsky TD, Swanson CJ, Smith KE, Zhong H, Borowsky B, Seeman P, Branchek T, and

Gerald CP (2007) The Trace Amine 1 receptor knockout mouse: An animal model with

relevance to schizophrenia. Genes, Brain Behav 6:628–639.

Yasuno F, Suhara T, Ichimiya T, Takano A, Ando T, and Okubo Y (2004) Decreased 5-HT1A

receptor binding in amygdala of schizophrenia. Biol Psychiatry 55:439–444.


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Dow

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FOOTNOTES

At the time these studies were conducted, all authors were either employees of

Sunovion Pharmaceuticals or Psychogenics Inc. Some authors are inventors on patents

related to the subject matter.


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FIGURE LEGENDS

Figure 1. SEP-856 exhibits a predominantly antipsychotic-like signature in

SmartCube®. The chemical structures of SEP-856 (A) and its enantiomer SEP-855 (B).

The behavioral signature of SEP-856 (C) includes anxiolytic (yellow) and antipsychotic

(purple) components. In contrast, its enantiomer SEP-855 is largely behaviorally

inactive in mice, represented by the vehicle-like white bar (D). The behavioral platform

was established and validated with marketed CNS drugs, producing a library of drug-

class signatures. (E) Each of the 15 classes is represented by a different color, as

indicated (*antipsychotic (purple) and high-dose antipsychotic (dark purple);

**antidepressant (green) and *high-dose antidepressant (dark green)). N = 8-10

mice/group.

Figure 2. Functional effects of SEP-856 at TAAR1, 5-HT1A and D2 receptors. The

functional effects of SEP-856 were determined using HEK-293 cells expressing TAAR1

(A), 5-HT1A (B) or D2 (C) receptors. cAMP accumulation was determined for TAAR1

using the DiscoveRx HitHunter cAMP Assay. The inactive enantiomer of SEP-856

(SEP-855) was also tested along with the trace amines PEA and p-tyramine. Inhibition

of forskolin-stimulated cAMP levels was used for the 5-HT1A (n = 4) and D2 receptors;

cAMP levels were determined with the DiscoveRx HitHunter cAMP XS+ assay or the

Cisbio HTRF®, cAMP assay. The D2 receptor was also studied using the PathHunter β-

arrestin recruitment assay. Representative traces are shown for A and C.

Figure 3. SEP-856 demonstrates antipsychotic- and antidepressant-like activity

without inducing catalepsy. (A) Oral SEP-856 administration dose-dependently

reduced PCP-induced hyperactivity in C57BL/6J mice (1-way ANOVA + Tukey’s post

hoc test, * p < 0.05 vs. Veh/PCP, #p < 0.05 vs Veh/Veh). Clozapine and PCP were

dosed at 1 mg/kg and 5 mg/kg i.p., respectively. (B) PPI measured in C57BL/6J mice

was significantly increased by Haloperidol (i.p.) and SEP-856 at 3, 10 and 30 mg/kg

(p.o.) compared to their respective vehicle controls (10 % DMSO for Haloperidol and

20% cyclodextrin for SEP-856; 1-way ANOVA + Tukey’s post hoc test, *# p < 0.05). (C)

Social interaction deficits induced by subchronic PCP treatment (2.5 mg/kg, s.c., twice

daily for 5 days) in Sprague-Dawley rats were significantly attenuated by acute


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Clozapine (2.5 mg/kg, i.p.) and SEP-856 (p.o) treatment at all doses tested (1-way

ANOVA + Tukey’s post hoc test, * p < 0.05 vs. PCP/Veh, #p < 0.05 vs Veh/Veh). (D)

Acute Sertraline (20 mg/kg, i.p.) and 1, 3 and 10 mg/kg SEP-856 (p.o.) dosing reduced

immobility time in the FST in Balb/CJ mice compared to vehicle controls (1-way ANOVA

+ Tukey’s post hoc test, * p < 0.05 vs. Veh). (E) In contrast to Haloperidol (i.p.), acute

SEP-856 (p.o.) treatment did not induce catalepsy in the mouse (C57BL/6J) bar test as

seen by the lack of increase in time spent holding the bar compared to vehicle (2-way

ANOVA + Tukey’s post hoc, *p < 0.05 vs. Veh). Abbreviations: CLZ, clozapine; SRT,

sertraline; PCP, phencyclidine; PPI, prepulse-inhibition; FST, forced-swim test. N = 8-

12/group. Data are shown as mean ± s.e.m.

Figure 4. SEP-856 decreases REM sleep in rats. (A) Acute, oral administration of

SEP-856 led to a dose-dependent reduction in cumulative REM sleep (A), increase in

latency to REM (B) and a decrease in REM/NREM ratio (C) in Sprague-Dawley rats

over the 6-hour recording period (1-way repeated-measures ANOVA + two-tailed t-test,

*p < 0.05 vs. Veh). Dosing occurred in the middle of the inactive phase, at the beginning

of Zeitgeber time 7. The positive control Caffeine (10 mg/kg, p.o. CAF) produced similar

effects on REM time and latency without altering the REM/NREM ratio. Additional

parameters are presented in Supplemental Figure 4. N = 7. Data are shown as mean ±

s.e.m.

Figure 5. SEP-856 does not exhibit significant occupancy at D2 receptors in non-

human primates. Representative MR (for anatomic reference) and mean summed PET

images, before and after administration of SEP-856 are shown from top to bottom for

one animal. Axial, sagittal, and coronal slices are shown from left to right. Regions of

interest, including the caudate, putamen and globus pallidus, were manually outlined.

Average [18F]Fallypride PET images over 180 minutes of acquisition are presented at

baseline and following bolus i.v. administration of SEP-856 (7.25 mg/kg). The

occupancy was estimated using the BPND-derived Simplified Reference Tissue Model

(SRTM). SUVr, relative standardized uptake value.


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Figure 6. SEP-856 binds to 5-HT1A receptors in rat brain slices. (A) Autoradiographs

of coronal brain sections depicting binding of the 5-HT1A agonist [3H]-8-OH-DPAT. The

highest receptor binding was observed in the septum and throughout the cortex

(illustrated by increasing intensity in yellow to red colors). (B) SEP-856 displaced [3H]-8-

OH-DPAT in a concentration-dependent manner, with an IC50 of 619 nM and 791 nM in

the cortex and septum, respectively (n = 4 brains, 2 sections/brain). Sections were

incubated with 2 nM [3H]-8-OH-DPAT and 10 µM serotonin for NSB (non-specific

biding), 0.1% DMSO for total binding or SEP-856 (0.1 - 32 µM). Data are shown as

mean ± s.e.m.

Figure 7. SEP-856 inhibits firing of DRN and VTA neurons in vitro. Representative

whole-cell patch-clamp recordings from DRN and VTA brain slices of C57BL/6J mice

are shown on the left, and quantification histograms on the right. Compound-induced

effects were determined based on significant changes in membrane potential (mV)

and/or firing rate (Hz) and expressed relative to baseline (Δ membrane potential and %

frequency change). DRN neurons were classified as sensitive or insensitive to the 5-

HT1A agonist 8-OH-DAPT. (A) SEP-856 induced inhibitory responses in the majority of

spontaneously active, 8-OH-DAPT-sensitive DRN neurons. This inhibitory effect was

absent in 4/5 neurons the presence of the 5-HT1A antagonist WAY-635 (B) but persisted

in 5/6 neurons in the presence of the TAAR1 antagonist EPPTB (C). (D) Based on

significant changes in membrane potential and/or firing rate, approximately half of the

spontaneously-active neurons in the VTA were inhibited by SEP-856 treatment when

compared to baseline. This effect persisted in presence of WAY-635 (E, n = 2 neurons)

but was reduced in the presence of EPPTB (F, n = 2 neurons). Histograms show

neuronal responses following administration of SEP-856 alone (control, A and D) and in

the presence of WAY-635 (B and E) or EPPTB (C and F). Solid horizontal bars indicate

the timeframe of compound administration to the slice. Two-tailed t-test, * p < 0.05. Data

are shown as mean ± s.e.m.


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Figure 8. SEP-856 inhibits firing of DRN in vivo and partially inhibits PCP-induced

hyperactivity through 5-HT1A receptors. (A) SEP-856 (2 mg/kg, i.v.) decreased single-

unit discharges of DRN neurons in anesthetized Sprague Dawley rats (n = 2). (B) The

most significant inhibition was observed during the first 10 minutes after dosing, with

discharge rates returned to baseline levels ~20 minutes later (48-53 minutes after start

of recording). (C, D) Neuronal DRN firing rates were completely suppressed by a higher

dose of SEP-856 (5 mg/kg, i.v., two-tailed t-test, *p < 0.05, n = 2), and this inhibition was

subsequently reversed by WAY-635 (0.08 mg/kg; n = 1). (E) Pretreatment of WAY-635

(1 mg/kg, i.p.) partially attenuated the ability of SEP-856 (3 mg/kg, p.o.) to reduce PCP-

induced hyperactivity in C57BL/6J mice (1-way ANOVA + Tukey’s post hoc test, +p <

0.05, *p < 0.05 vs. Veh/Veh/PCP, #p < 0.05 vs Veh/Veh/Veh; n = 9-10/group). Data are

shown as mean ± s.e.m.


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MAIN TABLES

Table1. Receptor affinities.

Panel screens of up to 105 radioligand binding assays and 34 enzyme assays were performed

and the affinity of SEP-856 was determined for receptors at which >50% inhibition was seen at

10 μM. Details of radioligands and incubation conditions are listed in the Material and Methods

section and the targets in the screening panels are listed in the Supplemental Information. Data

are shown as mean ± s.e.m. (n ≥ 3, n =1 for 5-HT2B).

Receptor Ki (µM)

D2s 21.3 ± 8.2 5-HT1A 0.284 ± 0.056 5-HT1B 1.90 ± 1.72 5-HT1D 1.13 ± 0.21 5-HT2A 17.25 ± 4.0 5-HT2B 1.1 5-HT2C 2.45 ± 0.82 5-HT7 0.031 ± 0.003 α2A 0.59 ± 0.06 α2B 1.9 ± 0.10


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Table 2. In vitro functional profile of SEP-856.

The functional effects of SEP-856 were determined for receptors at which >50% inhibition was

seen in the panel screens. The in vitro pharmacology studies were run in both agonist and

antagonist modes. The specific assays are listed in the Materials and Methods section. Data are

shown as mean ± s.e.m. (n ≥ 3).

Receptor Agonist Antagonist EC50 (µM) % Emax IC50 (µM) % inhibition

TAAR1 0.140 ± 0.062 101.3 ± 1.3 NE NE 5-HT1A 2.3 ± 1.40 74.7 ± 19.6 NE NE 5-HT1B 15.6 ± 11.60 26.6 @ 10M NE NE

5-HT1D 0.262 ± 0.09 57.1 ± 6.0 NE NE 5-HT2A >10 29.3 @ 10M NE NE

5-HT2C 30 ± 4.5 63.3 ± 3.1 NE NE 5-HT7 6.7 ± 1.32 41.0 ± 9.5 NE NE α2A >10 39.4 ± 4.2 NE NE α2B NE NE NE NE D2L (cAMP) 10.44 ± 4.0 23.9 @ 10M >100 34.0 ± 1.16

D2L (arrestin recruitment) 8.02 27.1 @ 10M >100 <25 @ 100 µM

NE = no effect (<30% @ 30 µM


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MAIN FIGURES

Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 7


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Figure 8


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Supplemental Information

SEP-363856, a novel psychotropic agent with a unique, non-D2 receptor

mechanism of action

Nina Dedic*, Philip G. Jones*, Seth C. Hopkins, Robert Lew, Liming Shao, John E.

Campbell, Kerry L. Spear, Thomas H. Large, Una C. Campbell, Taleen Hanania, Emer

Leahy, Kenneth S. Koblan

*both authors contributed equally to the work

The Journal of Pharmacology and Experimental Therapeutics

2

Supplemental Material and Methods

SmartCube® System for Behavioral Phenotypic Screening

The SmartCube® system is a mouse-based behavioral screening platform that

combines proprietary hardware, computer vision algorithms and machine learning based

data mining tools (Roberds et al., 2011; Alexandrov et al., 2015; Shao et al., 2016). In

order to create a reference database of therapeutic class signatures, various doses of

clinically approved CNS drugs used to treat schizophrenia, depression, anxiety and

other psychiatric disorders were screened. During a 45-minute automated test session,

where mice are presented with multiple challenges, the behavioral responses of adult

male C57Bl/6 mice (Taconic, Germantown, NY) treated with vehicle or test compounds

were captured and analyzed using computer vision software and proprietary algorithms.

During each test session over 2000 features are captured and the behavioral responses

following treatment were compared to the database of reference drugs. SEP-856 was

dissolved in vehicle (5% Pharmasolve; 30% PEG 200/ PEG 400/ propylene glycol; 65%

saline) and administered i.p. (10 ml/kg) at dose levels of 0.1, 0.3, 1 and 10 mg/kg.

Testing was initiated 15 minutes post-dosing.

PCP-Induced Hyperactivity

PCP-induced hyperactivity was assessed in open field chambers (27.3 x 27.3 x 20.3

cm; Med Associates Inc., St Albans, VT) surrounded by infrared photo beams (16 x 16 x

16) to measure horizontal and vertical activity. Distance travelled was measured from

horizontal beam breaks. Adult, male C57Bl/6J mice (Jackson Laboratories, Bar Harbor,

ME) were acclimatized to the experimental testing room for at least 1 hour prior to

3

testing. Mice were treated with vehicle (p.o.), SEP-856 (0.3, 1 and 3 mg/kg, p.o.) or

clozapine (positive control; 1 mg/kg, i.p.) and placed in the open field chambers for 30

min measurement of baseline activity. Mice were then injected with either water or PCP

(5 mg/kg, i.p.) and placed back in the open field chambers for a 60 min session. SEP-

856 was dissolved in 20% hydroxypropyl-beta-cyclodextrin and clozapine in 10%

DMSO. PCP was dissolved in sterile injectable water. The dosing volume for all

treatments was 10 ml/kg. The open field chambers were cleaned following each test.

Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test (p <

0.05).

Prepulse Inhibition (PPI)

The acoustic startle measures an unconditioned reflex response to external auditory

stimulation. PPI, consisting of an inhibited startle response (reduction in amplitude) to

an auditory stimulation following the presentation of a weak auditory stimulus or

prepulse, has been used to assess deficiencies in sensory-motor gating, such as those

seen in schizophrenia. Mice were placed in the PPI chambers (Med Associates) for a 5-

minute session of white noise (70 dB) habituation after which the test session was

automatically started. The session started with a habituation block of 6 presentations of

the startle stimulus alone, followed by 10 PPI blocks of 6 different types of trials. Trial

types are: null (no stimuli), startle (120 dB), startle plus prepulse (4, 8 and 12 dB over

background noise i.e. 74, 78 or 82 dB) and prepulse alone (82 dB). Trial types were

presented at random within each block. Each trial started with a 50 ms null period

during which baseline movements are recorded. There was a subsequent 20 ms period

4

during which prepulse stimuli were presented and responses to the prepulse measured.

After further 100 milliseconds the startle stimuli were presented for 40 milliseconds and

responses recorded for 100 milliseconds from startle onset. Responses were sampled

every ms. The inter-trial interval was variable with an average of 15 seconds (range

from 10 to 20 seconds). In startle alone trials the basic auditory startle was measured,

and in prepulse plus startle trials the amount of inhibition of the normal startle was

determined and expressed as a percentage of the basic startle response (from startle

alone trials), excluding the startle response of the first habituation block.

Adult, male C57Bl/6J mice (Jackson Laboratories, Bar Harbor, ME) were acclimatized

to the experimental room for at least 1 hour prior to testing. SEP-856 (0.3, 1, 3, 10 and

30 mg/kg, p.o.) was formulated in 20% hydroxypropyl-beta-cyclodextrin (vehicle) and

haloperidol (1 mg/kg, i.p.) in 10% DMSO. The dosing volume for all treatments was 10

ml/kg and animals were dosed 30 min prior to testing. The PPI enclosures were cleaned

following each test. Data were analyzed using one-way ANOVA followed by Tukey’s

post-hoc test (p < 0.05).

PCP-Induced Deficits in Social Interaction

For five days prior to test, male Sprague-Dawley rats (~ 150 g on arrival from Harlan

Laboratories, IN) were injected twice daily with either PCP (2 mg/kg; s.c.) or saline

(s.c.). On day 6, after acute pretreatment (30 min) with either water (p.o.), clozapine (2.5

mg/kg, i.p.) or SEP-856 (1, 3 and 3 mg/kg, p.o.) a pair of unfamiliar rats, receiving the

same treatment were placed in a white plexiglass open field arena (24" x 17" x 8") and

allowed to interact with each other for 6 minutes. Social interactions (‘SI’) included:

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sniffing the other rat; grooming the other rat; climbing over or under or around the other

rat; following the other rat; or exploring the ano-genital area of the other rat. Passive

contact and aggressive contact were not considered a measure of social interaction.

The time the rats spent interacting with each other during the 6-minute test was

recorded by a trained observer blinded to drug treatment and condition. The social

interaction chambers were thoroughly cleaned after each test session. Twenty animals

were tested in each group for a final number of ten interactions. PCP, clozapine and

SEP-856 were dissolved in saline, 5%PEG/5%Tween80 in saline, and sterile injectable

water respectively. All treatments were administered at a dosing volume of 1ml/kg. Data

were analyzed using one-way ANOVA followed by Tukey’s post-hoc test (p < 0.05).

Forced Swim Test

The forced swim test consisted of one 6-minute session of forced swimming in

individual opaque cylinders (15 cm tall x 10 cm wide, 1000 ml beakers) containing fresh

tap water at a temperature of 23 2 C and a depth of 12 cm (approximately 800 ml) for

each test animal. The time the animal spent immobile was recorded over the 6-minute

trial. Every one minute, the cumulative immobility time was recorded from the start of

the session and noted on the study data record sheet. Immobility was defined as the

postural position of floating in the water. The animals are generally observed with the

back slightly hunched and the head above water with no movements or with small

stabilizing movements of the limbs. Sometimes the back is arched with the animal

stretched across the sides of the beaker, and in this posture, immobility was recorded

only if the animal was not struggling. Adult, male BalbC/J mice (Jackson Laboratories,

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Bar Harbor, ME) were tested 60 minutes post administration of vehicle (sterile injectable

water; p.o.), or SEP-856 (0.3, 1, 3 and 10 mg/kg, p.o.) and 30 minutes post sertraline

(positive control; 20 mg/kg, i.p.) injection. All treatments were formulated in sterile

injectable water and administered at a dosing volume of 10 ml/kg. Data are represented

as the time the mice spent immobile during the 6-minute trial. Data were analyzed using

one-way ANOVA followed by Tukey’s post-hoc test (p < 0.05).

Catalepsy

Catalepsy was assessed using the bar test in adult, male C57Bl/6J mice (Jackson

Laboratories, Bar Harbor, ME). The front paws of a mouse were placed on a horizontal

metal bar raised 2” above a Plexiglas platform and time was recorded for up to 30

seconds per trial. The test ended when the animal’s front paws returned to the platform

or after 30 seconds. The test was repeated three times and the average of the trials is

reported as the intensity index of catalepsy. Mice were acclimatized to the experimental

room for at least one hour prior to testing. Catalepsy was assessed at 30 minutes and

90 minutes following vehicle (20% hydroxypropyl-beta-cyclodextrin, p.o.), haloperidol (1

mg/kg, i.p.) or SEP-856 (100 mg/kg, p.o.) administration. Haloperidol and SEP-856

were dissolved in 20% hydroxypropyl-beta-cyclodextrin and 10% DMSO respectively

and administered at a volume of 10 ml/kg. At the end of each trial, the apparatus was

thoroughly cleaned with 70% ethanol. Data were analyzed using two-way ANOVA

followed by Tukey’s post-hoc test (p < 0.05).

7

Electroencephalograph (EEG) Recordings

Seven male Sprague-Dawley rats (300 ± 25 g; Charles River, Wilmington, MA) were

implanted with chronic recording devices for continuous recordings of EEG,

electromyograph (EMG), core body temperature (Tb), and locomotor activity (LMA) via

telemetry. Under isoflurane anesthesia (1–4%), a dorsal midline incision on top of the

head and a midventral incision along the linea alba through the peritoneum were made.

Sterile miniature transmitters (F40-EET, Data Sciences Inc., St Paul, MN) were inserted

through the peritoneal incision and sewn to the musculature with a single stitch of silk

suture (4-0). Four biopotential leads from the transmitters were inserted subcutaneous

into the neck and head region. Two holes were drilled through the skull, one at –5.0 mm

AP and 4.0 mm ML and the other at 2.0 mm AP and 2.0 mm ML from bregma. The two

biopotential leads used as EEG electrodes were inserted into the holes and affixed to

the skull with dental acrylic. The two biopotential leads used as EMG electrodes were

sutured into the neck musculature. The incision was closed with suture (silk 4-0) and

antibiotics were administered topically. Pain was relieved by a long-lasting analgesic

(buprenorphine) administered intramuscularly postoperatively. After surgery, animals

were placed in a clean cage and observed until they recovered. EEG, EMG, Tb, and

LMA were recorded via telemetry using DQ ART 4.1 software (Data Sciences Inc., St

Paul, MN). Animals were acclimated to the handling procedures and were given two

separate 1 ml administrations of vehicle by oral gavage, one 7 days and the other 3

days before the first experimental day. Following completion of the data collection,

expert scorers determined states of sleep and wakefulness in 10 second (s) epochs by

examining the recordings visually using NeuroScore software (Data Sciences Inc., St

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Paul, MN). All doses of SEP-856 (1, 3 and 10 mg/kg), caffeine (10 mg/kg), and vehicle

were administered by oral gavage with a minimum of 3 days between successive

treatments. Doses were administered at the start of Zeitgeber hour 7 (ZT7; i.e. 6 hours

after light on) and the following 6 hours of continuous EEG and EMG recordings were

analyzed. EEG and EMG data were scored visually in 10 second epochs for wakes (W),

REM, and non-REM (NREM). Scored data were analyzed and expressed as time spent

in each state per hour. Latency to NREM onset for each rat was calculated from the

time of drug administration to the first six continuous 10 second epochs scored as

NREM. Latency to REM onset for each rat was calculated from the time of drug

administration to the first three continuous 10 second epochs scored as REM.

Cumulative time spent in W, NREM, and REM, as well as the REM:NREM ratios, were

calculated for the 6-hour recording period. Tb and LMA (counts per minute) were

analyzed as mean values per hour (hourly means).

In Vivo Microdialysis

Male Sprague-Dawley rats (240-325 g; Harlan, Frederick, MD) were allowed to

acclimate to the facility for at least 5 days before surgery.

Microdialysis experiment: Two days prior to test article administration, rats were

anesthetized with a mixture of ketamine/xylazine (70 mg/kg/6 mg/kg, i.m) and placed in

a stereotaxic instrument. Microdialysis probes (CMA/12; 4 mm membranes) were then

implanted in the striatum and/or prefrontal cortex. The coordinates from bregma for

striatum were +0.9 mm anterior-posterior, +3 mm medio-lateral; -7.5 mm dorso-ventral

and for prefrontal cortex, +3.4 mm anterior-posterior, -0.6 mm medio-lateral; -5.5 mm

9

dorso-ventral. Probes were secured to the skull with cranioplastic cement (Henry

Schein) and three screws. Once conscious, rats received a single dose of ketoprofen

(10 mg/kg, s.c) for analgesia. 24 to 28 hours after the surgery, rats were placed under

light isoflurane anesthesia in order to connect the microdialysis probes to the perfusion

pumps using sterile polyethylene tubing. Rats were then placed in microdialysis

chambers (with free access to food and water) and artificial cerebrospinal fluid (aCSF;

CMA) was perfused through the probes at a flow rate of 0.5 µL/min overnight.

Approximately 14-16 hours later, on the day of test article administration, the probe

perfusion flow rate was increased to 1.5 µL/min and a 2-hour equilibration period was

allowed before sample collection began. Microdialysate samples were collected every

30 minutes using tubes containing 10 µL of formic acid (0.5M) in refrigerated fraction

collectors, to prevent monoamine degradation. Four baseline samples were collected at

30-minute intervals over a 2-hour period before rats were dosed orally (2 mL/kg, via

gavage) with SEP-856 or vehicle. SEP-856 was administered at 3, 10 and 30 mg/kg

p.o. in saline. Eight samples were collected at 30-minute intervals over a 4-hour period

after test article administration. Animals were awake and freely moving throughout the

experiment. Rats were returned to their home cage at the end of the microdialysis

experiment and sacrificed by decapitation within 72 hours. Brains were immediately

removed and frozen using isopentane in dry ice and stored at -80ºC until histological

verification of probe placement.

Sample analysis: Monoamine levels in perfusate samples from either brain area were

analyzed by HPLC-EC detection within 24 hours of the microdialysis experiment. On

10

every experimental day, HPLC instruments were calibrated using freshly prepared

standards containing known concentrations of dopamine (DA) and serotonin (5HT) (0;

0.05; 0.1; 1; 2 and 5 pg/µL of aCSF). For each concentration, 45 µL of standards in

aCSF were added to 10 µL of formic acid (0.5M) in order to reproduce the dialysate

sample conditions. DA and 5HT levels in the striatum were measured by transferring the

dialysate samples to an autosampler (ESA, Inc. Model 540). 27 µL were injected onto a

Capcell Pak column (250 × 1.5 mm, 3 µm particle size; ESA MD-160). DA and 5HT

were eluted using a mobile phase consisting of 150 mM ammonium acetate and 140 µM

EDTA in 15% methanol and 5% acetonitrile, pH 6.0. DA and 5HT were detected with a

glassy carbon target analytical cell (ESA 5041) at a potential of 220mV using a

Coulochem III detector (ESA). Chromatography data was acquired on a PC and

analyzed using the EZ Chrome Elite software (Agilent technologies). DA and 5HT were

measured in dialysate samples from the prefrontal cortex using the Alexys 100 system

(Antec Leyden). Samples were transferred onto an autosampler (AS 100Antec Leyden)

and a 30 µL injection was split equally between 2 columns. DA and 5HT were eluted

with a C18 column (50 × 1 mm, 3 µm particle size; Antec Leyden ALB-105). The mobile

phase for either column consisting in 50 mM phosphoric acid, 8 mM KCl, 0.1 mM EDTA

and 500 mg/L OSA in 4 to 7% MeOH, pH 6.0. DA and 5HT were detected with a 0.7

mm glassy carbon electrode cell (VT-03, Antec Leyden) at a potential of 300 mV.

Chromatography data were acquired on a PC and analyzed using the Alexys software

(Antec Leyden).

11

In Vivo Pharmacokinetic Studies

Pharmacokinetic studies were conducted in male ICR mice (21.2 to 24.4 g; Shanghai

Laboratory Animal Center), male Sprague-Dawley rats (212 to 235 g; Shanghai

Laboratory Animal Center) and male rhesus monkeys (~ 3.75 years of age). In all

studies SEP-856 was formulated in phosphate buffered saline (pH 7 – 7.4) for oral or

i.v. administration to animals that were fasted overnight. To determine brain penetration,

mice or rats were dosed orally with 10 mg/kg SEP-856 and blood and brain collected at

various timepoints from 15 min to 24 hours (n = 3/timepoint) after drug administration.

Blood samples were collected by cardiac puncture following euthanasia by CO2

inhalation, placed into K2EDTA tubes and centrifuged at 8,000 rpm for 6 minutes at 40C,

and the plasma extracted and frozen at -80oC. Brains were removed, placed on ice and

rinsed with saline prior to freezing at -80oC. The PK properties of SEP-856 were

determined in rats dosed with 10 mg/kg (i.v.) or 50 mg/kg (p.o.) SEP-856 and in rhesus

monkeys dosed with 5 mg/kg SEP-856 (i.v. or p.o.). Serial blood samples were

collected at various timepoints ranging from 5 min to 24 hours post dose (n = 3

subjects). Concentrations of SEP-856 were determined by high performance liquid

chromatography with mass spectrometric detection. The lower limits of quantification

were 2.5 ng/ml for plasma and 5 ng/g for brain and pharmacokinetic parameters were

calculated using WinNonlin Pro version 5.0 or 5.2 (Pharsight Corporation, USA). Any

concentrations that were below the limit of quantification were omitted from the

calculation of pharmacokinetic parameters in individual animals.

12

In Vitro Autoradiography

In vitro autoradiography was used to determine the effects of SEP-856 on [3H]-8-OH-

DPAT binding to 5-HT1A receptors in brain sections of adult Sprague-Dawley rats

(Harlan, Frederick, MD). Briefly, slide mounted rat brain sections (12 μm) consisting of

prefrontal cortex (PFC), cortex (motor and somatosensory), septum, striatum, dorsal

hippocampus and hypothalamus were preincubated for 30 minutes in 50 mM tris buffer

(pH 7.5) at room temperature. Sections were then incubated in the same buffer

containing 4 mM CaCl2; 0.1% ascorbic acid pH 7.5 containing 2 nM [3H]-8-OH-DPAT in

the absence (total binding) and presence of SEP-856 (100 nM, 1 uM or 10 µM) for one

hour at room temperature (22 -25 °C). Non-specific binding was defined by 10 μM 5-HT.

Following incubation, slices were briefly rinsed, then washed in ice cold incubation

buffer for 2 x 10 minutes. Brain sections were then rinsed in ice cold H2O and dried

under a stream of cool air. Rat brain sections were imaged by placing the tissue

sections in a Biospace β-Imager for 6 hours. Afterwards, [3H]-8-OH-DPAT binding in the

absence and presence of SEP-856 was quantified using software provided by Biospace

and the effect of SEP-856 on [3H]-8-OH-DPAT binding to 5-HT1A receptors was

determined. In a follow up experiment, the above experimental design was repeated

using rat brain sections containing cortex and septum. Following image quantification,

Graphpad Prism software was used to determine the IC50 of SEP-856.

D2 Receptor Occupancy in Rats

The occupancy of D2 receptors after i.p. administration of SEP-856 was evaluated in

male Sprague-Dawley rats (216-232 g; Harlan, Frederick, MD). SEP-856 (10 mg/kg, 2

13

ml/kg) or vehicle (20% HPβCD) were administered i.p. at t = 0. [3H]-raclopride (60

µCi/kg) was administered i.v. at t = 30 min and rats were killed by rapid decapitation at t

= 60 min. Brains were frozen in isopentane (previously cooled on dry ice) and stored at -

80ºC until required for cryosectioning. Blood samples were processed to obtain plasma

samples which were then stored at -80ºC until required for exposure analysis. For

cryosectioning, brains were removed from the -80ºC freezer and allowed to thaw to

-20ºC. Coronal sections (20 µm) of the striatum (region of interest) and cerebellum

(reference region) were cut and thaw mounted onto glass microscope slides. Slide

mounted tissue sections (of striatum and cerebellum) were placed in the Biospace β-

Imager and imaged for 6 hours. Images were quantitated using software (Betavision

plus) provided by Biospace and a signal:noise (striatum:cerebellum) value was

determined for each animal. The percent receptor occupancy was determined using the

following equation:

%RO = 100 x [(average S:NVehicle) – S:NSubject] / [(average S:NVehicle) –1]

A satellite group of rats was also used for determination of SEP-856 exposures in the

plasma and brain. Rats received SEP-856 (10 mg/kg, 2 ml/kg, i.p.). 60 minutes later,

rats were euthanized by CO2 inhalation. Rats were then decapitated, and brains and

plasma samples collected. Brain and plasma samples were analyzed for drug

concentration using LC/MS analysis.

D2 Receptor Occupancy in Nonhuman Primates

Positron Emission Tomography (PET) imaging was conducted in two nonhuman

primate female baboons (Papio anubis; ~18 kg) to study the effect of SEP-856 on D2

14

receptors: one dose (~7.25 mg/kg) was tested in duplicate. Prior to injection, quality

control of the radiopharmaceutical was performed to ensure purity, identity, strength and

sterility. A blockade protocol design was used to estimate the receptor occupancy

where SEP-856 was administered 30 minutes prior to [18F]-fallypride injection. A

baseline study (no blocking agent) was also conducted with [18F]-fallypride for each

animal. The animals were fasted for 18–24 hours before the study and were

anesthetized with intramuscular ketamine at 10 mg/kg and glycopyrrolate at 0.01 mg/kg

(at 2 hours prior to radiopharmaceutical injection for the imaging studies), transferred to

the PET camera for the imaging studies, and intubated with an endotracheal tube for

continued anesthesia with 2.5% isoflurane administered through a rebreathing circuit.

Body temperature was kept at 37 °C using a heated water blanket. Vital signs, including

heart rate, blood pressure, respiration rate, oxygen saturation and body temperature,

were monitored every 10 to 20 min during the study. An intravenous line was placed

and used for injection of the radiopharmaceutical [18F]-fallypride and the test article

SEP-856 for the blockade studies. For the latter, the animal was injected with SEP-856

as a bolus over 5 min at T = 0 min and with the radiotracer [18F]-fallypride at T = 30

minutes. Following the intravenous injection of [18F]-fallypride as a bolus over 3 min at T

= 30 minutes, a series of 45 dynamic 3D PET scans were obtained continuously on a

Biograph mCT camera over three hours (T = 30 – 210 minutes) as follows: 6 x 30

seconds, 3 x 1minutes, 2 x 2 minutes, 34 x 5 minutes. The dynamic series were

subsequently reconstructed using iterative reconstruction with corrections for random,

scatter, and attenuation provided by the camera manufacturer. Reconstructed PET

image data volumes were transferred to the image processing PMOD software package

15

(PMOD Technologies, Zurich, Switzerland) where the images were realigned onto the

monkey MR to apply a volume of interest (VOI) template comprising the following

regions: caudate, putamen, globus pallidus and cerebellum. Average activity

concentration (kBq/cc) within each VOI was determined and time activity curves (TAC)

were generated for each study, depicting the regional brain activity concentration over

time (total uptake = specific plus non-displaceable). Time activity curves were

expressed in SUV units (g/mL) by normalizing by the animal weight and the injected

dose. The non-invasive reference region models SRTM, SRTM2 and Logan

noninvasive using the cerebellum as reference region were applied to the regional time

activity curves with the PMOD software to determine the binding potential BPND for

each region mentioned above. For SRTM2, k’2 was estimated by doing a coupled fit

across the caudate, putamen and globus pallidus. For Logan, the k’2 obtained from the

SRTM2 coupled fit was used. The occupancy was determined using the binding

potential BPND as follows: Occ = ((BPNDBaseline – BPNDDrug)/BPNDBaseline). Similar

results were obtained with all three non-invasive reference region models (SRTM,

SRTM2 and Logan noninvasive). Thus, only the values obtained with the SRTM model

are shown in the results.

Whole-Cell Patch Clamp Recordings in the DRN and VTA

Male C57BL/6 mice (4-16 weeks,15-35 g) were humanely killed by terminal anesthesia

with isoflurane, cervical dislocation, and decapitation. The brain was then removed and

300 μm thick sagittal slices containing the VTA and/or the DRN were sectioned using a

Leica VT1000S. After brain removal and throughout slicing the tissue was submerged in

16

ice cold (< 4°C) ‘high sucrose’ artificial cerebrospinal fluid (aCSF) of the following

composition (in mM): Sucrose, 154; KCl, 1.9; KH2PO4, 1.2; CaCl2, 0.1; MgCl2, 3.6;

NaHCO3, 26; D-glucose, 10; L-ascorbic acid, 0.3; equilibrated with 95% O2-5% CO2.

Once slices were cut, they were transferred to a beaker containing ‘standard’ aCSF and

left at room temperature for a minimum of one hour before commencing

electrophysiological recordings. After this period, individual slices were transferred to a

custom-built recording chamber continuously perfused with ‘standard’ aCSF at a rate of

4–10 ml/min. ‘Standard’ aCSF composition (in mM): NaCl, 127; KCl, 1.9; KH2PO4, 1.2;

CaCl2, 2.4; MgCl2, 1.3; NaHCO3, 26; D-glucose, 10; equilibrated with 95% O2-5% CO2.

Whole-cell patch-clamp recordings were performed at room temperature using the

‘blind’ version of the patch-clamp technique with either Axopatch 1D or Multiclamp 700B

amplifiers. Patch pipettes were pulled from thin-walled borosilicate glass with

resistances of between 3 and 8 MΩ when filled with intracellular solution of the following

composition (mM): potassium gluconate, 140; KCl, 10; EGTA-Na, 1; HEPES, 10;

Na2-ATP, 2, 0.3 GTP with pH and osmolarity compensated with KOH and sucrose,

respectively. Recordings were monitored on an oscilloscope and a PC running Axon

pClamp software and digitized at 2-10 kHz. At the beginning of each whole-cell patch-

clamp recording, a current/voltage (IV) relationship was performed to identify active

conductances in the recorded neuron, before testing the effects of SEP-856 and

TAAR1/5-HT1A receptor ligands. Changes in electrophysiological parameters including

membrane potential and neuronal firing rate were analyzed using Axon pClamp and

Microsoft Excel software to measure the effects of test compounds. To attempt neuronal

phenotype characterization, DRN neurons were classified as being either sensitive or

17

insensitive to administration of the 5-HT1A receptor agonist 8-OH-DPAT. In addition,

after the determination of repeatable responses, administration of SEP-856 was

repeated in the presence of the selective TAAR1 antagonist EPPTB and/or the selective

5-HT1A receptor antagonist WAY-100635. Compound concentrations were chosen

based on the results of pilot concentration-response whole-cell patch clamp recordings

in the VTA and DRN. Changes in neuronal properties are presented as mean ± S.E.M

and, in some cases, calculated as a percentage of control by calculating the mean of

the normalized response. Where appropriate, statistical comparisons have been

performed using the paired student’s t-test, with P < 0.05 taken to indicate statistical

significance.

In Vivo Extracellular Single-Unit Recordings in the DRN

Male Sprague-Dawley rats (275-400 g; Harlan, IN) were anesthetized with Urethane

(initial dose at 1.2 to 1.6 g/kg, i.p.) and were surgically implanted with two catheters, one

for femoral vein (drug administration) and one for femoral artery (blood sampling). The

animals were then mounted on a stereotaxic apparatus (David Kopf instrument) in a flat

skull position. Proper surgical anesthesia was maintained throughout the experiment by

administration of supplemental doses of the anesthetic. Core temperature was

maintained at 37⁰C by a heating pad. Borosilicate glass micropipette electrodes (3 mm

OD, 2 mm ID, Sutter Instrument) were pulled by PE-22 micro-electrode puller (Narishige

Group) and then filled with 0.5% sodium acetate in 2% Pontamine Skyblue (Sigma).

The electrodes had in vitro impedances of 1-3 MΩ. To gain access to dorsal raphe

nucleus recording site the micro-electrode was advanced by a single axis in vivo

18

micromanipulator (Scientifica, United Kingdom) mounted on Kopf stereotaxic holders.

One burr hole was drilled on the skull with stereotaxic coordinate of AP-7.8 mm, lateral

0.8 mm. The dura was carefully removed to expose the cortical surface. The recording

electrode was inserted into brain through the hole at a 10-degree angle towards midline

and advanced to reach the target coordinate of raphe nucleus (5.1-6.1 mm below the

brain surface).

Extracellular single-unit activities were amplified (x1000), filtered (low pass 3 KHz and

high pass at 300 Hz), displayed on the oscilloscope and stored in a computer equipped

with the Spike 2 analysis system (Cambridge Electronic Design, UK) for off-line

analysis. The recorded neurons location was histologically confirmed. Based on

previous reports, the neurons which met the following criteria were included for the

study: Slow firing rate (0.1 to 5 Hz), exhibiting a long duration (2-4 ms), single or

bursting patterned action potentials with biphasic or triphasic extracellular waveforms

(Aghajanian et al., 1978; Clifford et al., 1998; Hajós et al., 2007). The baseline firing

activity of the neuron was recorded for at least 10 min prior to the compound

administration. SEP-856 was tested at 1, 2, and 5 mg/kg by i.v. injection. After clear

inhibitory effects were observed (3-5 minutes after compound administration), WAY-

100635 (0.08 mg/kg, i.v.) was given to determine whether it could antagonize the

inhibitory effect of SEP-856. This dose range was previously reported to reverse the

inhibitory effects of 8-OH-DPAT (Martin et al., 1999). All test substances were dissolved

in sterile saline and administered i.v. at a dose volume of 1 ml/kg. Blood samples were

taken 30 min following compound administration. At the end of each experiment, the

recording site was marked by the microiontophoresis of pontamine skyblue (-20 µA, 15

19

min). Each rat was then given an overdose of urethane. The brains were immediately

removed, were frozen on dry ice, and were cut into 40 µM thick coronal sections using a

cryostat. The sections were mounted on gelatin-coated slides and stained with cresyl

violet in order to determine the location of the recording sites.

In Vitro Binding Studies

Equilibrium radioligand binding was performed using the following incubation conditions:

5-HT1A: Membranes from HEK-293 cells expressing the human recombinant 5-HT1A

receptor were incubated in the presence of 0.3 nM [3H]-8-OH-DPAT for 60 minutes at

22°C.

5-HT1B: Rat cerebral cortex membranes were incubated with 0.1 nM [125I]-CYP for 120

minutes at 37°C.

5-HT1D: Membranes from CHO cells expressing the rat recombinant 5-HT1D receptor

were incubated in the presence of 1 nM [3H]-5-HT for 60 minutes at 22°C.

5-HT2A: Membranes from HEK-293 cells expressing the human recombinant 5-HT2A

receptor were incubated in the presence of 0.1 nM [125I]-(±)DOI for 60 minutes at 22°C.

5-HT2B: Membranes from HEK-293 cells expressing the human recombinant 5-HT2B


5-HT2C: Membranes from HEK-293 cells expressing the human recombinant 5-HT2C


5-HT7: Membranes from HEK-293 cells expressing the human recombinant 5-HT7

receptor were incubated in the presence of 4 nM [3H] LSD for 120 minutes at 22°C.

20

D2: Membranes from HEK-293 cells expressing the human recombinant D2 receptor

were incubated in the presence of 0.3 nM [3H]-methylspiperone for 60 minutes at room

temperature.

2A: Membranes from HEK-293 cells expressing the human recombinant 2A receptor

were incubated in the presence of 0.3 nM [3H]-methylspiperone for 60 minutes at room

temperature.

21

Supplemental Figures

Supplemental Figure 1. SmartCube® behavioral signatures of typical and atypical antipsychotic

drugs in mice. (A) Behavioral class signature color key representing 15 classes (*antipsychotic

(purple) and high-dose antipsychotic (dark purple); **antidepressant (green) and *high-dose

antidepressant (dark green)). (B) The predominant purple color is characteristic of an

antipsychotic signature as shown for different doses of risperidone, haloperidol, clozapine and

quetiapine (administered i.p. 15 min before testing, n = 6-8/dose).

22

Supplemental Figure 2. SEP-856 exhibits good brain penetrance and rapid absorption in

rodents. SEP-856 was administered orally and/or intravenously to male (A) ICR mice and (B)

Sprague-Dawley rats. Plasma and brain samples were taken from n = 3 animals at each of the

time points indicated. The brain-to-plasma AUC ratios for the 10 mg/kg dose ranged from 2.58

to 3.54 and 2.1 to 3.9 in mice and rats, respectively. Data are shown as mean ± s.e.m.

23

Supplemental Figure 3. Effect of SEP-856 on baseline locomotion and the acoustic startle

response in C57Bl/6J mice. (A) At the highest tested dose (3 mg/kg), oral SEP-856

administration led to a slight reduction in overall activity during the open field test (one-way

ANOVA F(5, 59) = 5.5, p = 0.0003; Tukey’s post-hoc test, * p < 0.05 vs. Veh (second bar from the

left)). Clozapine (CLZ) tested at 1 mg/kg i.p. had no significant effect on locomotion. First

vehicle (20% cyclodextrin, i.p.); second vehicle (20% cyclodextrin, p.o.). (B) The acoustic startle

response, measured during the PPI test, was not significantly affected by SEP-856 (p.o.)

compared to the respective vehicle control. In contrast, Haloperidol (i.p.) produced a significant

reduction in the mean startle response (one-way ANOVA for the first experiment: F(4, 42) = 8.5, p

< 0.0001; second experiment: F(5, 49) = 11.5, p < 0.0001; Tukey’s post-hoc test, * p < 0.05 vs.

Veh; n = 8-10/group). Vehicle for haloperidol (10% DMSO, i.p.), vehicle for SEP-856 (20%

cyclodextrin, p.o.). Data are shown as mean ± s.e.m.

24

Supplemental Figure 4. Effects of SEP-856 on sleep/wake architecture in Sprague-Dawley rats.

(A) Acute, oral administration of SEP-856 led to a significant increase in cumulative wake time

at 10 mg/kg over the 6-hour recording period (one-way repeated-measures ANOVA + two-tailed

t-test, *p < 0.05 vs. Veh). Cumulative NREM (B), latency to NREM (C), locomotor activity (D)

and body temperature (E) were not significantly affected by SEP-856 during the 6-hour

recording period (two-way repeated-measures ANOVA + two-tailed t-test, *p < 0.05 vs. Veh).

The positive control caffeine (CAF, 10 mg/kg, p.o.) produced an expected increase in

cumulative wake time, decrease in cumulative NREM time, increase in the latency to NREM as

well as an increase in locomotion and body temperature. Dosing occurred in the middle of the

resting phase, at the beginning of Zeitgeber time 7. N = 7. Data are shown as mean ± s.e.m.

25

Supplemental Figure 5. SEP-856 binds to 5-HT1A receptors in rat brain slices. (A)

Autoradiographs of coronal brain sections depicting binding of the 5-HT1A agonist

[3H]-8-OH-DPAT. SEP-856 displaced [3H]-8-OH-DPAT in a concentration-dependent manner, in

all brain regions tested. N = 4 brains, 2 sections/brain. Data are shown as mean ± s.e.m.

26

Supplemental Figure 6. Effects of SEP-856 on DRN firing. (A) Summary of the general

properties of neurons recorded from the DRN. DRN neurons were classified based on their

electrophysiological properties and response to administration of the 5-HT1A receptor agonist

[3H]-8-OH-DPAT (DPAT). (B) Representative current/voltage relationship (left) of a DRN neuron

in which DPAT induced a marked membrane hyperpolarization (right). (C) Representative

current/voltage relationship (left) of a DRN neuron in which DPAT was without effect on

membrane potential (right). Example of a current-clamp recording (D) and current/voltage

relationship (E) of a quiescent, DPAT-sensitive DRN neuron in which SEP-856-induced

membrane hyperpolarization. (F) Current-clamp recording of the DRN neuron shown in D and E

during DPAT administration. SEP-856 had little effect in most DPAT-insensitive, quiescent (G)

and spontaneously active (H) DRN neurons. (I, J) Quantifications of SEP-856 response (based

on changes in membrane potential (mV) and/or firing rate (Hz) and expressed relative to

baseline) in DPAT-insensitive DRN neurons. Solid bars indicate the timeframe of compound

administration to the slice. Two-tailed t-test, * p < 0.05. Data are shown as mean ± s.e.m.

27

Supplemental Figure 7. Effects of SEP-856 on VTA firing. (A) Summary of the general

properties of neurons recorded from the VTA. (B) Representative current/voltage relationship

and current-clamp recording of a VTA neuron in which SEP-856 was without an effect. Solid bar

indicates the timeframe of SEP-856 administration to the slice.

28

Supplemental Figure 8. SEP-856 inhibits firing of DRN in vivo through 5-HT1A. (A) Single-unit

discharges in DRN neurons were abolished following administration of the 5-HT1A agonist 8-OH-

DPAT (i.v.). The inhibition of firing was reversed by subsequent administration of the selective

5-HT1A antagonist WAY-100635 (i.v., n = 1). (B) Rate histogram showing partial inhibitory

effects of SEP-856 (1 mg/kg, i.v.) on single unit discharge of dorsal raphe nucleus. The

inhibitory effect was time dependently reversed. Subsequent administration of SEP-856 at 2

mg/kg, i.v. resulted in a complete inhibition in discharge which was fully reversed by

WAY-100635 (i.v., n = 1). Abbreviations: WAY-100635 (WAY-635).

29

Supplemental Figure 9. Oral SEP-856 administration does not alter extracellular serotonin

(5-HT) and dopamine (DA) levels in the striatum and prefrontal cortex (PFC) of male

Sprague-Dawley rats. All treatments were administered at time = 0, following 90 minutes of

baseline-sample collection. Monoamine levels are expressed as percent change from baseline.

(A) SEP-856 (p.o.) at all doses tested did not affect extracellular DA levels in the striatum (two-

way repeated measures ANOVA: treatment effect F3, 509 = 1.3, p = 0.1; time x treatment

interaction F(33,509) = 1.34, p = 0.1; n = 11-13/group) and PFC (two-way repeated measures

ANOVA: treatment effect F(3, 343) = 0.85, p = 0.5; time x treatment interaction F(33,343) = 1.4, p =

0.3; n = 8-10/group). (B) Similarly, 5-HT release in the striatum and PFC was not affected by

oral SEP-856 treatment compared to vehicle control (two-way repeated measures ANOVA:

striatum - treatment effect F(3, 240) = 3.0, p = 0.05; time x treatment interaction F(33,240) = 1.1, p =

0.35; n = 5-9/group / PFC - treatment effect F(3, 91) = 3.5, p = 0.06; time x treatment interaction

F(33,91) = 0.9, p = 0.6; 3-4/group). Data are shown as mean ± s.e.m.

30

Supplemental Tables

Supplemental Table 1. Cerep Bioprint Targets

A1 ETB M2 5-HT6

A2A GABAA M3 5-HT7

A2B GABAB(1b) M4

A3 glucagon NK1 sst1

1A AMPA NK2 sst4

1B kainate Y1 GR

2A NMDA N neuronal

-BGTX-insensitive (42) ER

2B glycine

(strychnine-insensitive)

N muscle-type AR

2C CXCR4 (DOP) TR (TH)

TNF− (KOP) UT

2 CCR2 (MOP) VPAC1 (VIP1)

3 H1 PPAR V1a

AT1 H2 PAF V2

AT2 H3 PCP Ca2+ channel (L, dihydropyridine site)

APJ (apelin) H4 EP2 Ca2+ channel

(L, diltiazem site)

(benzothiazepines)

BZD I1 FP Ca2+ channel

(L, verapamil site)

(phenylalkylamine)

BB3 BLT1 (LTB4) IP (PGI2) Ca2+ channel (N)

B2 CysLT1 (LTD4) LXR SKCa channel

CB1 MCH1 PCP Na+ channel (site 2)

CB2 MC1 5-HT1A Cl- channel (GABA-gated)

CCK1

(CCKA)

MC3 5-HT1B norepinephrine transporter

CCK2

(CCKB)

MC4 5-HT1D dopamine transporter

CRF1 MT1 (ML1A) 5-HT2A GABA transporter

D1 MT3 (ML2) 5-HT2B 5-HT transporter

D2S MAO-A 5-HT2C

D3 motilin 5-HT3

ETA M1 5-HT4e

31

Supplemental Table 2. Cerep Enzyme Screen Targets

COX1 HIV-1 protease p38α kinase

COX2 neutral endopeptidase acetylcholinesterase

PDE2A MMP-1 COMT

PDE3A MMP-2 xanthine oxidase/ superoxide O2-

scavenging

PDE4D MMP-9 ATPase (Na+/K+)

PDE5 (non-selective) Abl kinase 5-HT transporter

PDE6 (non-selective) CDK2 (cycE) p38α kinase

ACE ERK2) (P42mapk) acetylcholinesterase

ACE-2 FLT-1 kinase (VEGFR1) COMT

BACE-1 (β-secretase) Fyn kinase xanthine oxidase/ superoxide O2-

scavenging

caspase-3 IRK (InsR) ATPase (Na+/K+)

caspase-9 Lyn A kinase (h) 5-HT transporter

32

Supplemental Table 3. Ricerca Receptor Screen Targets

Adenosine A1 Estrogen ERα N-Formyl Peptide Receptor

FPR1

Tachykinin NK3

Adenosine A2A Estrogen ERβ N-Formyl Peptide Receptor-Like

FPRL1

Thyroid Hormone

Adenosine A3 GPR103 Neuromedin U NMU1 TRH

Adrenergic α1A GABAA, Chloride Channel,

TBOB

Neuromedin U NMU2 TGF-β

Adrenergic α1B GABAA, Flunitrazepam, Central Neuropeptide Y Y1 Transporter, Adenosine

Adrenergic α1D GABAA, Muscimol, Neuropeptide Y Y2 Transporter, Choline

Adrenergic α2A GABAB1A Neurotensin NT1 DAT

Adrenergic α2C Gabapentin Nicotinic Acetylcholine Transporter, GABA

Adrenergic β1 Galanin GAL1 Nicotinic Acetylcholine α1,

Bungarotoxin

Transporter, Monoamine

Adrenergic β2 Galanin GAL2 Nicotinic Acetylcholine α7,

Bungarotoxin

NET

Adrenergic β3 Glucocorticoid 314541 NPBW2/GPR8 SERT

Adrenomedullin AM Glutamate, AMPA Opiate δ1 (OP1, DOP) TNF, Non-Selective

Adrenomedullin AM2 Glutamate, Kainate Opiate κ(OP2, KOP) Urotensin II

Androgen Glutamate, NMDA, Agonism Opiate μ(OP3, MOP) Vanilloid

Angiotensin AT2 Glutamate, NMDA, Glycine Orphanin ORL1 VIP1

APJ Glutamate, NMDA,

Phencyclidine

Phorbol Ester Vasopressin V1A

Atrial Natriuretic Factor (ANF) Glutamate, NMDA, Polyamine PDGF Vasopressin V1B

Bombesin BB1 Glycine, Strychnine-Sensitive Potassium Channel [KATP] Vasopressin V2

Bombesin BB2 Growth Hormone Secretagogue

(GHS)

Potassium Channel [SKCA] Vitamin D3

Bombesin BB3 Growth Hormone Secretagogue

(GHS)

Potassium Channel hERG

Bradykinin B1 Histamine H1 Progesterone PR-B

Bradykinin B2 Histamine H2 Prostanoid CRTH2

Calcitonin Histamine H3 Prostanoid DP

Calcium Channel L-Type,

Benzothiazepine

Histamine H4 Prostanoid EP2

Calcium Channel L-Type, Dihydropyridine

Imidazoline I2 Prostanoid EP4

Calcium Channel L-Type,

Phenylalkylamine

Inositol Trisphosphate IP3 Purinergic P2X

Calcium Channel N-Type Interleukin IL-1 Purinergic P2Y 314632

Cannabinoid CB1 Interleukin IL-6 Retinoid X RXRα

Chemokine CCR1 Leukotriene, BLT (LTB4) Ryanodine RyR3

Chemokine CCR2B Leukotriene, Cysteinyl CysLT1 5-HT1A

Chemokine CCR4 Leukotriene, Cysteinyl CysLT2 5-HT2B

Chemokine CCR5 Melanocortin MC1 5-HT2C

Chemokine CX3CR1 Melanocortin MC3 5-HT3

Chemokine CXCR2 (IL-8RB) Melanocortin MC4 5-HT4

Colchicine Melanocortin MC5 5-HT5A

Corticotropin Releasing Factor

CRF1

Melatonin MT1 5-HT6

Dopamine D1 Melatonin MT2 Sigma σ1

Dopamine D2S 10 μM -1 Sigma σ2

Dopamine D3 252200 Motilin Somatostatin sst1

Dopamine D4 Muscarinic M1 Somatostatin sst2

Dopamine D5 Muscarinic M2 Somatostatin sst3

Endothelin ETA Muscarinic M3 Somatostatin

Endothelin ETB Muscarinic M4 Somatostatin sst5

Epidermal Growth Factor (EGF) Muscarinic M5 Tachykinin NK1

33

Supplemental Table 4. Ricerca Enzyme Screen Targets

Catechol-O-Methyltransferase (COMT) PDE2

Cholinesterase, Acetyl, ACES PDE2A

Monoamine Oxidase MAO-A PDE3

Monoamine Oxidase MAO-B PDE3A

Nitric Oxide Synthase, Endothelial (eNOS) PDE4

Nitric Oxide Synthase, Inducible (iNOS) PDE4A1A

Nitric Oxide Synthase, Neuronal (nNOS) PDE5

PDE1 PDE5A

PDE10A2 PDE6

PDE1A

34

Supplemental Table 5. Pharmacokinetic properties of SEP-856.

Male ICR mice, Sprague-Dawley rats and rhesus macaques were dosed with SEP-856 by p.o. and/or i.v. administration. Parameters

were derived from mean plasma or brain concentrations for n = 3 animals per dose route. Data are shown as mean or mean ± s.d.

Parameters

Mouse

Rat

Monkey

p.o. i.v. p.o. i.v. p.o.

Plasma Brain Plasma Plasma Plasma Brain Plasma

Dose (mg/kg) 10 10 5 5 10 10 5 5

AUC0-t (ng*h/ml or ng*h/g) 7256 22442 3275 ± 567 1910 ± 144 4955 15102 6563 ± 2202 4708 ± 1694

AUC0-∞ (ng*h/ml or ng*h/g) 7273 22483 3306 ± 588 1930 ± 144 5352 16854 6677 ± 2173 3754

CL (L/h/kg) - - 1.54 ± 0.26 - - - 0.797 ± 0.223 -

Vss (L/kg) - - 2.57 ± 0.1 - - - 3.59 ± 1.96 -

MRT(0-∞ (h) 1.95 2.06 1.41 ± 0.22 1.68 ± 0.03 3.01 3.54 3.33 ± 1.15 5.90

t1/2 (h) 0.847 0.808 1.17 ± 0.16 1.24 ± 0.1 2.10 2.33 3.14 ± 1.26 3.03

tmax (h) 0.50 0.25 0.083 0.42 ± 0.14 0.25 0.25 0.083 6.00 ± 2.83

Cmax (ng/ml or ng/g) 2854 ± 298 7972 ± 2908 2578 ± 110 1056 ± 173 1750 ± 369 3762 ± 1324 2191 ± 194 431 ± 104

Bioavailability (%) - - 58 – 120 71.4 ± 1.59

35

Supplemental Table 6. Plasma and brain exposure to SEP-856 in mice and rats following single

oral administration in different behavioral tests. Data are shown as mean ± s.d.

SEP-856 (mg/kg) Plasma (ng/ml) Brain (ng/g)

Mouse PCP

hyperactivity test1

0.3 4.8 (2.5) 42.6 (0.9)

1 13.1 (7.3) 90.1 (42.6)

3 85.6 (50.2) 367.3 (177.0)

Mouse PPI/startle2

0.3 5.6 ± 1.8 79.1 ± 22.1

1 20.3 ± 3.7 255.8 ± 50.2

3 181.3 ± 73.8 711.9 ± 89.7

10 781.0 ± 169.4 2962.5 ± 373.3

30 1965.0 ± 308.6 6368.8 ± 1560.9

Rat PCP social

interaction test3

1 56.6 ± 10.5 380 ± 20.1

3 112.4 ± 58.9 868.8 ± 458.4

10 280.5 ± 173.2 1939.5 ± 1152.8

1Samples were collected from n = 4 mice per group at the end of behavioral testing, approximately 90 minutes after

SEP-856 administration

2 Samples were collected from n = 4 mice per treatment group at the end of behavioral testing, approximately 55

minutes after SEP-856 administration 3 Samples were collected from n = 4 rats per treatment group at the end of behavioral testing, approximately 40

minutes after administration of SEP-856

36

Supplemental Table 7. SEP-856 occupancy of D2 receptors measured with [3H]-raclopride in

rats.

In vivo occupancy of i.p. SEP-856 (10 mg/kg) at D2 receptors was assessed in male

Sprague-Dawley rats. [3H]-raclopride was administered i.v. 30 minutes post SEP-856/vehicle

dosing. Striatal and cerebellar (reference region) brain sections were assessed 30 min later

using autoradiography. Data are shown as mean ± s.e.m; n = 6/group.

Treatment (i.p.) Signal:Noise % RO Plasma (ng/ml) Brain (ng/g) B:P

Vehicle 5.7 ± 0.1 0 ± 8.5 NA NA NA

10 mg/kg 5.7 ± 0.3 12.6 ± 6.4 1321.7 ± 55.8 7858.3 ± 284.7 6.0 ± 0.2

37

Supplemental Table 8. In vivo occupancy of SEP-856 at D2 receptors measured with

[18F]-fallypride-PET in nonhuman primates (Papio anubis).

[18F]-fallypride binding potentials (BPND) were determined at baseline and following SEP-856

treatment using the non-invasive reference region model SRTM. BPND were subsequently used

to estimate the receptor occupancy in several brain regions. SEP-856 was administered i.v. 30

minutes prior to [18F]-fallypride injection. 3D PET scans were obtained continuously over 3 hours

(T = 30-210 minutes). Data are shown as mean ± s.e.m.

Brain region [18

F]-fallypride BPND

Receptor Occupancy (%)

Plasma (ng/ml)

Baseline ~ 7.25 mg/kg 60 min 180 min

Caudate 16.6 ± 5.7 15.2 ± 5.8 9.1 ± 3.3

2850 ± 250 1765 ± 125 Putamen 20.3 ± 4.8 19.4 ± 5.9 6.2 ± 7.0

Globus Pallidus 8.3 ± 2.2 7.7 ± 2.7 9.6 ± 8.8

38

References

Aghajanian GK, Wang RY, Baraban J, Serotonergic and non-serotonergic neurons of the dorsal

raphe: reciprocal changes in firing induced by peripheral nerve stimulation Brain Res 1978 169-

175.

Alexandrov V, Brunner D, Hanania T, and Leahy E (2015) High-throughput analysis of behavior

for drug discovery. Eur J Pharmacol 750:82–89.

Clifford EM, Gartside SE, Umbers V, Cowen PJ, Hajós M, and Sharp T (1998)

Electrophysiological and neurochemical evidence that pindolol has agonist properties at the 5-

HT1A autoreceptor in vivo. Br J Pharmacol 124:206–212.

Hajós M, Allers KA, Jennings K, Sharp T, Charette G, Sík A, and Kocsis B (2007)

Neurochemical identification of stereotypic burst-firing neurons in the rat dorsal raphe nucleus

using juxtacellular labelling methods. Eur J Neurosci 25:119–126.

Martin LP, Jackson DM, Wallsten C, and Waszczak BL (1999) Electrophysiological comparison

of 5-hydroxytryptamine1A receptor antagonists on dorsal raphe cell firing. J Pharmacol Exp

Ther 288:820–826.

Roberds SL, Filippov I, Alexandrov V, Hanania T, and Brunner D (2011) Rapid, computer vision-

enabled murine screening system identifies neuropharmacological potential of two new

mechanisms. Front Neurosci 5:103.

Shao L, Campbell UC, Fang QK, Powell NA, Campbell JE, Jones PG, Hanania T, Alexandrov V,

Morganstern I, Sabath E, et al. (2016) In vivo phenotypic drug discovery: applying a behavioral

assay to the discovery and optimization of novel antipsychotic agents. MedChemComm

7:1093–1101.

1521-0081/70/3/549–620$35.00 https://doi.org/10.1124/pr.117.015305PHARMACOLOGICAL REVIEWS Pharmacol Rev 70:549–620, July 2018Copyright © 2018 by The Author(s)This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.

ASSOCIATE EDITOR: JEFFREY M. WITKIN

Trace Amines and Their ReceptorsRaul R. Gainetdinov, Marius C. Hoener, and Mark D. Berry

Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg, Russia (R.R.G.); Skolkovo Institute of Science andTechnology (Skoltech), Moscow, Russia (R.R.G.); Neuroscience, Ophthalmology, and Rare Diseases Discovery and Translational Area,pRED, Roche Innovation Centre Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland (M.C.H.); and Department of Biochemistry,

Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada (M.D.B.)

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551II. Vertebrate Trace Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555

A. b-Phenylethylamine, p-Tyramine, and Related Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5551. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555

a. Regulation of Aromatic L-Amino Acid Decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555b. Other Sources of b-Phenylethylamine, p-Tyramine, and Related Compounds . . . . . . . 560

i. Microbiota-Derived Trace Amines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560ii. Food-Derived Trace Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561

2. Degradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5613. Storage and Passage across Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5614. Cellular Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

a. Indirect Sympathomimetic Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562b. b-Phenylethylamine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562c. p-Tyramine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564d. Tryptamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564e. Octopamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565

5. b-Phenylethylamine, p-Tyramine, and Tryptamine in Human Disorders . . . . . . . . . . . . . . . 565B. Isoamylamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566C. Trimethylamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566D. O-Methyl and N-Methyl Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568E. 3-Iodothyronamine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568

1. Cardiovascular Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5692. Metabolic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5693. Thermoregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5694. Other Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570

F. Polyamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570G. Putative Other Trace Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571

III. Invertebrate Trace Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571A. Synthesis and Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571B. Storage and Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571C. Octopamine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572D. Tyramine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572E. Physiologic Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572

1. Octopamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5722. Tyramine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573

IV. Trace Amine–Associated Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573

Address correspondence to: Raul R. Gainetdinov, Institute of Translational Biomedicine, St. Petersburg State University,Universitetskaya Emb. 7-9, 199034 St. Petersburg, Russia. E-mail: [email protected]

This work was supported by the Russian Science Foundation [Grant 14-50-00069 (to R.R.G.)] and the Research and DevelopmentCorporation of Newfoundland and Labrador and Memorial University of Newfoundland (to M.D.B.).

R.R.G., M.C.H., and M.D.B. contributed equally to this work.https://doi.org/10.1124/pr.117.015305.

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A. Evolution of Trace Amine–Associated Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577B. Trace Amine–Associated Receptor 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578

1. Pharmacology of Trace Amine–Associated Receptor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584a. Trace Amine–Associated Receptor 1 Gene Conservation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 584b. Expression of Trace Amine–Associated Receptor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584c. Trace Amine–Associated Receptor 1 Ligands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588

i. Development of Selective Agonists and Partial Agonists . . . . . . . . . . . . . . . . . . . . . . . . . 589ii. Development of N-(3-Ethoxyphenyl)-4-(1-Pyrrolidinyl)-3-(Trifluoromethyl)Benzamide, the First Selective Antagonist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589

d. Signal Transduction and Molecular Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590i. Adenylyl Cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590ii. G Protein–Coupled Inwardly Rectifying Potassium Channels . . . . . . . . . . . . . . . . . . . . 590iii. Heterodimerization with the D2-Like Dopamine Receptor. . . . . . . . . . . . . . . . . . . . . . . 590iv. b-Arrestin 2 and Biased Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592v. Other Signaling Cascades . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592

2. Central Nervous System Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592a. Cellular Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592

i. Dopaminergic Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592ii. Serotonergic Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594iii. Glutamatergic Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594

b. Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595i. Schizophrenia and Bipolar Disorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595ii. Cognitive Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595iii. Depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595iv. Sleep, Wake, and Narcolepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596v. Addiction and Compulsive Behaviors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596vi. Feeding Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597

3. Effects in the Periphery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598a. Diabetes and Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598b. Immunomodulatory Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598c. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599d. Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599

C. Other Tetrapod Trace Amine–Associated Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5991. Trace Amine–Associated Receptor 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5992. Trace Amine–Associated Receptor 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6003. Trace Amine–Associated Receptor 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6004. Trace Amine–Associated Receptor 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6005. Trace Amine–Associated Receptor 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6016. Trace Amine–Associated Receptor 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6017. Trace Amine–Associated Receptor 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6018. Trace Amine–Associated Receptor 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602

ABBREVIATIONS: 3-MT, 3-methoxytyramine; 3IT, 3-iodothyronamine; 5-HT, 5-hydroxytryptamine; AADC, aromatic L-amino aciddecarboxylase; AKT, protein kinase B; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AOC3, amine oxidase, coppercontaining 3; ASIC, acid-sensing ion channel; COMT, catechol-O-methyltransferase; CPP, conditioned place preference; D1R, D1-likedopamine receptor; D2R, D2-like dopamine receptor; DAT, dopamine transporter; DMT, N,N-dimethyltryptamine; DRL, differentialreinforcement of low response rate; DRN, dorsal raphe nuclei; EAAT-2, excitatory amino acid transporter 2; EPPTB, N-(3-ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl)benzamide (also known as RO5212773); FMO3, flavin monooxygenase 3; GLP-1, glucagon-like peptide 1;GPCR, G protein–coupled receptor; GSK3b, glycogen synthase kinase; HEK-293, human embryonic kidney 293; IUPHAR, InternationalUnion of Basic and Clinical Pharmacology; KO, knockout; L-687,414, (3S,4S)-3-amino-1-hydroxy-4-methylpyrrolidin-2-one; MAO, monoamineoxidase; MDMA, 3,4-methylenedioxymethamphetamine; MHC, major histocompatibility complex; NMDA, N-methyl-D-aspartate; NREM,nonrapid eye movement; OAMB, octopamine receptor in mushroom bodies; OCT, octopamine; OCT2, organic cation transporter 2; OE,overexpressing; PCP, phencyclidine; PEA, b-phenylethylamine, 2-phenylethylamine; phMRI, pharmacological magnetic resonance imaging;PKA, protein kinase A; PKC, protein kinase C; PNMT, phenylethanolamine-N-methyl transferase; PYY, peptide YY; RO5166017, (S)-4-((ethyl(phenyl)amino)methyl)-4,5-dihydrooxazol-2-amine; RO5203648, (S)-4-(3,4-dichlorophenyl)-4,5-dihydrooxazol-2-amine; RO5256390,(S)-4-((S)-2-phenylbutyl)-4,5-dihydrooxazol-2-ylamine; RO5263397, (S)-4-(3-fluoro-2-methylphenyl)-4,5-dihydrooxazol-2-amine; S18616, (S)-spiro[(1-oxa-2-amino-3azacyclopent-2-ene)-4,29-(89-chloro-19,29,39,49-tetrahydronaphthalene)]; SKF-82958, 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SSAO, semicarbazide-sensitive amine oxidase; TA, trace amine receptor; TAAR, trace amine–associated receptor; TMAO, trimethylamine-N-oxide; TRP, tryptamine; TYR, p-tyramine; UGT, uridine diphosphate glucuronosyltransferase;VAP-1, vascular adhesion protein-1; VTA, ventral tegmental area.

550 Gainetdinov et al.

D. Trace Amine–Associated Receptors in Teleost and Other Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602E. Trace Amine–Associated Receptors in Olfaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603

1. Trace Amine–Associated Receptor 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6042. Trace Amine–Associated Receptor 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6043. Trace Amine–Associated Receptor 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6044. Trace Amine–Associated Receptor 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6045. Trace Amine–Associated Receptors 7, 8, and 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6056. Teleost Olfactory Responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605

V. Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606A. Better Understanding of the Physiologic Role(s) of Trace Amine–Associated Receptors

and Their Endogenous Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6061. Trace Amine–Associated Receptor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6062. Other Trace Amine–Associated Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606

B. Development of Selective Trace Amine–Associated Receptor 1 Ligands as Therapeutics . . . 6061. Schizophrenia and Bipolar Disorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6062. Addiction and Compulsive Behaviors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6073. Metabolic Syndromes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608

C. Trace Amine–Associated Receptors in Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608D. Trace Amine–Associated Receptors, Nutrition, the Microbiome, and Health . . . . . . . . . . . . . . . 609

VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610

Abstract——Trace amines are endogenous com-pounds classically regarded as comprising b-phenyl-ethyalmine, p-tyramine, tryptamine, p-octopamine,and some of their metabolites. They are also abundantin common foodstuffs and can be produced and de-graded by the constitutive microbiota. The ability touse trace amines has arisen at least twice duringevolution, with distinct receptor families present ininvertebrates and vertebrates. The term “trace amine”was coined to reflect the low tissue levels in mammals;however, invertebrates have relatively high levels wherethey function like mammalian adrenergic systems, in-volved in “fight-or-flight” responses. Vertebrates expressa family of receptors termed trace amine–associatedreceptors (TAARs). Humans possess six functionalisoforms (TAAR1, TAAR2, TAAR5, TAAR6, TAAR8, andTAAR9), whereas some fish species express over 100.With the exception of TAAR1, TAARs are expressed in

olfactory epithelium neurons, where they detect diverseethological signals including predators, spoiled food,migratory cues, and pheromones. Outside the olfactorysystem, TAAR1 is the most thoroughly studied and hasboth central and peripheral roles. In the brain, TAAR1acts as a rheostat of dopaminergic, glutamatergic, andserotonergic neurotransmission andhas been identifiedas a novel therapeutic target for schizophrenia, depression,and addiction. In the periphery, TAAR1 regulatesnutrient-induced hormone secretion, suggesting itspotential as a novel therapeutic target for diabetesand obesity. TAAR1 may also regulate immuneresponses by regulating leukocyte differentiation andactivation. This article provides a comprehensivereview of the current state of knowledge of the evolution,physiologic functions, pharmacology, molecularmechanisms, and therapeutic potential of trace aminesand their receptors in vertebrates and invertebrates.

I. Introduction

Although vertebrate receptors showing a high selec-tivity for trace amines have been known for approxi-mately 15 years (Borowsky et al., 2001; Bunzow et al.,2001), research and interest in the endogenous com-pounds now known as trace amines dates back almost150 years. The term “trace amine” itself appears to havebeen coined in the early 1970s by Alan Boulton and hiscolleagues (Boulton, 1974) as a way to distinguish agroup of endogenous vertebrate monoamines from theirmore abundant close structural relatives, the catechol-amine and indoleamine neurotransmitters (Fig. 1). Theoriginal intent of the term “trace” was to emphasizethe low endogenous tissue concentrations present(,10 ng/g; 100 nM), at levels that are at least 100-fold

below those of the corresponding neurotransmitters(Berry, 2004). With the close structural similarity tothe monoamine neurotransmitters a central drivingforce behind much of the prereceptor research, the termtrace amine subsequently became synonymous with justa small group of endogenousmonoamines—in particular,b-phenylethylamine (PEA), p-tyramine (TYR), trypt-amine (TRP), and p-octopamine (OCT), the compoundsthat have the most obvious similarity to the wellestablished monoamine neurotransmitters (Fig. 1).

The earliest known reports of the presence of acompound with a chemical composition consistent withone of the trace amines (PEA) is from work in thelaboratory of Marceli Nencki during the late 1870s (seeGrandy, 2007), aimed at better understanding thebacterial processes of putrefaction and fermentation.

Trace Amines and Their Receptors 551

During these studies, PEA was shown to be a product ofbacterial decomposition of both gelatin and egg white,due to the anaerobic decarboxylation of L-phenylala-nine. PEA was subsequently found to be produced aspart of the decomposition processes of various otheranimal-derived proteins, along with the first reports ofits presence in fermented foodstuffs. This ready pro-duction of trace amines by microbiota has often beenoverlooked in more recent years; as described in sub-sequent sections, however, recent increased interest inthe role of host-microbiome interactions and dietarycomponents in health and disease suggests that suchproduction is likely to gather renewed interest.The initial demonstrations of biologic effects of PEA

and TYR are intricately linked to the very origins of thefield of pharmacology. The isolation of both PEA andTYR from biologic sources such as rotting horsemeatand ergot-contaminated grains (the original use of theterm ergotamine was to describe TYR) led to thepioneering studies of George Barger, George S.Walpole,Sir Henry Hallett Dale, and Alfred J. Clark (Barger andWalpole, 1909; Barger and Dale, 1910; Clark, 1911),demonstrating pronounced blood pressure–elevatingeffects of the purified extracts. Although interest inthe compounds continued and their presence wasconfirmed in every species in which they were examined(see Berry, 2004), research into the trace aminesgradually faded away as interest in the more abundantmonoamine species norepinephrine, epinephrine, dopa-mine and serotonin surged. As the new discipline ofpharmacology developed and the chemical basis ofneurotransmission became accepted, the following cri-teria for endogenous compounds to be consideredneurotransmitters were developed: 1) the presence ofthe compound and its biosynthetic enzymes limited tothe sites at which application of the exogenous chemical(at physiologic concentrations) elicits responses; 2)release of the compound occurs on nerve stimulationwith no (or minimal) release in the absence of nervestimulation; 3) exogenous application of physiologicconcentrations mimics the effects of nerve stimulation;and 4) responses to nerve stimulation and exogenouschemical application are affected in the same mannerby pharmacological agents. Unfortunately, at least invertebrate systems, none of the trace amines met most,if any, of these criteria, and the compounds becameincreasingly viewed as little more than metabolicby-products.With the growing use of psychotropic drugs in the

1960s, and amphetamine-based compounds in particu-lar, a resurgence of interest in the trace amines wasseen. Although researchers had struggled to demon-strate responses to PEA or TYR at endogenous tissueconcentrations, both were well established to haveindirect sympathomimetic properties at supraphysio-logic concentrations (Fuxe et al., 1967), effects that wereshared with the new amphetamine-based drugs of

abuse. Furthermore, amphetamine (and its derivatives)has a strong structural similarity to the trace amines,particularly PEA (Fig. 2). As such, PEA and TYR, to alesser extent, became of interest as potential “endoge-nous amphetamines” (Borison et al., 1975; Janssenet al., 1999). This also powered an interest in the traceamines as possible biomarkers and etiologic factors forpsychiatric disorders, and extensive tabulations ofchanges in the levels of endogenous trace amines andtheir metabolites in various body fluids were compiled(Davis, 1989). Although most groups were content torely on the amphetamine-like, indirect sympathomi-metic effects of trace amines as a mechanistic explana-tion for observed effects, a small number of researchersbegan a careful examination of the neuropharmacologyof more physiologically relevant concentrations. Mostnotable among this latter group were those affiliatedwith the Neuropsychiatry Research Unit in Saskatoon,Canada, under the directorship of Alan A. Boulton, anda number of their studies are described in later sections.Although a number of effects were observed, this secondphase of studies in vertebrates stalled due to the lack ofa selective receptor target through which the observedeffects could be mediated. In contrast, TYR and OCTwere established as bona fide invertebrate neurotrans-mitters during the same time period, with selectivereceptors identified (Morton and Evans, 1984; Roederand Gewecke, 1989; Han et al., 1998; Consoulas et al.,1999). Thus, by the early 1990s, trace amine researchwas essentially restricted to invertebrate systems.

The picture changed again in 2001 when a family ofvertebrate G protein–coupled receptors (GPCRs) wasidentified, a subset of which showed high selectivity forPEA, TYR, and OCT (Borowsky et al., 2001; Bunzowet al., 2001). Interestingly, the receptors were found tobe evolutionarily distinct from the invertebrate TYRand OCT receptors (Lindemann et al., 2005), indicatingthat the ability to detect trace amines has arisen at leasttwice during evolution. This resurrected interest in thevertebrate trace amine system. As detailed elsewhere(Berry et al., 2017), however, the new family of receptorshas posed a number of unique challenges that haveslowed progress and dissuaded many from establishing(and funding) dedicated trace amine research programs.A brief history of the discovery of this family ofreceptors, and their subsequent naming as traceamine–associated receptors (TAARs), is provided inIV. Trace Amine–Associated Receptors. Notwithstandingthe difficulties, the last 15 years have seen a number ofadvances that have identified trace amines and theirreceptors as novel targets for the pharmacotherapy ofvarious disorders, as well as being novel sites for environ-mental chemical interactions leading to behavioral ecologyeffects. Althoughanumber of excellent reviews focusing onindividual subareas of trace amine pharmacology, partic-ularly in relation to TAAR1, have been published (Grandy,2007; Sotnikova et al., 2008; Jing and Li, 2015; Lam et al.,


2015; Liberles, 2015; Pei et al., 2016; Berry et al., 2017), acomprehensive review of all aspects of trace aminepharmacology is lacking. This article aims to provide such

a comprehensive overview of the current state of theknowledge of trace amine systems throughout the body,in both vertebrates and invertebrates.

Fig. 1. Relationship of archetypal trace amines to the monoamine neurotransmitters.


Before we begin, however, it is worthwhile to discussthe definition of the term “trace amine.” As describedabove, this term is generally accepted to comprise thegroup of compounds formed when the tyrosine hydrox-ylase or tryptophan hydroxylase step of catecholamineand indoleamine neurotransmitter synthesis is omit-ted. The pharmacological probing of the TAAR family invarious species, however, indicates that this is far toonarrow a definition. A variety of other endogenousamines that function as selective agonists at one ormore TAAR family members are present in body fluidsat low levels (Fig. 3) and these compounds are oftenassociated with metabolic routes that are distinct fromthose of the compounds traditionally called traceamines. Such compounds include the endogenous thy-roid hormone metabolite 3-iodothyronamine (3IT)(Scanlan et al., 2004; Dinter et al., 2015c), the catechol-amine neurotransmitter metabolites 3-methoxytyramine(3-MT) and normetanephrine (Bunzow et al., 2001;Sotnikova et al., 2010), trimethylamine (Ferrero et al.,2012; Wallrabenstein et al., 2013; Li et al., 2015),isoamylamine (Liberles and Buck, 2006; Ferrero et al.,2012), the polyamines putrescine and cadaverine(Hussain et al., 2013), and possibly agmatine, spermine,

and spermidine (Saraiva et al., 2016). In addition, theN-methylated metabolites of both PEA and TYR, N-methylphenylethylamine and N-methyltyramine, arealso TAAR agonists (Lindemann and Hoener, 2005), asis the N-methyl metabolite of TRP N,N-dimethyltryp-tamine (DMT); although in this latter instance, thisshows a strong species dependence (Simmler et al.,2016). With a receptor family bearing the name traceamine now present, we propose that a formalizedworking definition of the trace amine term be adopted.The rather broad substrate tuning that the TAARfamily exhibits (described in detail in subsequentsections) complicates the development of a clear defini-tion. For example, both dopamine and serotonin showpartial agonistic activity at TAAR1 at physiologicallyrelevant concentrations (Lindemann et al., 2005) butwould not be regarded as trace amines per se. Thesituation is further complicated by TAARs only beingpresent in vertebrate systems, whereas invertebrateshave receptors that are selectively activated by TYRand OCT but are distinct from TAARs and are muchmore closely related to vertebrate adrenergic receptors(Roeder, 2005; Lange, 2009). Indeed, as will be dis-cussed below, TYR and OCT are thought to fulfill the

Fig. 2. Structures of synthetic TAAR1 ligands.


role of adrenergic neurotransmission in invertebrates, asituation that is distinctly different from their roles invertebrate species. Correspondingly, invertebrate en-dogenous levels of TYR and OCT are thought to be fargreater than is the case for vertebrates (Roeder, 2016).Furthermore, as will be discussed in subsequent sec-tions, some “endogenous” ligands for TAARs may bereliant on the constitutive microbiota for their pro-duction. We therefore propose that a working definitionof the term trace amine should recognize the evolution-ary separation of identical signaling molecules betweenvertebrates and invertebrates, and it should takeaccount of both the generally low vertebrate tissuelevels as well as a selective interaction with one ormore TAARs. This is an area in which engagementbetween the International Union of Basic and ClinicalPharmacology (IUPHAR) nomenclature committee andthose active in the trace amine field would be advanta-geous. For the purposes of this article, and as a startingpoint for future discussions, we suggest that traceamines be defined as follows: a trace amine is an aminethat is endogenously present in vertebrate tissues and/or bodily fluids at concentrations ,50 ng/g tissue (≲500nM) and selectively binds to one ormore TAARs at theseconcentrations.

II. Vertebrate Trace Amines

A. b-Phenylethylamine, p-Tyramine, andRelated Compounds

1. Synthesis. The archetypal trace amines are syn-thesized after initial decarboxylation of precursoramino acids (Fig. 4). This pathway is directly analogousto the synthesis of the monoamine neurotransmitters,with the trace amines being the end product if thetyrosine hydroxylase (EC 1.14.16.2) or tryptophanhydroxylase (EC 1.14.16.4) steps of neurotransmittersynthesis are omitted. As such, PEA, TYR, and TRP canbe formed directly by the action of aromatic L-aminoacid decarboxylase (AADC; EC 4.1.1.28) on L-phenylal-anine, L-tyrosine, and L-tryptophan, respectively(Boulton and Wu, 1972, 1973; Saavedra, 1974;Snodgrass and Iversen, 1974; Silkaitis and Mosnaim,1976; Dyck et al., 1983). Both m- and o-isoforms oftyramine have also been identified but have rarely beenstudied, and they are present in even smaller quantitiesthan the p-isoforms (Boulton, 1976; Davis, 1989). OCTand p-synephrine can subsequently be formed by thesequential action of dopamine-b-hydroxylase (EC1.14.17.1) (Boulton and Wu, 1972, 1973) and phenyl-ethanolamine-N-methyl transferase (PNMT; EC2.1.1.28). The trace amines can also undergo N-meth-ylation by action of the enzymes PNMT or indolethyl-amine N-methyltransferase (EC 2.1.1.49) to generateadditional TAAR ligands, N-methylphenylethylamine,N-methyltyramine, N-methyltryptamine, and, at leastin some species, DMT (Fig. 4).

The similarity to monoamine neurotransmitter syn-thesis has often led to the synthesis of trace amines beingreported as neuronal. However, it should be borne inmind that AADC expression is not restricted to neuronalcells. AADC is present in a number of other cell types,including glia (Li et al., 1992b; Juorio et al., 1993), bloodvessels (Li et al., 2014), and cells of the gastrointestinaltract (Lauweryns and Van Ranst, 1988; Vieira-Coelhoand Soares-da-Silva, 1993), kidney (Christenson et al.,1970; Lancaster and Sourkes, 1972; Aperia et al., 1990;Hayashi et al., 1990), liver (Bouchard and Roberge, 1979;Ando-Yamamoto et al., 1987;Dominici et al., 1987), lungs(Lauweryns and Van Ranst, 1988; Linnoila et al., 1993),pancreas (Lindström and Sehlin, 1983; Furuzawa et al.,1994; Rorsman et al., 1995), and stomach (Lichtenbergeret al., 1982). In such cells, it can reasonably be expectedthat AADC will convert any precursor amino acidspresent into the corresponding trace amine(s). Thephysiologic function of AADC in non-neuronal tissue isgenerally poorly understood but it does provide amechanism for the local production of ligands for TAARsthat are localized to non-neuronal tissue, and investiga-tion of possible colocalization of AADC with TAARs is anarea for future studies. Furthermore, a distinct group ofneurons that contain AADC, but not tyrosine hydroxy-lase or serotonin, are present in the mammalian centralnervous system (Jaeger et al., 1983, 1984; Fetissov et al.,2009; Kitahama et al., 2009). These D-neurons offer apotential trace aminergic neuronal system.

Although AADC is widely accepted as the vertebratesynthetic enzyme for PEA, TYR, and TRP, the precursoramino acids are in fact rather poor substrates for AADC.Indeed, the Km values for decarboxylation of L-phenylal-anine, L-tyrosine, and L-tryptophan approach the limitsof solubility of each in aqueous media (Christensonet al., 1970; Juorio and Yu, 1985a). Although this ismarkedly, and selectively, improved both in vitro andin vivo by the presence of organic solvents (Lovenberget al., 1962; Juorio and Yu, 1985a,b), this does raisequestions about how PEA, TYR, and TRP are beingsynthesized in vivo. Substrate-selective regulation ofAADC has been reported (Bender and Coulson, 1972;Sims and Bloom, 1973; Sims et al., 1973; Rahman et al.,1981; Siow and Dakshinamurti, 1985), along with anumber of splice variants of the enzyme (O’Malley etal., 1995; Rorsman et al., 1995; Chang et al., 1996;Vassilacopoulou et al., 2004). Whether one or more ofthese exhibits enhanced selectivity for the production ofPEA, TYR, and/or TRP requires systematic investiga-tion. Furthermore, L-phenylalanine, L-tyrosine, and L-tryptophan are all also substrates for additional aminoacid decarboxylase enzymes (Table 1), although the roleof these putative additional sources of PEA, TYR, andTRP synthesis has not yet been investigated.

a. Regulation of Aromatic L-Amino Acid Decarboxylase.As described above, AADC is found in both neuronal andnon-neuronal cells and alternative splicing of exons


Fig. 3. Structures of new members of the trace amine family. Based on demonstrated high-affinity binding to individual TAARs and low endogenousconcentrations, the compounds shown are proposed as new members for inclusion in the trace amine family.


Fig. 4. Endogenous synthetic and metabolic routes for trace amines. DBH, dopamine-b-hydroxylase; INMT, indolethylamine N-methyltransferase;PAH, phenylalanine hydroxylase; TH, tyrosine hydroxylase.


1 and 2 within the 59-untranslated region has beenestablished as allowing for distinct control of neuronaland non-neuronal expression (Albert et al., 1992; Ichinoseet al., 1992; Hahn et al., 1993; Sumi-Ichinose et al., 1995).A variety of transcription factor binding sites have beenidentified within both the neuronal (Chireux et al., 1994;Aguanno et al., 1995) and non-neuronal (Aguanno et al.,1996) promoter regions through which tissue-selectiveexpression could occur. A variant on this alternativesplicing, inwhich the non-neuronal variantwas spliced tothe neuronal acceptor site, has also been suggested in Gcells of the rat stomach antrum (Djali et al., 1998), whichmay indicate cell type–selective plasticity in the control ofAADC expression. Recently, a number of cis-actingpolymorphisms of AADC have been identified withputative clinical relevance (Li and Meltzer, 2014;Eisenberg et al., 2016), and disease-associated AADCcoding variants are also known (Graziano et al., 2015;Kojima et al., 2016; Montioli et al., 2016).The functional significance of alternative splicing

within the coding region of AADC is poorly defined. Asplice variant lacking exon 3 has been reported to bewidely expressed in both neuronal and non-neuronaltissue (O’Malley et al., 1995; Chang et al., 1996), withthis shorter variant suggested to be devoid of bothL-DOPA and L-5-hydroxytryptophan decarboxylatingactivities (O’Malley et al., 1995). An even shorter variant,lacking exons 11–15, has also been reported to beexpressed in non-neuronal tissues (Vassilacopoulouet al., 2004), although enzyme activity of this variantwas not examined. Additional coding splice variantswere also reported to be present in pancreatic b cells(Rorsman et al., 1995), although again the functionalityof these putative alternative forms does not appear tohave been further investigated. Whether activity towardsubstrates other than L-DOPA and L-5-hydroxytrypto-phan is lost, or even enhanced, is unknown, but theapparent widespread expression of an ostensibly non-functional variant seems unlikely, and a role of one ormore splice variants in selective trace amine synthesiscould provide an answer to this paradox.Although it is not a rate-limiting step in the synthesis

of catecholamine and indoleamine neurotransmit-ters under physiologic conditions, AADC activity isregulated in a biphasic manner. Both early changes,consistent with alterations in phosphorylation status,and delayed, longer-lasting changes in enzyme expres-sion have been reported (Buckland et al., 1992,1996, 1997; Hadjiconstantinou et al., 1993; Berryet al., 1996). Multiple consensus phosphorylation sitesfor protein kinase A (PKA) (Young et al., 1993;Duchemin et al., 2000), protein kinase C (PKC)(Young et al., 1994; Zhu et al., 1994), protein kinase G(Duchemin et al., 2010), calmodulin-dependent kinaseII (Hadjiconstantinou et al., 2010), and proline-directedkinase (Hadjiconstantinou et al., 2010) are present,and both site-directed mutagenesis of individual

phosphorylation sites (Hadjiconstantinou et al., 2010)or selective activation/inhibition of individual proteinkinases (Young et al., 1993, 1994; Zhu et al., 1994;Duchemin et al., 2000, 2010) alters AADC activity.Direct evidence for phosphorylation of AADC by PKA(Duchemin et al., 2000) and protein kinase G(Duchemin et al., 2010) has been provided, althoughPKC does not appear to directly increase phosphoryla-tion despite the presence of consensus recognition sites(Duchemin et al., 2000).

In the retina, AADCactivity is increased in response tolight (Hadjiconstantinou et al., 1988) or selective antag-onism of a2-adrenergic receptors (Rossetti et al., 1989) orD1-like dopamine receptors (D1Rs) (Rossetti et al., 1990).Consistentwith this, light stresswas recently reported toincrease retinal PEA levels (de la Barca et al., 2017). Incontrast, D1R agonists decrease both basal and light-induced AADC activity (Rossetti et al., 1990). Similarresponses to dopamine receptor ligands have also beenobserved in various rodent brain regions (Zhu et al.,1992, 1993, 1994; Hadjiconstantinou et al., 1993; Choet al., 1997, 1999; Neff et al., 2006). Regulation of AADCby serotonergic receptors (Neff et al., 2006) and N-methyl-D-aspartate (NMDA) glutamatergic receptors(Hadjiconstantinou et al., 1995; Fisher et al., 1998) hasalso been reported. There is also some evidence for AADCregulation associated with systemic lupus erythemato-sus (Bengtsson et al., 2016), Parkinson’ disease (Gjeddeet al., 1993), and schizophrenia (Reith et al., 1994). Inexperimental animals, regulation of AADC after spinalcord injury has also been reported (Li et al., 2014;Wienecke et al., 2014; Azam et al., 2015).

In each of the above cases, the reported changes inAADC activity are normally rather modest (approxi-mately 30%) and insufficient to change endogenousdopamine levels (Berry et al., 1994; Cho et al., 1999).Such treatments have, however, been shown to changeboth PEA and TYR levels (Juorio, 1979; Juorio et al.,1991; Berry, 2004) and can generally be summarized asfollows: treatments that increase monoamine neuro-transmitter receptor activation decrease PEA/TYR syn-thesis, whereas treatments that decrease receptoractivation increase PEA/TYR synthesis. In this respect,it is important to note that reports of changes in AADCactivity have almost exclusively used only L-DOPA as asubstrate. Whether greater changes in the activity ofAADC toward other substrates (particularly L-phenyl-alanine, L-tyrosine, and/or L-tryptophan) occurs is un-known, although as described above there is evidencethat substrate-dependent regulation of AADC is possi-ble (Rahman et al., 1981; Juorio and Yu, 1985a,b; Siowand Dakshinamurti, 1985). Kinetic studies have report-ed alterations in AADC Vmax in response to pharmaco-logical agents (Zhu et al., 1992; Young et al., 1994;Duchemin et al., 2010) and both Vmax and Km inresponse to site-directed mutagenesis of consensusphosphorylation sites (Hadjiconstantinou et al., 2010).


TABLE 1Decarboxylated products of common amino acids, their known TAAR ligand status, and putative physiologic effects

Putative enzymes were obtained from the Brenda-enzymes.org database. Only those known or predicted to be present in eukaryotes are shown. See the text for citations,with the exception of entries with footnotes.

Amino Acid Decarboxylated Amine Known PutativeEnzyme TAAR Ligand Physiologic Functions

Alanine Ethylamine Valinedecarboxylase(EC 4.1.1.14)

Arginine Agmatine Argininedecarboxylase(EC 4.1.1.19)

TAAR13d, TAAR13e, andTAAR14d agonist

Putative neurotransmitter.Regulates variousneurotransmitter receptors,regulates ion channels, inhibitsnitric oxide synthase, downregulatesmatrix metalloproteases, preventsadvanced glycation end productformation, and activates NADPHoxidase

Ornithinedecarboxylase(EC 4.1.1.17)

Asparagine 3-aminopropanamide NoAspartic acid 3-amino propanoic acid (b-alanine) Aspartate

1-decarboxylase(EC 4.1.1.11)

Precursor for carnosine. Possibleprecursor for pantothenic acid(vitamin B5). Agonist at glycinereceptorsArginine

decarboxylase(EC 4.1.1.19)

Glutamatedecarboxylase(EC 4.1.1.15)

Tyrosinedecarboxylase(EC 4.1.1.25)

Cysteine Cysteamine Valinedecarboxylase(EC 4.1.1.14)

Precursor of homotaurine.Treatment of cystinosis

Glutamic acid GABA Glutamatedecarboxylase(EC 4.1.1.15)

No Neurotransmitter

Argininedecarboxylase(EC 4.1.1.19)


Glutamine 4-aminobutanamide (GABamide) No GABA uptake inhibitor. Putativeepidermal growth factor receptorantagonist

Glycine Methylamine No Implicated in cardiovascularcomplications of diabetes

Histidine Histamine Histidinedecarboxylase(EC 4.1.1.22)

TAAR13a and TAAR13d agonist Neurotransmitter. Paracrinehormone

AADC (EC 4.1.1.28)Phenylalanine


Isoleucine 2-methyl-1-butylamine Methioninedecarboxylase(EC 4.1.1.57)

Putative ligand at TAAR1 andTAAR3a

Food additive

Valinedecarboxylase(EC 4.1.1.14)

Leucine Isoamylamine (3-methyl-1-butylamine; isopentylamine)

Methioninedecarboxylase(EC 4.1.1.57)

TAAR3, TAAR12h, and TAAR16fagonist

See text for details


Lysine Cadaverine Lysinedecarboxylase(EC 4.1.1.18)

TAAR13c and 13d agonist. Agonistat unidentified rat TAARs



Ornithinedecarboxylase(EC 4.1.1.17)

(continued)


b. Other Sources of b-Phenylethylamine, p-Tyramine,and Related Compounds.i. Microbiota-Derived Trace Amines. There is a

growing recognition of the role of the commensal micro-biota in health and disease, including neurologic andpsychiatric disorders (Dinan and Cryan, 2015; Funget al., 2017), cancer and its chemotherapy (Alexanderet al., 2017; Roy and Trinchieri, 2017), metabolic disor-ders (Sonnenburg and Bäckhed, 2016; Brunkwall andOrho-Melander, 2017), and immune disorders (Hondaand Littman, 2016; Thaiss et al., 2016; Fung et al., 2017;Luo et al., 2017). Prokaryotes contain a large array of

decarboxylase enzymes, many of which include L-aminoacids in their substrate profile (Zheng et al., 2011;Nelsonet al., 2015). Indeed, the production of PEA, TYR, andTRP by commensal prokaryotes has been established(Marcobal et al., 2006; Irsfeld et al., 2013;Williams et al.,2014; Yang et al., 2015a), and bacterial production ofthese compounds was the original basis of Nencki’sstudies on putrefaction and fermentation (see Grandy,2007). With TAARs established to be present throughoutthe body, it is expected that the role of trace amines andtheir receptors inmediating host-microbiota interactionswill become a growing area of interest.

TABLE 1—Continued

Amino Acid Decarboxylated Amine Known PutativeEnzyme TAAR Ligand Physiologic Functions

Methionine 3-methylthiopropylamine Methioninedecarboxylase(EC 4.1.1.57)

Interacts with enzymes inpolyamine metabolism


Ornithine Putrescine Ornithinedecarboxylase(EC 4.1.1.17)

TAAR13d agonist Spermine/spermidine precursor

Lysinedecarboxylase(EC 4.1.1.18)


Phenylalanine PEA AADC(EC 4.1.1.28)

TAAR1, TAAR4, and TAAR12hagonist


Phenylalaninedecarboxylase(EC 4.1.1.53)




Proline Pyrrolidine Argininedecarboxylase(EC 4.1.1.19)

Fish attractantb

Serine Ethanolamine Argininedecarboxylase(EC 4.1.1.19)

Membrane phospholipid headgroup. Sclerosing agent

Threonine 1-aminopropan-2-ol(isopropanolamine)

No Present in salivac

Tryptophan TRP AADC (EC 4.1.1.28) TAAR1 and TAAR10b agonist See text for detailsPhenylalanine


Tyrosine TYR AADC (EC 4.1.1.28) TAAR1 and TAAR4 agonist See text for detailsTyrosine


Phenylalaninedecarboxylase(EC 4.1.1.53)

Valine Isobutylamine(2-methyl-1-propylamine)


Found in various foodstuffs.Activates olfactory/vomeronasalorgan. Putative puberty-accelerating pheromoneMethionine


aLindemann and Hoener (2008).bHarada (1985).cSugimoto et al. (2013).


ii. Food-Derived Trace Amines. PEA, TYR, andTRP, as well as other endogenous trace amines, havelong been recognized as being abundant in commonlyingested foods (Coutts et al., 1986). Although thispresence is often viewed from the perspective ofbacterial-induced food spoilage (Naila et al., 2010),foods in which anaerobic fermentation is part of theirproduction are enriched in trace amines (Toro-Funeset al., 2015; Gardini et al., 2016; Pessione and Cirrin-cione, 2016). Aged cheeses, fermented meats, red wine,soy products, and chocolate are well established asbeing enriched in one or more of PEA, TYR, and TRP(Coutts et al., 1986).Another rich source of food-derived trace amines is

seafood, from molluscs and crustaceans to fish, wherehigh concentrations of agmatine, cadaverine, OCT,PEA, putrescine, spermidine, spermine, TRP, andTYR are found (An et al., 2015; Biji et al., 2016). Infact, the name octopamine originates from octopus fromwhere it was first isolated (Erspamer, 1952). Althoughunder normal conditions the concentrations of food-derived trace amines in the body are unlikely to reachsufficient levels to activate TAARs other than thoseexpressed in the stomach (e.g., TAAR1 and TAAR9;Ohta et al., 2017) and certainly do not exert indirectsympathomimetic actions, there are conditions in whichthis may occur. Individuals who adversely react to traceamine–rich nutrients such as seafood, cheese, and wineare known (Finberg andGillman, 2011; Biji et al., 2016),although the molecular basis of the sensitivities are notestablished in all cases. Genetic deficiency in mono-amine oxidases (MAO)-A/B, which are key trace amine–metabolizing enzymes, are known to significantly in-crease urinary trace amine concentrations and pressorresponses to TYR (Lenders et al., 1996). Furthermore,the well known “cheese effect” in patients treated withMAO-A inhibitors is explained by pronounced accumu-lation of TYR, sufficient to indirectly elevate bloodnorepinephrine concentrations to the point of inducinghypertensive crisis, severe migraine, and even death(Finberg and Gillman, 2011). With the growing accep-tance of TAARs by the wider scientific community, therole of nutrient-derived TAAR ligands in health anddisease is likely to become a growing area of nutritionalbiochemistry and food science interest and to overlapwith studies of the molecular basis of host-microbiotainteractions. Indeed, the possibility that food-derivedtrace amines can activate host TAARs has now begun tobe recognized (Ohta et al., 2017).A variety of herbal extracts and brews that are (or

have been) used in various cultures as medicines, forreligious ceremonies, or for their psychotropic effectsare also reported to either contain high levels of traceamines or to alter their concentrations within the body,with such changes implicated in the subsequent phys-iologic responses. For example, N-methyltyramine hasbeen identified in Ginkgo biloba preparations (Könczöl

et al., 2016). Various trace amine compounds have alsobeen suggested to be present in Chinese herbal medi-cines (Zhang et al., 2016) and in a variety of religiousherbal brews (Leonti and Casu, 2014), including aya-huasca where DMT is thought to be one of the majoractive constituents (Riba et al., 2003).

2. Degradation. Catabolism occurs primarily viaMAO (EC 1.4.3.4), with PEA being a highly selectivesubstrate for MAO-B (Yang and Neff, 1973). Littleselectivity is shown toward either MAO-A or MAO-B byother compounds (Philips and Boulton, 1979; Durdenand Philips, 1980). All compounds have also beenreported to be substrates for the enzyme variously knownas semicarbazide-sensitive amine oxidase (SSAO; EC1.4.3.21, formerly EC 1.4.3.6), vascular adhesionprotein-1 (VAP-1), or amine oxidase, copper containing3 (AOC3) (Elliott et al., 1989). Catabolism via cyto-chromeP450 isozymes has also been reported (Niwa et al.,2011), but this is thought to be a very minor route underphysiologic conditions (Yu et al., 2003). Interconversionof the trace amines readily occurs (Fig. 4), includ-ing via methylation of the primary amine (Lindemannand Hoener, 2005) generating both N-methyl and N,N-dimethyl derivatives, presumably via indolethylamineN-methyltransferase for TRP and via PNMT for PEA,TYR, and OCT (Fig. 4). Some of these metabolites act onone or more TAARs in their own right, although this isspecies dependent with some such as DMT (Simmleret al., 2016). PEA can also be acted on directly bydopamine-b-hydroxylase generating phenylethanol-amine (Saavedra and Axelrod, 1973; Danielson et al.,1977), which also has been reported to be a full, albeitweak, agonist at human TAAR1 (Wainscott et al., 2007).Interconversion with the catecholamine and indole-amine neurotransmitters (e.g., TYR conversion to dopa-mine) was originally not thought to occur (Dyck et al.,1983), although this may be an outcome of the limitedcytochrome P450–mediated metabolism at least in somespecies (Hiroi, 1998; Bromek, 2010).

3. Storage and Passage across Membranes. Unliketheir neurotransmitter structural analogs, PEA, TYR,and TRP do not appear to be stored. All three readilydiffuse across synthetic lipid bilayers in the absence oftransporters, with diffusion half-lives of 15 seconds or less(Berry et al., 2013). Consistent with this, PEA passageacross the blood-brain barrier occurs by a mechanismconsistent with passive diffusion (Mosnaim et al., 2013),whereas TYR passage across intestinal epithelial cells alsoappears to be diffusionmediated (Tchercansky et al., 1994).Release of PEA from the whole brain (Henry et al., 1988),striatal slices (Dyck, 1989), or synaptosomes (Berry et al.,2013) is not increased by K+-induced depolarization.Similar results were also obtained with TYR (Henryet al., 1988; Berry et al., 2013), with only a small increasein release in response to K+-induced depolarization ob-served in brain slices (Dyck, 1989). Likewise, no effectof depolarization on TRP release from striatal slices was


present (Dyck, 1989). The lack of effect of K+-induceddepolarization strongly suggests that these compoundsare neither stored in synaptic vesicles nor released byexocytosis, presumably indicating “release” by simplediffusion across the membrane, consistent with previousreports of a lack of reserpine-sensitive vesicular storage(Juorio et al., 1988). In contrast, veratridine-induced de-polarization does increase the release of TYR from striatalslices (Dyck, 1989), although whether similar effects occurwith PEA and/or TRP was not studied. Whether thisrepresents a subset of synaptic vesicles that TYR (andpossibly other trace amines) can access requires furtherinvestigation. Consistent with a lack of vesicular storage,however, the half-life of the endogenous pool of PEA andTRPhas beenestimated to be less than30 seconds (Durdenand Philips, 1980). OCT has also been reported to have amarkedly higher turnover rate than either norepinephrineor dopamine (Molinoff and Axelrod, 1972).As recently discussed, the above-noted lack of effect of

depolarization on PEA, TRP, and, to a less clear extent,TYR release from neuronal preparations is difficult toexplain thermodynamically (Berry et al., 2016). Even if“release” is by diffusion, membrane depolarizationshould increase this due to the subsequent increase inthe electrochemical gradient. The lack of such an effectmay indicate the presence of a membrane transporterregulating the synaptic levels of PEA, TYR, and TRP.Consistent with such a conclusion, PEA transport intorabbit erythrocytes was previously reported to involvean unknown transporter (Blakeley and Nicol, 1978), aswas transport across Caco-2 cells (Fischer et al., 2010).Through the use of pharmacological probes, organiccation transporter 2 (OCT2; Slc22A2) was recentlyidentified as a likely transporter of TYR in rat brainsynaptosomes, exhibiting a Kt approximately equal to100 nM (Berry et al., 2016), in reasonable agreementwith estimates of endogenous TYR concentrations(Berry, 2004). OCT2 is widely regarded as a polyspecifictransporter (Couroussé and Gautron, 2015) that inter-acts with a wide variety of therapeutics (Kido et al.,2011; Hacker et al., 2015), not dissimilar to the broadsubstrate tuning reported for TAAR1 (see IV.B.1.c.Trace Amine–Associated Receptor 1 Ligands). Should arole for OCT2 in regulating trace amine membranepassage at physiologically relevant concentrations bevalidated, this would implicate the trace amine/TAARsystem as a new molecular target to be considered withrespect to adverse drug interactions for therapeuticsthat interact with the OCT2 transporter.In combination, the above studies suggest that the

archetypal trace amines readily cross cell membranesby diffusion but that their levels are further regulatedby one ormore transporters. The current understandingof the synthesis, metabolism, and transport of PEA andTYR in neurons is summarized in Fig. 5.4. Cellular Effects. Effects that are clearly demon-

strated to be due to interaction with one or more TAARs

are described in detail in subsequent sections dedicatedto the individual TAAR subtypes. A number of cellularresponses were described prior to the identification ofTAARs, however, and these are summarized in thefollowing paragraphs. Whether these effects are due toan interaction with TAARs is often unknown, althoughin many cases responses are strikingly similar tocurrent thoughts on the physiologic function of TAARs.

a. Indirect Sympathomimetic Responses. The TAAR1ligandsPEAandTYR inparticular have long been knownto possess indirect sympathomimetic, amphetamine-likeeffects (Fuxe et al., 1967; Baker et al., 1976; Raiteri et al.,1977; Parker and Cubeddu, 1988). Such effects, whichinvolve inhibition of reuptake processes and the displace-ment of monoamine neurotransmitters from their stores,generally require PEA/TYR concentrations of at least10 mM (Baker et al., 1976; Raiteri et al., 1977), which isthought to be at least 100-fold in excess of physiologicconcentrations (see Berry, 2004). Amphetamine-like be-havioral stereotypy is also induced by high-dose (.25mg/kg) PEA/TYR administration (Dourish, 1982; Stoff et al.,1984). Although they are less studied, similar indirectsympathomimetic responses to OCT have also beenreported (Raiteri et al., 1977; Parker and Cubeddu,1988). As previously described, such results led tosuggestions that trace amines, and PEA in particular,serve the role of an endogenous amphetamine (Borisonet al., 1975; Janssen et al., 1999). The initial pharmaco-logical characterization of TAARs provided a furthertwist to this hypothesis, with various amphetamine-derived compounds (Fig. 2) showing agonist activity atTAAR1 (Bunzow et al., 2001). Focusing exclusively on theso-called amphetaminergic-like effects does a consider-able disservice to trace aminergic physiology, however,and subsequent pharmacological profiling of TAAR1(described in detail in IV.B. Trace Amine–AssociatedReceptor 1) indicates that this is an oversimplification.Amphetamine-type drugs are rather promiscuous intheir actions, and it is far more accurate to describeamphetamines as including trace amine–like effects aspart of their spectrum of activity than to describe traceamines as being endogenous amphetamines. Consistentwith this, differences in the aversive stimulus proper-ties elicited by PEA and amphetamine were describedas far back as the 1980s, such that unlike amphetamine(and other drugs of abuse), PEA does not induce aconditioned taste aversion response (Greenshaw, 1984;Greenshaw et al., 2002).

b. b-Phenylethylamine. Either iontophoretic orintra-arterial administration of low doses of PEA hasbeen reported to cause changes in neuronal activity.Although the doses employed were insufficient to alterbaseline neuronal activity, responses to norepineph-rine (Paterson, 1988, 1993; Paterson and Boulton, 1988),electrical stimulation of the locus coeruleus (Paterson,1993), dopamine (Jones and Boulton, 1980b), or syn-thetic dopamine agonists (Paterson et al., 1991) are all


modulated by PEA. Such effects do not require thepresence of endogenous norepinephrine or dopamine(Paterson, 1988, 1993), nor are they mimicked by theexogenous application of dopamine (Jones and Boul-ton, 1980b). Furthermore, selective elevation of endog-enous PEA levels through inhibition of MAO-B givesthe same responses as exogenous PEA administration(Paterson et al., 1991; Berry et al., 1994) and effects arereversed by the subsequent selective reduction of PEAlevels through partial inhibition of AADC. Thesemodulatory responses show selectivity, with neuronalresponses to serotonin (Paterson and Boulton, 1988),acetylcholine (Paterson and Boulton, 1988; Paterson,

1993), glutamate (Paterson and Boulton, 1988; Berryet al., 1994), and GABA (Paterson, 1988; Patersonand Boulton, 1988; Berry et al., 1994) remainingunaffected.

Similarly, dopamine-mediated effects on membranefluidity are potentiated by the presence of low concen-trations of PEA (Harris et al., 1988), although PEAalone shows no effect on membrane fluidity (Harriset al., 1988; Knight and Harris, 1993). These responseswere suggested to represent a PEA-mediated change inboth microtubule protein conformation and polymeri-zation (Knight andHarris, 1993), although this does notappear to have been further studied.

Fig. 5. Current understanding of the interplay between the TAAR1 and dopamine systems. b-PEA and TYR are synthesized in dopaminergicterminals. Unlike dopamine, they are not stored within synaptic vesicles and readily diffuse across plasma membranes. Reuptake into presynapticterminals appears to be aided by OCT2. TAAR1 has a predominantly intracellular location, in which both pre- and postsynaptic effects are possible.Whether a transporter contributes to postsynaptic uptake of trace amines is as yet unknown. TAAR1 may translocate to the cell surface afterheterodimerization with D2R, an effect that promotes preferential D2R signaling through the Gi signal transduction cascade rather than b-arrestin2 pathway. TAAR1 interactions with D1R do not occur. A complex system of crosstalk between the dopamine and TAAR1 system is present.Presynaptic activation of D2R, via the Gi signal transduction cascade, results in inhibition of PEA/TYR synthesis. Extracellular metabolism ofdopamine by COMT generates 3-MT, an agonist at TAAR1 that like extracellular PEA and TYR can activate the TAAR1/D2R heteromer complex bothat pre- and postsynaptic membranes. Dotted blue arrows indicate molecular movement, solid green arrows indicate receptor-mediated stimulation, andsolid red lines indicate receptor-mediated inhibition. DOPAC, 3,4-dihydroxyphenylacetic acid; L-Phe, L-phenylalanine; L-Tyr, L-tyrosine; PAA,phenylacetic acid/4-hydroxyphenylacetic acid; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter 2.


Chronic elevation of PEA has been reported to inducea downregulation of D1R but not D2-like dopaminereceptors (D2Rs), as well as both b1- and b2-adrenergicreceptors (Paetsch and Greenshaw, 1993; Paetsch et al.,1993). The mechanism by which such regulation occursis unknown. Acutely, PEA only interacts with D1R onceconcentrations approach millimolar levels (Berry,2011). Since brain levels of PEA were only reported tobe increased 10-fold (Paetsch et al., 1993), a directinteraction with D1R is somewhat unlikely. Further-more, the effect on b-adrenergic receptors was not seenin all brain regions, arguing against a response second-ary to direct interaction of PEAwith b-adrenoceptors. Itis possible that these effects of chronic PEA elevationsare secondary to increased synaptic monoamine con-centrations after indirect sympathomimetic-like effectsof PEA increasing transporter-mediated outflow(Sotnikova et al., 2004). Indeed, Sotnikova et al. ob-served that rapid, short-acting effects of PEA onextracellular dopamine levels were absent, and corre-sponding locomotor activation and stereotypies werenot observed, if the dopamine transporter (DAT) wasknocked out. Rather, spontaneous activity manifestedby the DAT knockout (KO) mice during exposure to anovel environment was significantly inhibited for anextended period of time by PEA (Sotnikova et al., 2004).This indicates that, similar to previously observedresponses with amphetamine (Gainetdinov et al.,1999), PEA can exert a paradoxical calming action ondopamine-mediated hyperactivity via mechanisms me-diated by other monoamines, possibly also involvinginteraction with TAAR1 (Sotnikova et al., 2009).Aside from a regulation of neurotransmitter recep-

tors, PEA has been reported to stimulate gastrinsecretion from G cells of the antrum of rat stomach(Dial et al., 1991), an effect shared with other endoge-nous TAAR1 ligands TYR and TRP. This is particularlyinteresting, given subsequent studies showing TAAR1-mediated effects on nutrient-induced hormone secretionfrom the gastrointestinal tract (see IV.B.3. Effects in thePeriphery). Production of PEA (as well as TYR and TRP)has also been reported to enhance the ability ofStaphylococcus (Luqman et al., 2018) and Enterococcus(Fernández de Palencia et al., 2011) species to adhere toepithelial cells, promoting subsequent internalizationand enterocyte cytokine secretion. Finally, PEA hasbeen proposed to be a tiger pheromone (Brahmacharyand Dutta, 1979).c. p-Tyramine. TYR has in general been reported to

have similar effects to PEA. Potentiation of neuronalresponses to both dopamine and norepinephrine hasbeen seen after low-level iontophoretic administrationof eitherm-tyramine (Jones and Boulton, 1980b) or TYR(Jones and Boulton, 1980b; Jones, 1983). As seen withPEA, selectivity of responses is present with no effect ofTYR on responses to GABA (Jones and Boulton, 1980b),glutamate, or serotonin (Jones, 1982b, 1983). As before,

the low-level supplemental iontophoretic application ofdopamine (Jones and Boulton, 1980b) was unable torecapitulate the responses observed to TYR.

Both PEA and TYR have been reported to be releasedfrom activated platelets (D’Andrea et al., 2003), anobservation that takes on added interest in light ofTAAR-mediated chemotactic responses of leukocytes(see IV.B.3.b. Immunomodulatory Effects). Interest-ingly, species-dependent effects of TYR on leukocyteand erythrocyte chemotaxis and aggregation were de-scribed as far back as the early 1920s (Wolf, 1921, 1923).In this regard, it is interesting to note that TYRproduction by Enterococcus species found in cheese isenhanced by conditions that simulate passage throughthe gastrointestinal tract (Fernández de Palencia et al.,2011). As described above, such production promotesadherence of microbes to enterocytes (Fernández dePalencia et al., 2011; Luqman et al., 2018) and maymodulate subsequent cytokine signaling by the entero-cytes (Fernández de Palencia et al., 2011). TYR, alongwith TRP, has also recently been reported to regulateindividual isoforms of neuronal acid-sensing ion chan-nels (ASICs) (Barygin et al., 2017), an effect seen as avoltage-dependent inhibition of opening and a voltage-independent potentiation of closing of the ASIC1aisoform.

d. Tryptamine. Compared with PEA and TYR,electrophysiological responses to TRP are somewhatmore complicated. Although TRP potentiates inhibitoryserotonin responses, excitatory serotonin responses areeither unaffected or converted to inhibitory responses inthe presence of low-level TRP application (Jones andBoulton, 1980a; Jones, 1982b). Similar effects of TRPare seen on cortical neuron responses to electricalstimulation of the raphe nuclei. Here, the excitatorycomponent of biphasic responses was markedly reducedby the presence of TRP, whereas the inhibitory compo-nent remained unaffected (Jones, 1982b). Corticalneuron excitation in response to raphe stimulation isnormally followed by a long-lasting decrease in basalfiring, and this secondary decrease was potentiated bythe presence of TRP (Jones, 1982b). Such effects of TRPon electrical stimulation were not mimicked by the low-level iontophoretic application of serotonin itself (Jones,1982b). In contrast, TRP application is without effect onacetylcholine-induced responses (Jones and Boulton,1980a). Similarly, serotonin-induced contraction ofsmooth muscle is decreased in the presence of TRP atconcentrations below those that mimic the effect ofserotonin (Frenken and Kaumann, 1988), an effectsuggested to be due to an increase in the proportion of5-hydroxytryptamine 2a (5-HT2a) receptors that are inthe agonist high-affinity state (Frenken and Kaumann,1988). Such an effect suggests a TRP-mediated regula-tion of receptor–G protein coupling mechanisms. Asindicated above, TRP has also been reported to regulateASIC1a ion channels (Barygin et al., 2017) and to


promote the adherence of staphylococci species togastrointestinal epithelial cells (Luqman et al., 2018).e. Octopamine. Although OCT was first synthesized

in 1910 (Barger and Dale, 1910), it was not identified intissue extracts until 1952 (Erspamer, 1952), when itwas found to be a component of the salivary gland ofOctopus vulgaris. OCT was suggested to be a cotrans-mitter of norepinephrine over 40 years ago (Axelrod andSaavedra, 1977) and to possibly modify noradrenergicactivity through an interaction with sites distinct fromnorepinephrine and dopamine receptors (Hicks andMcLennan, 1978). Iontophoretic application of OCT inamounts insufficient to affect basal neuronal activitypotentiates both inhibitory and excitatory responses tonorepinephrine (Jones, 1982a). Unlike TYR and PEA,however, no effect was observed on responses to dopa-mine or serotonin (Jones, 1982a). As observed withother trace amines, low-dose norepinephrine applica-tion was unable to elicit a similar modification ofresponses (Jones, 1982a).5. b-Phenylethylamine, p-Tyramine, and Tryptamine

in Human Disorders. Historically, trace amines havebeen implicated in a diverse array of human disorders(Berry, 2007). Although there is a large body of litera-ture describing altered levels of PEA, TYR, TRP, andtheir derivatives in various pathologies, and extensivetabulations of such have previously been published(Davis, 1989), many of these studies were performedin small cohorts and findingswere not always confirmedin subsequent studies. Probably, the most convincingfindings are observed in diseases involving alteredbiogenic amine synthesis or metabolism such as phe-nylketonuria, in which PEA levels are increased due toelevated L-phenylalanine concentrations caused byphenylalanine hydroxylase (EC 1.14.16.1) deficiency(Reynolds et al., 1978). In patients with MAO-A/Bdeficiencies, a dramatic increase of PEA, TYR, andTRP is also found (Lenders et al., 1996). The contribu-tion of increased trace amine levels to the complex set ofneurologic, cognitive, and psychiatric manifestations ofthese disorders remains to be determined and is an areafor re-examination in light of the discovery of TAARs.There are several lines of evidence supporting a role

of trace amines in schizophrenia. The finding that thelevels of “an endogenous amphetamine” (PEA) is in-creased in the urine of some patientswith schizophrenialed to a general hypothesis that PEA plays a critical rolein schizophrenia pathogenesis (Janssen et al., 1999).Increased levels of DMT, a potent hallucinogen that isproduced endogenously in humans, have also beenreported (Jacob and Presti, 2005). It should be notedin this respect, however, that althoughDMT is a TAAR1agonist in common laboratory rodent species, this doesnot appear to be the case in humans (Simmler et al.,2016). This is perhaps not surprising, given that thecurrent evidence indicates that TAAR1 is primarilytuned toward primary amines (Ferrero et al., 2012).

Whether one of the tertiary amine-tuned TAAR5–TAAR9 receptors is capable of binding DMT is an areafor future studies.

Multiple studies have shown an association of schizo-phrenia with key enzymes involved in the synthesis anddegradation of trace amines, particularly AADC andcatechol-O-methyl transferase (COMT; EC 2.1.1.6)(Børglum et al., 2001; Shifman et al., 2006). Finally, adecrease in the number of brain D-neurons, groups ofneurons that contain AADC but do not express tyrosinehydroxylase or tryptophan hydroxylase and thus repre-sent potential trace aminergic neurons, has been re-ported in postmortem schizophrenia brains (Ikemotoet al., 2003).

Similarly, links between trace amines and drugabuse/dependence have long been noted, again basedlargely on the “PEA as an endogenous amphetamine”hypothesis. The discovery of TAARs, and in particularTAAR1, has considerably strengthened these links, andthe role of TAAR1 in various drug abuse paradigms isdescribed in detail in IV.B.2.b.v. Addiction and Com-pulsive Behaviors. Prior to the establishment of the roleof TAAR1, PEA was proposed to be involved in theneural mechanisms of reward and reinforcement(Greenshaw, 1984; Shannon and Thompson, 1984), aswell as increases in brain PEA levels reported inresponse to D9-tetrahydrocannabinol (Sabelli et al.,1974). Alterations in the activity of the trace aminesynthetic enzyme AADC have also been reported inalcohol-dependent patients (Tiihonen et al., 1998),along with single nucleotide polymorphisms of AADC(Ma et al., 2005) and the chromosomal region containingthe AADC gene (Wang et al., 2005) being linked tonicotine dependence. The dopaminergic system, and inparticular D2Rs, is well established to be important inreward circuitry and addiction. The A1 allele of theD2R, which has been linked with alcohol (Blum et al.,1990; Noble, 2003), nicotine (Spitz et al., 1998; Bierutet al., 2000), opioid (Lawford et al., 2000), psychostimu-lant (Persico et al., 1996; Noble, 2003), and polysub-stance (Comings et al., 1991, 1994; O’Hara et al., 1993)abuse as well as pathologic gambling (Comings et al.,1996), has also been reported to be associated with anincrease in AADC activity (Laakso et al., 2005).

A cohesive theory has also been put forth for a role ofPEA in affective disorders, whereby deficits in PEA areassociated with depression, whereas elevated PEA isassociated with mania symptoms (Karoum et al., 1982;Davis, 1989; Sabelli and Javaid, 1995). Decreased levelsof PEA are observed in patients with depression (Wolfand Mosnaim, 1983), whereas clinically effective MAOinhibitors are known to elevate trace amine levels(Sabelli and Mosnaim, 1974; Sabelli et al., 1996), andelevated PEA levels have been suggested to underlie theantidepressant effects of exercise (Szabo et al., 2001).An association of unipolar and bipolar depression withCOMT and AADC genes has also been reported


(Børglum et al., 2001; Shifman et al., 2006). Changes inPEA levels have also been found in patients withattention deficit hyperactivity disorder, for which am-phetamine (a TAAR1 agonist) remains one of the mosteffective treatments (Baker et al., 1991).A role for trace amines in Parkinson disease has also

been suggested, with both COMT and MAO inhibitorsbeing used in clinical practice (Espinoza et al., 2012).Intriguingly, levels of the extracellular dopamine me-tabolite and TAAR1 agonist 3-MT are significantlyincreased after chronic L-DOPA treatment (Rajputet al., 2004) and may contribute to L-DOPA–induceddyskinesia (Sotnikova et al., 2010). 3-MT levels are alsomarkedly increased in patients with pheochromocy-toma and are the most sensitive biomarker forthis catecholamine-related disorder (Lenders andEisenhofer, 2017). A role for trace amines in migraineand cluster headaches (D’Andrea et al., 2004) and food-related headache attacks (Smith et al., 1970) has beenspeculated, although certainly with respect to food-induced migraines there is sparse supportive evidence(Berry, 2007).Recent unbiased metabolomics studies indicated that

elevated fecal PEA levels are present in patients withCrohn disease (Jacobs et al., 2016; Santoru et al., 2017),an effect that was suggested to contribute to the abilityto discriminate these patients from healthy controls(Santoru et al., 2017). Similarly elevated TYR levelswere reported in ulcerative colitis fecal samples(Santoru et al., 2017). Such studies are generallyconsistent with previous links between inflammatorybowel disorder pathology and altered L-phenylalanineand L-tyrosine metabolism (Burczynski et al., 2006;Jansson et al., 2009). Potential roles of trace amines inanxiety disorders, eating disorders, epilepsy, and Reyesyndrome have been also been sporadically suggested(see Berry, 2007). Elevated urinary PEA levels areassociated with general stress responses in both hu-mans and rodents (Paulos and Tessel, 1982; Snoddyet al., 1985; Grimsby et al., 1997) and this couldcertainly be a confound in interpreting elevations ofPEA in disease conditions.

B. Isoamylamine

Although isoamylamine was identified as a biologi-cally active component of both decayingmeat and tissuesamples at the same time as PEA, TYR, and TRP(Barger and Dale, 1910), it has been far less studied insubsequent years, presumably due to less structuralsimilarity to subsequently established neurotransmit-ters (c.f. Figs. 1 and 3). Isoamylamine can be producedby the decarboxylation of leucine (Table 1) and is acomponent of commonly consumed fermented foodstuffs(Cunha et al., 2011; Bach et al., 2012; Coton et al., 2012).Although a variety of leucine decarboxylase activitieshave been described in prokaryotes (Coton et al., 2012),including some commensal gut microbiota (Haughton

and King, 1961), there is no known leucine decarbox-ylase enzyme in vertebrates (Table 1). Isoamylamineproduction could occur, however, via methionine decar-boxylase (EC 4.1.1.57) or valine decarboxylase (EC4.1.1.14), both of which include L-leucine in their sub-strate profile (Table 1).

The initial descriptions of MAO activity identifiedisoamylamine as a substrate (Blaschko et al., 1937;Pugh and Quastel, 1937) and further characterizationhas suggested that isoamylamine exhibits selectivitytoward MAO-B, similar to that of PEA (Peers et al.,1980). In addition, isoamylamine is also a substrate forSSAO/VAP-1/AOC3 (Elliott et al., 1989). Isoamylamineis an agonist at TAAR3 (Liberles and Buck, 2006), andTAAR3 and its responses are described in section IV.E.2Trace Amine–Associated Receptor 3. Isoamylamine isexcreted in male mouse urine (Nishimura et al., 1989)and may induce puberty in female mice (Nishimuraet al., 1989), suggesting a possible role as a pheromone,which would most likely require that a nonmicrobialsynthetic pathway is present. Others, however, havefailed to replicate the puberty-accelerating effect ofisoamylamine (Price and Vandenbergh, 1992).

Previously, isoamylamine was reported to share agastrin secretion-stimulating effect with PEA and TYR(Dial et al., 1991) and it has recently been reported toregulate gastrointestinal motility in a tetrodotoxin-sensitive manner (Sánchez et al., 2017), possibly in-dicating a neuron-mediated mechanism. In this regard,it is interesting to note that isoamylamine was sug-gested to be an endogenous neurally active compound atleast 40 years ago (Tashiro et al., 1974). More recently,cardiac levels of isoamylamine were reported to nega-tively correlate with septal wall thickness and to beincreased in patients suffering cardiac arrest (Meanaet al., 2016). Isoamylamine was also suggested to bebeneficial in an animal model of endotoxemia (Yenet al., 2016). The mechanism and importance of theseputative effects requires further study.

C. Trimethylamine

Although repeatedly identified as a component ofvarious human bodily fluids between themid-1850s andearly 1910s (see Mitchell and Smith, 2016), a physio-logic relevance of trimethylamine was not suggesteduntil the 1930s when an increase in its excretion wasreported to occur during menstruation (Ashley-Montagu, 1938), an effect that has subsequently beenconfirmed (Shimizu et al., 2007). Although trimethyl-amine levels have been associated with various diseaseconditions (Chhibber-Goel et al., 2016), are sexuallydimorphic (Gavaghan McKee et al., 2006), and a pro-posed male pheromone in mice (males having a geneticdeficiency in trimethylamine metabolism) (Li et al.,2013), there is no known endogenous synthetic path-way. Rather, trimethylamine production is thought tooccur as a by-product of prokaryotic degradation of


dietary choline, betaine, phosphatidylcholine, and L-carnitine (Zhang et al., 1999; Craciun and Balskus,2012; Zhu et al., 2014; Kalnins et al., 2015; Fennemaet al., 2016). Such well documented production by thecommensal microbiota, along with an established hostreceptor (TAAR5, see subsequent sections), makestrimethylamine an attractive candidate molecule forinvestigating the growing appreciation of the role of themicrobiota in health and disease. The apparent lack ofnonmicrobial production of trimethylamine raises ques-tions as to whether it is the true endogenous ligand forTAAR5 and offers an interesting parallel to the arche-typal trace amines TYR, PEA, and TRP, which werepreviously often consigned to the role of metabolicby-products (Berry, 2004). With a receptor that isselectively activated by trimethylamine now identified,a re-examination of whether trimethylamine produc-tion is solely dependent on the commensal microbiota iswarranted.Trimethylamine has been confirmed to be a high-

affinity agonist at TAAR5 from multiple species(Liberles and Buck, 2006; Ferrero et al., 2012; Liet al., 2013; Wallrabenstein et al., 2013; Zhang et al.,2013). Effects clearly demonstrated to be TAAR5 medi-ated are described in sections IV.C.4 Trace Amine-Associated Receptor 5 and IV.E.4 Trace Amine–Associated Receptor 5, although these are rather fewin number at present. The main interest in trimethyl-amine thus far has been with respect to its degradation.This primarily occurs via the enzyme flavin monoox-ygenase 3 (FMO3; EC 1.14.13.8), which is prevalent inhepatic tissue, including in humans (Fennema et al.,2016). This generates trimethylamine-N-oxide (TMAO),a compound that has been increasingly implicated asplaying a role in both cardiovascular and metabolicdisorders (Fennema et al., 2016; Zhang and Davies,2016). Interestingly, the Km for trimethylamine metab-olism by FMO3 is rather high (28 mM; Lang et al., 1998)and is approximately 100-fold in excess of the EC50

values of trimethylamine at TAAR5 from variousspecies (Berry et al., 2017). This raises the possibilityof previously unsuspected physiologic effects of nano-molar trimethylamine levels and that TAAR5 may beunder tonic activation. As described in later sections, atleast two receptor targets for trimethylamine are likelyto be present, since TAAR5 KO prevents low-dose butnot high-dose trimethylamine effects (Li et al., 2013).An evolutionary deletion of FMO3 in male mice

underlies their markedly increased urinary trimethyl-amine levels (Li et al., 2013). In humans, a geneticdeficiency in FMO3 leads to the condition of trimethy-laminuria (Humbert et al., 1970), in which largequantities of trimethylamine are excreted in sweat,urine, and exhaled air (Fennema et al., 2016). Kidneydamage (Bain et al., 2006; Chhibber-Goel et al., 2016)and gut dysbiosis (Fennema et al., 2016) can also lead totrimethylaminuria, as can invasion by pathologic

microflora associated with bacterial vaginosis or infec-tions of the oral cavity (Fennema et al., 2016; Zhang andDavies, 2016). The increased trimethylamine urinaryexcretion associated with menstruation (Mitchell andSmith, 2010) may reflect regulation of FMO3 by femalesex hormones (Coecke et al., 1998; Shimizu et al., 2007).Consistent with this, FMO3 activity is altered duringpregnancy (Hukkanen et al., 2005). Interestingly, tri-methylamine urinary levels were found to be very stablein a recent study in children, showing the least intra-individual variability during repeated sampling,whereas those of its metabolite TMAO were highlyvariable (Maitre et al., 2017), suggesting that trimethyl-amine levels aremore tightly controlled than those of itsprimary metabolite. This could indicate a physiologicrelevance of the parent compound. Trimethylamine hasalso been detected in the feces of at least one avianspecies (the black-bellied whistling duck) (Robackeret al., 2000), where it may play a role in eitherconspecific (Mueller et al., 2008) or heterospecific(Robacker et al., 2000) communication.

Trimethylaminuria is generally classified as a ratherbenign condition, except for the socially stigmatizingstrong fish odor associated with afflicted individuals,and this is the basis of the more common name for thedisorder—fish malodor syndrome. The pronouncedmalodor associated with trimethylaminuria does, how-ever, result in a severe loss of quality of life for affectedpersons (Fennema et al., 2016).

TMAO can also be produced by bacterial action ontrimethylamine through various oxygenase enzymes orby the nonenzymatic action of reactive oxygen species(Brown and Hazen, 2017). In addition, prokaryotes canconvert TMAO back to trimethylamine (Zhang et al.,1999). Alternatively, dehydrogenase enzymes from atleast some bacteria can convert trimethylamine intodimethylamine (Kim et al., 2001; Shi et al., 2005) withsubsequent conversions to methylamine and ammonia(Kim et al., 2001), although whether this ability isshared by commensal bacteria is unknown.

At high concentrations trimethylamine has beenreported to inhibit macromolecule synthesis, leadingto teratogenesis in mouse embryos due to inhibition ofreceptor-mediated precursor uptake (Guest and Varma,1992; Guest et al., 1994). This effect has been suggestedto be more pronounced in male offspring due totrimethylamine-mediated inhibition of testosteronesynthesis (Guest and Varma, 1993). FMO3 expressionis regulated by both glucagon and insulin, with insulindecreasing FMO3 levels in a sexually dimorphic man-ner (Miao et al., 2015), and dysregulated trimethyl-amine metabolism has been implicated in the pathologyof both obesity and diabetes (Dumas et al., 2006; Toyeet al., 2007), including in humans (Elliott et al., 2015).Trimethylamine levels have also been correlated to theonset of symptoms in the interleukin-10 KO mousemodel of inflammatory bowel disease (Murdoch et al.,


2008), although the basis of this relationship is unclear.As previously indicated, there is a growing body of workthat implicates the trimethylamine metabolite TMAOin cardiovascular disease (Koeth et al., 2013;Miao et al.,2015). Although few studies have examined for a role ofthe parent compound, one study has linked trimethyl-amine to atherosclerosis in patients with human im-munodeficiency virus infection (Srinivasa et al., 2015).Elevated trimethylamine levels have also been sug-gested in patients with a malignant peripheral nervesheath tumor (Fayad et al., 2014) and glomeruloscle-rosis (Hao et al., 2013), although the relevance of theseobservations to disease processes has not been deter-mined. High-altitude pulmonary edema has also beenreported to be associated with a decrease in plasmatrimethylamine levels (Luo et al., 2012), although therelevance of this beyond serving as a potential bio-marker is unknown.Trimethylaminuria has sporadically been reported to

be associated with epilepsy (McConnell et al., 1997;Pellicciari et al., 2011), and a lowering of the epilepto-genic threshold has been reported in experimentalanimals in response to trimethylamine (Gajda et al.,2003; Leniger et al., 2004; Sayyah et al., 2007; Nassiri-Asl et al., 2008), possibly as a consequence of theopening of gap junctions (Gajda et al., 2003; Bocianet al., 2011; Chang et al., 2013). That epileptic activitycould be controlled by a diet restricted in trimethyl-amine precursors (McConnell et al., 1997; Pellicciariet al., 2011) raises the possibility of a direct causal linkbetween elevated trimethylamine and a lowered epi-leptogenic threshold. Possibly related to this, trimethyl-amine has also been reported to increase the amplitudeof theta wave oscillations (Bocian et al., 2011). A varietyof psychiatric and behavioral disorders have beensuggested to coexist with trimethylaminuria, althoughhow many of these are either simply coincidental(McConnell et al., 1997) or secondary to the socialstigma associated with the pronounced halitosis andbody odor (Todd, 1979; Mitchell and Smith, 2001) isunclear. Some behavioral abnormalities have, however,been reported to be ameliorated with diets low intrimethylamine precursors (McConnell et al., 1997),indicating a possible link between trimethylamine me-tabolism and central neuronal function.Lower maternal urinary trimethylamine levels have

been reported to be associated with fetal growth re-tardation (Maitre et al., 2014), whereas maternalplasma trimethylamine levels may be a marker fordiscrimination of trisomy 18 from trisomy 21 (Bahado-Singh et al., 2013). Interestingly, FMO3 expression isnot turned on until after birth in humans and continuesto increase until adulthood (Fennema et al., 2016),suggesting a developmental component to thetrimethylamine/TMAO axis. Trimethylamine is presentin breast milk and its concentration in amniotic fluidincreases markedly during gestation (Lichtenberger

et al., 1991). Together, these studies raise the possibilityof an involvement of trimethylamine in programmingfetal and neonatal metabolism.

D. O-Methyl and N-Methyl Derivatives

The O-methyl metabolites of the catecholamineneurotransmitters (3-MT, 4-methoxytyramine,metanephrine, and normetanephrine) were identifiedas agonists at TAAR1 during the initial characteriza-tion of the receptor family (Bunzow et al., 2001), withthe effect of 3-MT being subsequently confirmed byothers (Sotnikova et al., 2010). Although very fewstudies have examined the physiologic effects of thesecompounds, a complex spectrum of locomotor behaviorsand the corresponding striatal intracellular signalingevents are induced by intracerebroventricular 3-MTadministration, and these are partially ameliorated inTAAR1-KO animals (Sotnikova et al., 2010). 3-MTlevels are increased in brains with Parkinson diseaseduring the development of dyskinesias after chronic L-DOPA administration (Rajput et al., 2004). As such,TAAR1 may be a target for improving the on-offphenomenon associated with long-term L-DOPA ther-apy. Such effects certainly point to at least 3-MT havingphysiologic roles beyond being a mere marker ofextracellular dopamine levels, and it is expected thatthe identification of TAAR1 as a receptor target for theO-methyl neurotransmitter metabolites will lead tofurther studies in these areas.

N-methyl metabolites of trace amines have also beenshown to be ligands for TAARs (Lindemann andHoener, 2005); however, with at least some of thesecompounds, this is species dependent (Simmler et al.,2016; Berry et al., 2017). N-methyltyramine is widelydistributed in plant species (Smith, 1977; Stohs andHartman, 2015), where its presence has been knownsince 1950 (Kirkwood andMarion, 1950). In addition,N-methylphenylethylamine has been identified in humanurine (Reynolds and Gray, 1978). Like the parentcompounds, at high concentrations, N-methyl traceamines have long been known to cause pronouncedpressor effects (Hjort, 1934), most likely due to eitherdirect or indirect sympathomimetic effects (Stohs andHartman, 2015). In addition, and at much lowerconcentrations, N-methyltyramine has been reportedto stimulate gastric (Yokoo et al., 1999) and pancreatic(Tsutsumi et al., 2010) secretions. DMT is perhaps themost widely studied of the N-methyl derivatives due toits potent hallucinogenic properties, which likely have apolypharmacological basis (Carbonaro and Gatch,2016). In addition to neural effects, DMT also appearsto possess immunomodulatory properties (Szabo, 2015).

E. 3-Iodothyronamine

Like the archetypal trace amines, 3IT is an endoge-nous compound found at nanomolar levels throughoutthe body in both rodents and humans (Scanlan et al.,


2004; Zucchi et al., 2014). Although it is often reportedas a derivative/metabolite of thyroid hormones (Hoefiget al., 2016), whether 3IT is indeed derived from either3,39,5-triiodothyronine or thyroxine after decarboxyl-ation and deiodination is a matter of some conjecture(Ackermans et al., 2010; Hoefig et al., 2011, 2015;Hackenmueller et al., 2012). The available evidenceindicates that 3IT is not formed by the action of AADC(Hackenmueller et al., 2012; Hoefig et al., 2012) butmaybe formed by ornithine decarboxylase (EC 4.1.1.17)(Hoefig et al., 2015). Once formed, 3IT is a substratefor MAOs, SSAO/VAP-1/AOC3, and deiodinases, result-ing in the production of 3-iodothyroacetic acid andthyronamine (Piehl et al., 2008; Wood et al., 2009;Saba et al., 2010; Hackenmueller and Scanlan, 2012;Laurino et al., 2015). In addition,N-acetyl,O-sulfonate,and glucuronide conjugates of 3IT have been reported tobe widely distributed throughout the body (Pietschet al., 2007; Hackenmueller and Scanlan, 2012).In 2004, 3IT was identified as a high-affinity agonist

at TAAR1 (Scanlan et al., 2004). This has subsequentlybeen verified by numerous laboratories (Mühlhauset al., 2014; Cöster et al., 2015; Chiellini et al., 2017),with 3IT also shown to act as an agonist at TAAR2(Babusyte et al., 2013; Cichero and Tonelli, 2017) and asan inverse agonist at TAAR5 (Dinter et al., 2015c). 3ITis promiscuous, however, and also interacts with highaffinity at a2-adrenoceptors (Regard et al., 2007; Dinteret al., 2015b), b-adrenergic receptors (Meyer andHesch,1983; Kleinau et al., 2011; Dinter et al., 2015a),muscarinic acetylcholine receptors (Laurino et al.,2016), transient receptor potential cation channel sub-family M member 8 ion channels (Khajavi et al., 2015;Lucius et al., 2016), various monoamine and organicanion transporters (Snead et al., 2007; Panas et al.,2010), and molecular target(s) within mitochondria,including the F1-F0ATP synthase (Cumero et al.,2012) and possibly complex III (Venditti et al., 2011).3IT also tightly binds to the plasma protein apolipopro-tein B-100 (Roy et al., 2012).The physiologic effects of 3IT are often, although not

exclusively, opposite to those of the thyroid hormones. Avariety of physiologic effects of 3IT have been reportedand a number of reviews of such effects have previouslybeen published to which the reader is referred forfurther details (Ianculescu and Scanlan, 2010; Zucchiet al., 2010, 2014; Piehl et al., 2011; Hoefig et al., 2016).Here, we will provide a brief overview of such effectswith the following caveat: the promiscuous nature of3IT makes it highly unlikely that all, or even most, ofthe physiologic effects described aremediated via one ormore TAARs. Where there is unequivocal evidence ofTAAR-mediated 3IT responses, these are discussed inthe subsequent sections focused on those individualreceptor subtypes. Furthermore, it was recently sug-gested that at least some of the effects previouslyascribed to 3IT may in fact be a function of one or more

of its metabolites (Hoefig et al., 2016; Laurino andRaimondi, 2017), adding a further level of complexity tothe elucidation of the physiologic relevance and phar-macological effects of 3IT.

1. Cardiovascular Effects. Negative inotropic andchronotropic effects are observed both in vivo and inisolated heart preparations after 3IT administration(Scanlan et al., 2004; Chiellini et al., 2007), possibly dueto an interference with release of Ca2+ from intracellu-lar stores (Ghelardoni et al., 2009). At higher concen-trations, a decrease in aortic flow and cardiac output isalso observed (Frascarelli et al., 2011).

2. Metabolic Effects. Effects on energy metabolismhave been seen after both intraperitoneal and intra-cerebroventricular administration of 3IT. Effects in-clude hyperglycemia (Regard et al., 2007; Klieveriket al., 2009; Manni et al., 2012), reduced insulin andincreased glucagon secretion (Regard et al., 2007;Manni et al., 2012), and ketonuria associated with aloss of body fat due to a switch from glucose to lipidmetabolism (Braulke et al., 2008). Changes in geneexpression consistent with increased lipolysis have alsobeen reported to occur in response to 3IT (Mariotti et al.,2014). Some of these responses were suggested to besecondary to altered neuropeptide Y secretion (Dhilloet al., 2009). Interestingly, TAAR1 activation hasseparately been reported to regulate secretion of theclosely related peptide YY (PYY) (Raab et al., 2015).Both increases (Dhillo et al., 2009) and decreases(Manni et al., 2012) in food intake have been observedin response to 3IT, effects that are dependent on thedose administered. Effects on in vivo energymetabolismand food consumption appear to have both central andperipheral components (Klieverik et al., 2009; Manniet al., 2012) and are almost certainly reflective of thepromiscuity of 3IT’s molecular targets (Regard et al.,2007).

3. Thermoregulation. Administration of 3IT inducesa rapid, long-lasting hypothermia, with no compensa-tory induction of shivering or piloerection (Scanlanet al., 2004; Doyle et al., 2007; Braulke et al., 2008).These effects are maintained in TAAR1-KO animals(Panas et al., 2010) and are proposed to be due to adecrease in basal metabolic rate (Braulke et al., 2008).Indeed, 3IT has been reported to induce a hibernation-like torpidity in lizards (Ha et al., 2017) and mice (Juet al., 2011). On the basis of the hypothermic effect, itwas hypothesized that 3IT may reduce infarct size afterischemic injury, an effect that was subsequently seen inthe middle cerebral artery occlusion model of stroke(Doyle et al., 2007). That effects were indeed due tohypothermia was suggested by the lack of a neuro-protective effect of 3IT in primary neuronal culturemodels and by a loss of in vivo protection when bodytemperature was maintained by use of a heating pad(Doyle et al., 2007). In contrast, in an isolated heartpreparation, protection against ischemia was still seen


(Frascarelli et al., 2011), an effect that occurred atconcentrations below those that induced changes incardiac output or aortic flow. This cardioprotectiveeffect appeared to be due to an activation of PKC andsubsequent opening of KATP channels.4. Other Effects. A modulation of pain pathways,

seen as an antinociceptive effect, has been reportedafter 3IT administration (Manni et al., 2013), an effectsuggested to be mediated after metabolism to3-iodothyroacetic acid, and subsequent modulation ofhistaminergic signaling (Laurino et al., 2015). Manniet al. (2013) have also reported procognitive effects of3IT. Regulation of sleep, and in particular a decrease innonrapid eye movement (NREM) sleep, has also beenreported after 3IT dosing (James et al., 2013). Whetherthis relates to the NREM-suppressing, wakefulness-promoting effects of TAAR1 activation (Black et al.,2017; Schwartz et al., 2017) is an open question.

F. Polyamines

Like the archetypal trace amines, polyamines arefound in all species (Miller-Fleming et al., 2015). Theyare traditionally viewed as consisting of cadaverine,putrescine, spermidine, and spermine. Polyamine me-tabolism is complex, not least because of their ability tointerconvert, and this has been detailed fully in numer-ous review articles (Miller-Fleming et al., 2015; Pegg,2016). Putrescine is primarily derived from L-ornithineby the action of ornithine decarboxylase (EC 4.1.1.17;Table 1). Putrescine itself can be converted to spermi-dine by spermidine synthase (EC 2.5.1.16), which inturn can be converted to spermine by spermine syn-thase (EC 2.5.1.22). Both spermidine synthase andspermine synthase are dependent on the action of S-adenosylmethionine decarboxylase (EC 4.1.1.50) togenerate decarboxylated S-adenosylmethionine as adonor of aminopropyl groups. Back conversion of sper-mine to spermidine, and spermidine to putrescine canoccur after N1-acetylation by spermidine/spermine N1-acetyltransferase, and subsequent oxidation by poly-amine oxidase (EC 1.5.3.11). A distinct spermine/spermidine N8-acetylation pathway is also present,which may generate substrates of MAO (Youdimet al., 1991). Cadaverine is produced by direct decar-boxylation of lysine through the action of lysine decar-boxylase (EC 4.1.1.18; Table 1).Although polyamines are usually considered distinct

from monoaminergic signaling systems, a putativediamine binding site was reported to be present in bothhuman TAAR6 and TAAR8 (Li et al., 2015). Further-more, at least in fish, olfactory detection of cadaverinewas shown to be dependent on TAAR13c (Hussain et al.,2013), with other teleost-specific TAAR isoforms alsorecognizing diamines (Table 2). Although these TAARisoforms are not found in terrestrial vertebrates, pan-TAAR-KO prevents the innate avoidance behaviorshown by mice in response to the presence of both

putrescine and cadaverine (Dewan et al., 2013). Whichmouse TAAR isoform(s) is responsible, however, re-mains unknown. Furthermore, whether polyaminesother than putrescine and cadaverine also act as ligandsat tetrapod TAARs is an area for systematic study, but itis perhaps quite likely that spermine and/or spermidinewill also interact with one or more TAARs, given theirclose structural homology to putrescine and cadaverineand similar ecological context to other TAAR ligandssuch as trimethylamine and isoamylamine.

The physiology of cadaverine and putrescine remainslargely a mystery, beyond their association with decay-ing carcasses due to bacterial degradation of proteina-ceous material and innate repulsive properties inmost species. The classic polyamines as a whole havebeen implicated in a variety of mammalian cellularprocesses, most notably in the regulation of cellgrowth and proliferation, cellular stress responses,ion channel function, and as allosteric regulators ofglutamatergic NMDA and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors(Miller-Fleming et al., 2015; Pegg, 2016). One otherpolyamine perhaps deserves mention in these regards.Agmatine is formed by decarboxylation of arginine(Table 1), primarily via arginine decarboxylase (EC4.1.1.19), and can itself be subsequently converted intoputrescine after elimination of urea by the enzymeagmatinase (EC 3.5.3.11). A variety of physiologic andpathologic functions have been ascribed to agmatine(Table 1) (Piletz et al., 2013), many of which bear astriking overlap with the reported effects of TAARs and/or trace amines. For example, agmatine has been shownto interact either directly or indirectly with the functionof a variety of neurotransmitter receptors, includingimidazoline receptors (Li et al., 1994; Raasch et al.,2001), NMDA receptors (Yang and Reis, 1999), andserotonin 5-HT2a receptors (Taksande et al., 2009).Agmatine also modulates the functions of KATP chan-nels (Shepherd et al., 1996) and ASICs (Li et al., 2010)and interacts with a variety of transporter proteins,including OCT2 (Gründemann et al., 2003), OCT3(Gründemann et al., 2003), andmitochondrial transportsystems (Grillo et al., 2007). Like the known TAARligands PEA, TYR, and isoamylamine, regulation ofgastrin secretion by agmatine has also been reported(Dial et al., 1991). This is far from an exhaustive list;agmatine has been reported to have a wide variety ofmolecular targets and physiologic effects (Molderingsand Haenisch, 2012; Piletz et al., 2013; Laube andBernstein, 2017), and binding to one ormore TAARmayjust be one of many mechanisms by which agmatine canexert its effects.

Although the molecular basis of its effects remainslargely unknown, agmatine dysregulation has beenimplicated in affective disorders, cognition, drugabuse/addiction, and metabolic disorders (Piletz et al.,2013). Although agmatine is known not to interact with


TAAR1 (Hu et al., 2009), it does interact with multiplezebrafish TAARs, including TAAR13c (Table 2)(Hussain et al., 2013). Putative interaction at mamma-lian TAAR subtypes is an area for future study,particularly in light of the presence of a diamine bindingpocket in TAAR6 and TAAR8 that is conserved withthat present in TAAR13c (Li et al., 2015).

G. Putative Other Trace Amines

The majority of TAARs remain orphan receptors withno known cognate ligand. Indeed, as described in sub-sequent sections, this is problematic with respect tofollowing current IUPHAR conventions for naming thereceptor family. From the preceding sections, however,it is readily apparent that a large number of TAARligands are the direct products of amino acid decarbox-ylation (Table 1), and this is perhaps a good place tostart the search for endogenous ligands of currentlyorphaned TAARs. With this in mind, Table 1 provides,for reference, the decarboxylated products of commonamino acids and, where known, their physiologic effectsand TAAR binding activity.

III. Invertebrate Trace Amines

TYR and OCT are now well established signalingmolecules in invertebrates (Downer et al., 1993;Kutsukake et al., 2000; Anton et al., 2006; Farooqui,2007; Kononenko et al., 2009). Unlike in vertebratespecies, however, they appear to serve the role of bonafide neurotransmitters in invertebrates, functioning asinvertebrate versions of vertebrate adrenergic systems(Roeder, 2005; Lange, 2009; Homberg et al., 2013).Indeed, in invertebrate nervous systems, TYR andOCT are present at high concentrations, raising thequestion as to whether they should continue to bereferred to as “trace” amines in these species (Roeder,2016). Consistent with the divergence of physiologicroles of the ligands, invertebrates possess dedicatedTYR and OCT receptors that are evolutionarily distinctfrom TAARs (see subsequent sections for further de-tails). As such, current evidence indicates that theability to use TYR and OCT has arisen at least twice

during evolution. This also necessitates that caution beexercised, because it makes a number of common modelsystems (particularly Caenorhabditis elegans andDrosophila) not suitable for translational research withtrace amines due to the independence of functionsand divergence of receptors. The evolutionary dis-tance between invertebrate OCT/TYR receptors andmammalian TAARs has made the invertebrate recep-tors an attractive target for the development of selec-tively acting insecticides, and some progress has beenmade in this area in recent years (Roeder et al., 1995,2003). Although it is far less studied, isoamylamine hasalso been suggested to be an endogenous neuronallyactive compound in invertebrates (Tashiro et al., 1974),whereas trimethylamine-induced release of intracellu-lar calcium stores has been reported in gastropodneurons (Willoughby et al., 2001).

A. Synthesis and Degradation

Although invertebrate synthesis and degradation ofTYR and OCT occurs by analogous pathways to those ofvertebrates, distinct enzymes are present (Blenau andBaumann, 2001; Roeder, 2005; Verlinden et al., 2010a).Dedicated tyrosine decarboxylase enzymes are found invarious invertebrate species (Livingstone and Tempel,1983; Ishida and Ozaki, 2012; McCoole et al., 2012;Christie et al., 2014) and, at least inDrosophila, distinctneuronal and non-neuronal isozymes exist (Cole et al.,2005). Although an AADC-like, DOPA decarboxylaseenzyme may also be present, this does not appear to acton L-tyrosine (Han et al., 2010). In addition to beingactive in its own right, TYR can be further acted upon bytyramine-b-hydroxylase enzymes to generate OCT(Monastirioti et al., 1996; Alkema et al., 2005;Nishimura et al., 2008; Châtel et al., 2013). Again,multiple isozymic forms are present in some species(Châtel et al., 2013). Formation of TYR from dopaminevia a dopamine dehydroxylase pathway is also possiblein some species (Walker and Kerkut, 1978; Roeder,2005).

Whereas vertebrate degradation of TYR and OCToccurs primarily by MAOs, the primary invertebratedegradative route is thought to be viaN-acetylation andN-methylation (Dewhurst et al., 1972; Roeder, 2005;Verlinden et al., 2010a), with sulfate, b-alanyl, glycosyl,and ɣ-glutamyl conjugations also possible (Maxwellet al., 1980; Wright, 1987; Sloley, 2004; Roeder, 2005).

B. Storage and Release

OCT release has been demonstrated to occur frominsect neuronal preparations in response to both elec-trical- and K+-induced depolarization (Morton andEvans, 1984; Orchard and Lange, 1987; Verlindenet al., 2010a), with typical neurotransmitter-like exo-cytotic release from synaptic vesicles (Consoulas et al.,1999). Released TYR and OCT are primarily removedfrom the synaptic cleft by dedicated transporter systems

TABLE 2Identified ligands at teleost TAARs

TAAR Ligand

TAAR10a SerotoninTAAR10b TRPTAAR12h PEA, isoamylamineTAAR12i 3-MTTAAR13a HistamineTAAR13c CadaverineTAAR13d Putrescine, histamine, cadaverine, agmatineTAAR13e AgmatineTAAR14d AgmatineTAAR16c N-methylpiperidine, N-methylpyrrolidineTAAR16e N,N-dimethylcyclohexylamineTAAR16f Isoamylamine


(Roeder and Gewecke, 1989; McClung and Hirsh, 1998;Gallant et al., 2003; Donly and Caveney, 2005), withsome evidence for species specificity in the transporterspresent (Roeder, 2005).

C. Octopamine Receptors

The first invertebrate OCT receptor to be identifiedwas found in the mushroom bodies of the D. mela-nogaster brain andwas namedOAMB (Han et al., 1998).More recently, it has been suggested that this receptorbe renamed OctaR, reflective of its homology to verte-brate a-adrenergic receptors (Evans and Maqueira,2005; Bayliss et al., 2013). The OAMB receptor iscoupled to Ca2+ accumulation (Zeng et al., 1996; Hanet al., 1998; Grohmann et al., 2003; Balfanz et al., 2005;Evans and Maqueira, 2005). Orthologs of OAMB havesubsequently been identified in Apis (Blenau andBaumann, 2001; Grohmann et al., 2003; Balfanz et al.,2014), Anopheles (Kastner et al., 2014), Schistocerca(Verlinden et al., 2010b), and Locusta (Hiripi et al.,1994; Ma et al., 2015), among other invertebratespecies. Whether the OAMB receptor is a true OCTreceptor or a mixed OCT/TYR receptor has been amatter of some recent conjecture (El-Kholy et al.,2015). Very recently, a putative novel receptor withhomology to a2-adrenoceptors was reported inDrosoph-ila, coupled to Gi-mediated inhibition of adenylyl cy-clase, and activated by both OCT and TYR (Qi et al.,2017).Subsequent to the OAMB receptor discovery, three

further invertebrate OCT receptors were identified(Oct1bR, Oct2bR, and Oct3bR; Evans and Maqueira,2005; El-Kholy et al., 2015), with strong homology to thevertebrate b1–3 adrenergic receptors, respectively(Maqueira et al., 2005). Like their vertebrate counter-parts, all three b-receptors are coupled to the cAMPsignal transduction cascade through activation ofadenylyl cyclase (Chang et al., 2000; Maqueira et al.,2005; Balfanz et al., 2014). A fourth b-like receptor hasalso been suggested to be present in honeybees (Hauseret al., 2006; Balfanz et al., 2014). For a more detailedreview of species-specific expression of the variousisoforms, the reader is referred to a number of excellentdedicated reviews on invertebrate OCT/TYR receptors(Hauser et al., 2006; Verlinden et al., 2010a).

D. Tyramine Receptors

Dedicated invertebrate TYR receptors were firstsuggested in 1990 (Arakawa et al., 1990; Saudouet al., 1990), with three GPCRs (TyrRI, TyrRII, andTyrRIII) subsequently identified. TyrRI is the leastselective of the three, recognizing both OCT and TYR(Robb et al., 1994; Reale et al., 1997), although in mostspecies there is a higher affinity for TYR (VandenBroeck et al., 1995; Evans and Maqueira, 2005;Bayliss et al., 2013). TyrRI is homologous to vertebratea2-adrenoceptors (Evans and Maqueira, 2005) and may

exhibit agonist-dependent coupling, since TYR wasreported to induce adenylyl cyclase inhibition (VandenBroeck et al., 1995; Blenau and Baumann, 2001),whereas OCT induces an increase in cytosolic Ca2+

(Robb et al., 1994; Evans and Maqueira, 2005; Beggset al., 2011). A second TyrRI isoform was very recentlyreported in the cockroach (Blenau et al., 2017).

TyrRII and TyrRIII show much greater selectivitytoward TYR over OCT (Cazzamali et al., 2005; Baylisset al., 2013), although TyrRIII also responds to a varietyof other amines (Bayliss et al., 2013). Orthologs ofTyrRII have been identified in multiple species(Cazzamali et al., 2005; Hauser et al., 2006; Huanget al., 2009; Bayliss et al., 2013; Wu et al., 2015),although TyrRIII has thus far only been identified inDrosophila. TyrRII activation is associated with arelease of Ca2+ from intracellular stores (Cazzamaliet al., 2005; Huang et al., 2009), whereas TyrRIII iscoupled to an inhibition of adenylyl cyclase (Baylisset al., 2013).

In addition to GPCRs, one ionotropic TYR receptorhas been reported in C. elegans (Pirri et al., 2009;Ringstad et al., 2009). The ligand-gated ion channelLGC-55 receptor is a TYR-gated Cl2 channel that canalso be activated by PEA (Safratowich et al., 2014) andis involved in the control of locomotor activity. Ortho-logs of LGC-55 have also been identified in othernematode species (Pirri et al., 2009).

E. Physiologic Responses

TYR and more particularly OCT have been impli-cated in a variety of physiologic responses in inverte-brates. Their receptors are not restricted to the nervoussystem but rather are distributed throughout the body(Saraswati et al., 2004; Roeder, 2005; Vierk et al., 2009;Ma et al., 2015). Although responses to TYR havegenerally received less attention, when both have beenstudied, TYR generally induces responses opposite tothose of OCT (Saraswati et al., 2004; Ma et al., 2015).Although some of the responses (in particular thosetoward OCT) are similar to those that have beenproposed to be mediated by TAARs in vertebrates, it isimportant to emphasize again that vertebrate TAARsand the invertebrate OCT/TYR receptors are evolution-arily very distant from each other (Gloriam et al., 2005;Lindemann et al., 2005; Hashiguchi andNishida, 2007).

1. Octopamine. Consistent with serving the role ofan invertebrate adrenergic system, OCT has beenshown to modulate both peripheral and central re-sponses in a manner consistent with a “fight-or-flight”response (Sotnikova and Gainetdinov, 2009). Amongthese effects are enhanced locomotion and flight muscleperformance (Pribbenow and Erber, 1996; Saraswatiet al., 2004; Fussnecker et al., 2006; Bloch and Meshi,2007; Vierk et al., 2009), both direct and indirectmobilization of fat stores (Roeder, 2005), as well asincreases in cardiac and respiratory rates (Zornik et al.,


1999). OCT-mediated regulation of immune responses(Huang et al., 2012) and a role in reproductive behav-ior have also been reported (Lee et al., 2003; Limet al., 2014). In the firefly, OCT is well established tounderlie the neural basis of light emission (Copelandand Robertson, 1982; Christensen et al., 1983;Hashemzadeh et al., 1985) and also regulates biolumi-nescent emissions in glow worms (Rigby and Merritt,2011). Modulation of sensory inputs by OCT is also wellestablished (Farooqui, 2007), with effects seen in re-lation to vision (Bacon et al., 1995; Scheiner et al., 2014),olfaction (Barron et al., 2002; Anton et al., 2006;Farooqui, 2007; Flecke and Stengl, 2009; Cassenaerand Laurent, 2012; Ma et al., 2015), hearing (Andréset al., 2016), taste (Scheiner et al., 2002; Pankiw andPage, 2003), and touch and proprioception (Büschgeset al., 1993; Skorupski, 1996).Within the central nervous system, OCT has been

established to play a role in sensitization responses(Sombati and Hoyle, 1984; Stelinski et al., 2003), as wellas learning and memory (Hammer, 1997; Farooquiet al., 2003; Evans and Maqueira, 2005), rewardcircuitry (Unoki et al., 2005), and feeding behavior(Yang et al., 2015b). A role for OCT in kin recognitionand other complex conspecific social behaviors withincolonies has also been shown (Schulz and Robinson,2001; Spivak et al., 2003; Lehman et al., 2006; Ma et al.,2015). Finally, OCT has been reported to play a role inmediating aggressive behaviors (Stevenson et al., 2005;Hoyer et al., 2008; Rillich et al., 2011).2. Tyramine. As previously described, although less

well studied, TYR effects generally appear to function-ally oppose those of OCT. As such, TYR-mediateddecreases in locomotion (Nagaya et al., 2002;Saraswati et al., 2004), flying behavior (Fussneckeret al., 2006; Vierk et al., 2009), and foraging (Barronet al., 2002; Schulz et al., 2003) have been seen. Roles insensory perception, particularly taste (Scheiner et al.,2002; Pankiw and Page, 2003) and olfaction (Kutsukakeet al., 2000; Anton et al., 2006; Kononenko et al., 2009;Ma et al., 2015), have also been ascribed to TYR. TYR-induced increases in the rate of habituation (Braun andBicker, 1992) and modification of complex group dy-namics (Ma et al., 2015) are reported to occur indepen-dently of the conversion of TYR to OCT.

IV. Trace Amine–Associated Receptors

High-affinity mammalian receptors for the arche-typal trace amines were long suspected. A saturable,high-affinity binding site for PEA was reported in1982 (Hauger et al., 1982), although this was sub-sequently suggested to in fact represent binding toMAO-B (Li et al., 1992a). Similarly saturable, high-affinity binding sites for TYR (Ungar et al., 1977;Vaccari, 1986) and TRP (Kellar and Cascio, 1982;Altar et al., 1986; McCormack et al., 1986; Perry,

1986; van Nguyen et al., 1989) were sporadicallyreported but were rarely verified or characterizedbeyond the initial reports. Even the identification ofinvertebrate high-affinity receptors for TYR and OCTdid little to help, with vertebrate homologs not present.

The receptor family now known as TAARs wasinitially discovered in 2001 by two separate groups(Borowsky et al., 2001; Bunzow et al., 2001). Borowskyet al. (2001) had been searching for novel 5-HT1–likereceptors, screening genomic DNA with primersdesigned to conserved regions of the sixth and seventhtransmembrane regions of known 5-HT receptors. Thenovel family of GPCRs they identified was initiallytermed trace amine receptors on the basis of the highaffinity for PEA and TYR of two of the family membersidentified. The authors proposed the new receptorfamily be abbreviated as TAx, with x being a numeralto designate individual isoforms (Table 3), although theindividual isoform identifiers were rarely subscripted insubsequent use. Bunzow et al. (2001), as part of theirsearch for novel catecholamine receptors, screenedcDNA obtained from rat pancreatic tumor cell linesusing degenerate primers designed against the con-served third and sixth transmembrane regions ofknown catecholamine GPCRs. The novel GPCR theyidentified was termed trace amine receptor 1 or TAR1,based on their pharmacological profiling. In retrospect,the choice of pancreatic cell lines was remarkablyprescient and was a factor that has subsequently beenlargely overlooked. Although many have struggled withthe low expression levels of TAARs in most tissues, thepancreas and particularly b cells have been shown to beuniquely abundant in their TAAR1 expression (Regardet al., 2007; Raab et al., 2015).

The identification of a family of trace amine receptorsgenerated considerable interest in large part because theinitial pharmacological profiling indicated that a numberof psychotropic agents exhibited high affinity for one ofthe family members (TAAR1) (Bunzow et al., 2001).Furthermore, both original reports localized the newtrace amine receptor family to human chromosome6q23.2 (Borowsky et al., 2001; Bunzow et al., 2001), areplicated putative susceptibility locus for schizophrenia(Cao et al., 1997; Kaufmann et al., 1998; Levinson et al.,2000) and mood disorders (Venken et al., 2005). As such,it is not too surprising that the greater abundance of atleast some of the receptor family in the pancreas becameoverlooked, and the field became devoted almost entirelyto the central nervous system effects of TAAR1, the onlyfunctional family member in humans with a clearlydemonstrated endogenous ligand. Unfortunately, therewas little consistency in nomenclature in these earlydays (see Table 3), with some researchers using thenomenclature of Bunzow, others that of Borowsky, andsome a third nomenclature system designated as TRAR(Duan et al., 2004). Not only were different nomencla-tures used, but isoform identification was not consistent,


TABLE 3Relationship of different nomenclature systems to TAAR families

TAARNomenclature

PreviousNomenclatures

MaximumFunctionalIsoforms

Possible Pseudogenes Ligand Tuning Notes

TAAR1 TRAR1, TA1,TAR1, TA1

1 2 Primary amines Two pseudogenes only reported in thebottlenose dolphin

The elephant shark is the only otherspecies with multiple isoformsreported (one functional, onepseudogene)

TAAR2 GPR58 2 2 (lesser hedgehog tennec),1 (multiple species)

Primary amines TAAR2 is the only tetrapod TAAR geneto contain multiple (2) exons

The chicken is the only species reportedto have two functional TAAR2 genes

TAAR3 GPR57P 1 1 Primary aminesTAAR4 TA2P, 5-HT4P,

TA2

5 2 Primary amines

TAAR5 PNR 2 1 Tertiary amines The horse is the only species reported tohave multiple functional isoforms

TAAR6 TRAR4, TA4 5 6 Tertiary aminesDiamine binding pocket

TAAR7 TA12 (TAAR7a) 16 4 Tertiary aminesTA15 (TAAR7d)TA14 (TAAR7e)TA13P (TAAR7f)TA9 (TAAR 7g)TA6 (TAAR7h)

TAAR8 TRAR5, TA5,TAR5, GPR102

3 3 Tertiary amines

TA11 (TAAR8a)Diamine binding pocket

TA7 (TAAR8b)TA10 (TAAR8c)

TAAR9 TRAR3, TA3,TAR3

7 1 Tertiary amines

TAAR E1 2 2 Novel family recently proposedTAAR M1 1 1 Novel family recently proposed. Only

present in marsupialsTAAR M2 9 2 Novel family recently proposed. Only

present in marsupialsTAAR M3 5 1 Novel family recently proposed. Only

present in marsupialsTeleost-

specificisoforms

Up to 24 species-specific pseudogenesare present but individualpseudogene isoforms haverarely been reported

TAAR10 5TAAR11 zTA1a 1TAAR12 zTA69

(TAAR12b)13

zTA71(TAAR12d)

zTA72(TAAR12f)zTA73

(TAAR12k)TAAR13 zTA64

(TAAR13b)5 Diamines (TAAR13c)

zTA65(TAAR13c)

zTA66(TAAR13e)

TAAR14 zTA70(TAAR14d)

12 Diamine binding pocketin some isoforms

zTA68(TAAR14i)zTA67

(TAAR14k)TAAR15 2TAAR16 zTA63

(TAAR16c)7

zTA62(TAAR16e)

zTA36(TAAR16f)zTA35

(TAAR16g)

(continued)


TABLE 3—Continued

TAARNomenclature




TAAR17 zTA48(TAAR17a)

3

zTA47(TAAR17b)

zTA49(TAAR17c)

TAAR18 zTA28(TAAR18d)

11

zTA61(TAAR18f)zTA27

(TAAR18g)zTA18

(TAAR18h)zTA19

(TAAR18i)zTA20

(TAAR18j)TAAR19 zTA54

(TAAR19c)22

zTA34(TAAR19d)

zTA59(TAAR19f)zTA33

(TAAR19g)zTA50

(TAAR19h)zTA31

(TAAR19i)zTA32

(TAAR19l)zTA51

(TAAR19o)zTA29

(TAAR19q)zTA30

(TAAR19s)zTA16

(TAAR19u)TAAR20 zTA44

(TAAR20a)30

zTA23(TAAR20a1)

zTA39(TAAR20b)

zTA21(TAAR20b1)

zTA45(TAAR20c)

zTA40(TAAR20d)

zTA38(TAAR20e)

zTA41(TAAR20f)zTA43

(TAAR20i)zTA53

(TAAR20j)zTA25

(TAAR20k)zTA57

(TAAR20l)zTA24

(TAAR20m)zTA42

(TAAR20o)zTA90+

(TAAR20p)zTA91+

(TAAR20q)

(continued)


resulting in the same receptor sometimes being givendifferent numerical designations in each system(Table 3). A standardized nomenclature system (TAAR)was proposed in 2005 based on a thorough examinationof gene sequences, phylogenetic relationships, and chro-mosomal organization (Lindemann et al., 2005). Thisclassification system has stood the test of time and beenindependently validated on numerous occasions(Hashiguchi and Nishida, 2007; Hussain et al., 2009;Libants et al., 2009; Vallender et al., 2010; Tessaroloet al., 2014; Azzouzi et al., 2015; Eyun et al., 2016; Gaoet al., 2017). Indeed, the TAAR nomenclature system(Table 3) is now used exclusively by those active in thefield. Despite this, controversy remains. The only familymember to be deorphanized by IUPHAR has beendesignated TA1 (Maguire et al., 2009; Alexander et al.,2015), presenting an uncomfortable situation in whichthose working in the field and the official nomenclaturecommittee are using different naming systems. TheIUPHAR recommendation is based on the conventionthat receptors be named for their endogenous ligand.There is, however, no consensus ligand for the TAARfamily as awhole (Lindemann et al., 2005; Ferrero et al.,2012), with some members (TAAR1–TAAR4) beingtuned to primary amines, whereas other family mem-bers (TAAR5–TAAR9) are tuned toward tertiaryamines (Ferrero et al., 2012). Some (TAAR6 and 8)may even be activated by diamines (Li et al., 2015)(Table 3). Furthermore, as discussed earlier, there is nostandard, accepted definition of what comprises a “traceamine.” With the IUPHAR TA designation prone toconfusion with the earlier nomenclature systems thatwere based on an incomplete phylogeny, we strongly

advocate for a renewed, open dialog between IUPHARand leading trace amine researchers to resolve thissituation.

In 2006, a new and surprising arm to trace amineresearch was identified when Stephen Liberles andLinda Buck showed that the TAAR family also actedas a new class of receptors for olfaction (Liberles andBuck, 2006). This attracted a whole new cadre ofresearchers who have contributed to significant ad-vancements of the TAAR field, in particular, withrespect to the identification of putative endogenousligands for family members other than TAAR1. Asdiscussed fully in subsequent sections, the one notableexception is TAAR1, which is evolutionarily the oldestmember of the family but the only family member withno role to play in the detection of olfactory cues (Liberlesand Buck, 2006). The identification of the role of TAARsin the detection of olfactory cues was not withoutcontroversy, however, as it was also reported that nofamilymember (other than TAAR1) was present outsideof the olfactory epithelium (Liberles and Buck, 2006;Carnicelli et al., 2010). This now appears to not be thecase (Table 4) and likely was due to the low expressionlevels of TAARs generally present, along with a lack ofsuitably sensitive and selective reagents at that time.This lack of research tools has been a recurring problemfor the TAAR field (Berry et al., 2017), although it isslowly beginning to be addressed.

The identification of putative endogenous ligands forTAARs other than TAAR1 has provided an addedimpetus to the elucidation of TAAR pharmacology. Thatmany of these ligands originate from ecologically sig-nificant sources, such as predators, spoiled food, and

TABLE 3—Continued

TAARNomenclature




zTA37(TAAR20r)

zTA56(TAAR20t)zTA55

(TAAR20u)zTA46

(TAAR20w)zTA26

(TAAR20x)zTA22

(TAAR20y)zTA52

(TAAR20z)TAAR21 6TAAR22 6TAAR23 15TAAR24 3TAAR25 12TAAR26 28TAAR27 6TAAR28 8

LampreyoutgroupsTAAR-like 25 17

GPR57, G protein–coupled receptor 57; GPR58, G protein–coupled receptor 58; PNR, putative neurotransmitter receptor.


even putative pheromones, has further spurred re-search efforts to understand the evolution of TAARs.Although there are still many open questions, throughthese research efforts a picture has emerged of TAARsbeing intricately linked to the detection of olfactory cuesassociated with species-specific survival mechanisms.That TAARs are also distributed throughout the body(Table 4) and at least some of their endogenous ligandsreadily cross cell membranes (Berry et al., 2013)provides an interesting perspective, which potentiallyallows the same molecule that provides the environ-mental cue to directly affect various physiologic process-es that could conceivably further aid in adaptationresponses. Rather than existing as distinct fields, it isexpected that in the upcoming years much closer linkswill be forged between the olfactory and nonolfactoryTAAR communities, which will further speed the fillingof current knowledge gaps with respect to the basicunderlying physiology of TAAR systems.

A. Evolution of Trace Amine–Associated Receptors

At present, the majority of evidence suggests that anancestral TAAR-like protein first emerged in lamprey(Gloriam et al., 2005; Hashiguchi and Nishida, 2007;Libants et al., 2009; Eyun et al., 2016), with a conservedTAAR signature motif appearing later, after the di-vergence of jawed vertebrates from jawless fish (Fig. 6).Others have, however, suggested that TAAR ancestralemergence only occurred after the divergence fromlamprey (Hussain et al., 2009; Tessarolo et al., 2014)largely because of the absence of the conserved TAARmotif from the lamprey receptors. Lamprey do innatelyavoid PEA sources (Imre et al., 2014), a response knownto be TAAR mediated in jawed vertebrates; althoughthe molecular basis of this lamprey avoidance behavioris unknown, it is potentially consistent with ancestralTAARs being present.Among mammals, the highest number of TAARs

identified thus far is in the flying fox, with 26 functionalgenes, whereas the bottlenose dolphin is the onlyvertebrate species known to contain no functional TAARgenes (Eyun et al., 2016). Like in other tetrapods,mammalian TAARs generally belong to nine subfamilies(TAAR1–TAAR9). Within these, repeated species-specific expansion, duplication, and pseudogenizationevents have occurred. This has resulted in great vari-ability in the total number of functional TAARs presentbetween species (Table 5), including the appearance ofspecies-specific isoforms (Lindemann et al., 2005;Vallender et al., 2010; Eyun et al., 2016). For example,four putative new subgroups of TAARs were recentlydescribed: TAAR E1, which is present in the commonshrew, hedgehog, lesser hedgehog tenrec, and Africanelephant; and TAARs M1–M3 found in two marsupialspecies, tammar wallaby and opossum (Eyun et al.,2016), consistent with previous suggestions ofmarsupial-specific TAARs (Vallender et al., 2010). These

new families appear to belong to the clade II, tertiaryamine-recognizing group of TAARs, and as such aremostclosely related to TAAR5–TAAR9 (Eyun et al., 2016). Ofthese putative new TAARs, species-specific variants ofTAAR E1, M2, and M3 were reported.

Two species appear rather unique (Table 5); thebottlenose dolphin, as previously described, has nofunctional TAAR genes, only possessing three pseudo-genes (Eyun et al., 2016). Meanwhile, the dog is the onlyknown species to express functional TAARs but noTAAR1, and its genome being represented with func-tional TAAR4 and TAAR5 plus a further two pseudo-genes (Vallender et al., 2010).

Interestingly, the number of TAARs appears to becorrelated with the number of olfactory receptors inmost species, with the exception of Canis, which hasmore than 800 olfactory receptors but only two func-tional TAARs, with the olfactory receptors likelycompensating for the majority of functions served byTAARs in other species (Eyun et al., 2016). There are upto seven functional genes in Xenopus (Mueller et al.,2008; Eyun et al., 2016), although the TAAR comple-ment of the true frogs (Rana sp.) has not been de-termined. This is a notable knowledge gap for thedetermination of the evolutionary relationships ofolfactory functions, with the amphibian olfactory sys-tem regarded as an evolutionary midpoint betweenteleosts and tetrapods (Duchamp-Viret and Duchamp,1997; Gliem et al., 2013).

The mammalian TAAR1–TAAR9 family seems tohave two distinct evolutionary patterns. Primaryamine-detecting clade I TAARs (TAAR1–TAAR4)(Ferrero et al., 2012) appear to be evolving under strongnegative (purifying) selection criteria, whereas thetertiary amine-detecting clade II TAARs (TAAR5–TAAR9) (Ferrero et al., 2012) show significant varia-tions in gene numbers and appear to evolve under theinfluence of positive selection pressures (Eyun et al.,2016). Similarly, clade III zebrafish TAARs have un-dergone strong positive selection (Hussain et al., 2009).Thus, the profiles of TAAR expression appear to beevolutionarily determined by adaptive responses; in-deed, there is now evidence that TAAR expression caneither be influenced by habitat changes or determinehabitat choice (Churcher et al., 2015; Fatsini et al.,2016), consistent with a role in the detection andresponse to migratory cues.

As expected from the above, the primary amine-detecting TAAR1–TAAR4 appear to be evolutionarilyolder, more conserved, and are generally represented bya single isoform in the majority of genomes, with theexception of TAAR4 (in which a low level of species-specific expansion has occurred) (Hussain et al., 2009;Eyun et al., 2016). In contrast, the tertiary amine-detecting mammalian TAARs (TAAR5–TAAR9) havearisen more recently with multiple species-specific iso-forms of each subtype present, with the exception of


TAAR5 (the oldest of the clade II genes) (Eyun et al.,2016). The only TAAR that is not expressed in theolfactory system (TAAR1) is the oldest member of thefamily, with TAAR4 the second oldest, and these twofamily members share considerable overlap in theirligand selectivity (Lindemann et al., 2005). Otherevolutionarily ancient members of the family (TAAR2,TAAR3, and TAAR5) are also thought to predate theorigin of amniotes (Eyun et al., 2016). All other mam-malian TAAR subfamilies are currently thought toderive from a single copy of one of these ancestralTAARs via gene duplication (Eyun et al., 2016). Theteleost-specific clade III TAARs are thought to be themost recent family members to have emerged (Hussainet al., 2009).

B. Trace Amine–Associated Receptor 1

TAAR1 is the best characterized of the TAAR familyand is currently regarded as the main target for PEAand TYR, although in this respect it should be notedthat TAAR4, a pseudogene in humans, is also a targetbut has rarely been studied. TAAR1 is expressed inbrain structures associated with psychiatric disorders,in particular in key areas where modulation of dopa-mine [ventral tegmental area (VTA)] and serotonin[dorsal raphe nuclei (DRN)] occurs (Table 4), and thepresence of TAAR1 variants was recently reported to beassociated with schizophrenia (John et al., 2017). In theperiphery, TAAR1 expression suggests putative roles inregulating immune system responses and controllingenergy metabolism. The current understanding of the

TABLE 4Cellular and tissue expression profile of TAARs

Mammalian TAAR Species Expression Method

TAAR1 Human,a–d mouse,b,e–l

ratj,m–pVTA,e,k substantia nigra,k DRN,e,k amygdala,e,t

rhinal cortices,e subiculum,e prefrontal cortex,n–p

nucleus accumbens,o hypothalamus,e,t preopticarea,e spinal trigeminal nucleus,e medullaryreticular nucleus,e nucleus of the solitary tract,e

area postrema,e pancreatic b cells,a,f,g,l stomach(D cellsi),a,f,i–k intestines (neuroendocrine cells,a,f

enterochromaffin mucosal cellsd,s),a,d,f,j,k,s whiteadipose tissue,l spinal cord,m testes,j

leukocytesb,c,h

TAAR1-KO/lacZ KI mouse,e,f

immunohistochemistry,a,c,o,p RT-PCR,b,g–j,l,m,s

RNA sequencing,i,k in situ hybridizationk,m

TAAR2 Human,b,q,r mouse,b,h,q,s

ratjOlfactory epithelium,q,r intestines,s testes,j

leukocytesb,hRT-PCRb,h–j,q–s

TAAR3 Mouse,q ratj Olfactory epithelium,q testesj RT-PCRq,j

TAAR4 Mouse,l,q ratj,m Olfactory epithelium,l,q trachea,l spinal cord,m

spleen,j testes,j musclejRT-PCR,j,l,m,q in situ hybridizationm

TAAR5 Human,q,r mouse,b,h,q,t

ratj,m,uOlfactory epithelium,q,r amygdala,t arcuate

nucleus,t ventromedial hypothalamus,t spinalcord,m intestines,u testes,j leukocytesb,h

RT-PCR,b,h,j,m,q,r,u in situ hybridizationt

TAAR6 Human,k,q,r,v mouse,h–j,l

ratj,mOlfactory epithelium,l,q,r amygdala,k,v

hippocampus,k,v basal ganglia,v frontal cortex,v

substantia nigra,v spinal cord,m intestines,s

testes,j kidney,k leukocytesh

RT-PCRh–l,q–s,v

TAAR7 Mousel,q Olfactory epitheliuml,q RT-PCRl,q

TAAR8 Human,k,q,r mouse,c,h,j,l

ratj,mOlfactory epithelium,q,r,t amygdala,k cerebellum,j

cortex,j spinal cord,m islets of Langerhans,l

intestines,j,l spleen,j,l testes,j heart,j lungs,j

kidney,j,k leukocytesh

RT-PCRc,h,j–m,q,r

TAAR9 Human,k,q,r,w mouse,h,i,l,s

ratmOlfactory epithelium,q,r,t pituitary gland,w skeletal

muscle,w spinal cord,m intestines,l,s spleen,l

kidney,k leukocytesh

RT-PCR,h,k–m,q–s Northern blotw

KI, knock-in; RT-PCR, reverse transcription polymerase chain reaction.aRaab et al. (2015).bNelson et al. (2007).cWasik et al. (2012).dKidd et al. (2008).eLindemann et al. (2008).fRevel et al. (2013).gRegard et al. (2007).hBabusyte et al. (2013).iAdriaenssens et al. (2015).jChiellini et al. (2012).kBorowsky et al. (2001).lRegard et al. (2008).mGozal et al. (2014).nEspinoza et al. (2015b).oLiu et al. (2018).pFerragud et al. (2017).qLiberles and Buck (2006).rCarnicelli et al. (2010).sIto et al. (2009).tDinter et al. (2015c).uKubo et al. (2015).vDuan et al. (2004).wVanti et al. (2003).


Fig. 6. Phylogenetic tree of TAARs. Sequences from the publications by Azzouzi et al. (2015), Gao et al. (2017), Eyun et al. (2016), Tessarolo et al. (2014), andVallender et al. (2010) were used. Using the jackhmmer program from the hmmer-3.1b1 software package (http://hmmer.org, Howard Hughes Medical Institute)and chimpanzee TAAR1 as a seed, we iteratively built a hidden Markov model (HMM) representing TAARs. All of the sequences were scored and aligned againstthis HMM (which has 339 match positions). Next, all sequences between 250 and 450 amino acids long, with at least 280 match positions to the HMM, and with astarting methionine were selected. We made the selection nonredundant, correcting apparent sequencing errors in a few cases where the different sources abovedisagreed. Based on the alignment of the selected sequences to the TAAR-HMM, protein distances were calculated using the protdist program from the phylipsoftware package version 3.69 (with default parameters, http://evolution.genetics.washington.edu/phylip.html, University of Washington). A tree was thengenerated using the neighbor program from the same package, with default parameters, and using human histamine receptor H2 as an outgroup.


http://hmmer.org

http://evolution.genetics.washington.edu/phylip.html

TABLE 5Species variation in TAAR isoforms

Some of the studies used incompletely characterized or draft genomes resulting in markedly lower TAAR numbers identified than in studies using more rigorous protocols.

Species FunctionalTAAR Pseudogenes Incompletely

Characterized Chromosomal/Scaffold Location References

EuarchontogliresHuman 6 3 Chromosome 6q23.1 Lindemann et al. (2005), Hashiguchi

and Nishida (2007), Hussain et al.(2009), Vallender et al. (2010),Tessarolo et al. (2014), Eyun et al.(2016), Gao et al. (2017)

6 25 37 Not

reported5 Not

reportedChimpanzee 3 6 1 (TAAR7 not

reported)Chromosome 5 Lindemann et al. (2005), Vallender

et al. (2010)3 5Orangutan 3 5 1 (TAAR7 not

reported)Vallender et al. (2010)

Rhesus macaque 6 2 1 (TAAR7 notreported)

Vallender et al. (2010)

Squirrel monkey 4 1 1 (TAAR7 notreported)


Common marmoset 3 5 1 (TAAR7 notreported)

Vallender et al. (2010)2 5

Cottontop tamarin 3 0 1 (TAAR7 notreported)


House mouse 15 1 Chromosome 10A4 Lindemann et al. (2005), Hashiguchiand Nishida (2007), Hussain et al.(2009), Vallender et al. (2010),Tessarolo et al. (2014), Eyun et al.(2016), Gao et al. (2017)

15 Notreported

Norway rat 17 2 Chromosome 1p12 Lindemann et al. (2005), Hussainet al. (2009), Vallender et al.(2010), Eyun et al. (2016)

17 1

LaurasiatheriaBottlenose dolphin 0 3 Scaffold_23789.1-1595,

scaffold_105514.1-521399Eyun et al. (2016)

Common shrew 9 3 1 Scaffold_152798.1-16186,scaffold_230199.1-67322,scaffold_230199.1-67323,scaffold_230199.1-67324,scaffold_234999.1-41078,scaffold_227500.1-50167,scaffold_227500.1-50168,scaffold_231179.1-93586,scaffold_231179.1-93587,scaffold_231179.1-93588,scaffold_167158.1-10409,scaffold_161956.1-9068,scaffold_193904.1-13319

Eyun et al. (2016)

Cow 21 8 Chromosome 9 Hussain et al. (2009), Vallender et al.(2010), Eyun et al. (2016), Gaoet al. (2017)

19 0 ChrUn004.45113 0 ChrUn004.3514 Not

reportedHorse 11 0 Scaffold_12.50000001-55000000 to

scaffold_12.50000001-55000014Vallender et al. (2010), Eyun

et al. (2016)11 4

Pig 9 0 Vallender et al. (2010)Dog 2 2 Chromosome 1 and chromosome Un Vallender et al. (2010), Eyun et al.

(2016)Hedgehog 6 4 2 Scaffold_315330.1-30722,

scaffold_322681.1-22522,scaffold_287976.1-9947,scaffold_332111.1-9337,scaffold_325288.1-13778,scaffold_364605.1-21850,scaffold_321546.1-13098,scaffold_184140.1-8575,scaffold_302268.1-8721,scaffold_183615.1-1222

Eyun et al. (2016)

Little brown bat 6 1 Scaffold_130715.1-10777,scaffold_130715.1-10778,scaffold_140852.1-155364,scaffold_140852.1-155365,scaffold_140852.1-155366,scaffold_140852.1-155367,scaffold_220.1-46453

Eyun et al. (2016)

(continued)


TABLE 5—Continued



Malayan flying fox 26 10 Scaffold_12831.1-39811,scaffold_12831.1-39812,scaffold_12831.1-39813,scaffold_12831.1-39814,scaffold_17466.1-19369,scaffold_17466.1-19370,scaffold_18837.1-16342,scaffold_18837.1-16343,scaffold_18973.1-15890,scaffold_19339.1-14625,scaffold_19339.1-14626,scaffold19375.1-14563,scaffold_19997.1-13482,scaffold_19997.1-13483,scaffold_20227.1-12945,scaffold_20465.1-15670,scaffold_20465.1-15671,scaffold_22299.1-9969,scaffold_24703.1-7136,scaffold_25351.1-11297,scaffold_25351.1-11298,scaffold_26592.1-7418,scaffold_29671.1-4951,scaffold_34747.1-4153,scaffold_35764.1-2782,scaffold_36915.1-2635,scaffold_38546.1-2466,scaffold_44184.1-2021,scaffold_8480.1-69282,scaffold_8480.1-69283,scaffold_8480.1-69284,scaffold_8949.1-64714,scaffold_8949.1-64715,scaffold_8949.1-64716,scaffold_8949.1-64717,scaffold_8949.1-64718

Eyun et al. (2016)

AfrotheriaAfrican elephant 9 3 3 Scaffold_7842.1-91204,

scaffold_7842.1-91205,scaffold_7842.1-91206,scaffold_7842.1-91207,scaffold_0.25000001-30000000,scaffold_7842.1-91209,scaffold_23946.1-26436,scaffold_23946.1-26437,scaffold_23946.1-26438,scaffold_3583.1-140411,scaffold_3583.1-140412,scaffold_3583.1-140413,scaffold_19337.1-58480,scaffold_19337.1-58481,scaffold_79621.1-6335

Eyun et al. (2016)

Lesser hedgehogtenec

9 7 1 Scaffold_96430.1-8246,scaffold_161156.1-8572,scaffold_231756.1-18732,scaffold_198886.1-11295,scaffold_167756.1-3881,scaffold_167122.1-3947,scaffold_256442.1-4595,scaffold_262922.1-7019,scaffold_229023.1-10280,scaffold_228176.1-10370,scaffold_10127.1-6004,scaffold_232273.1-5125,scaffold_47746.1-4410,scaffold_250749.1-11800,scaffold_234201.1-11315,scaffold_173561.1-5671,scaffold_187047.1-4403

Eyun et al. (2016)

XenarthraNine-banded

armadillo5 4 Scaffold_13715, scaffold_68458,

scaffold_106240, scaffold_58549,scaffold_181544, scaffold_31645,scaffold_31595

Eyun et al. (2016)

(continued)


TABLE 5—Continued



MarsupialiaOpossum 22 4 2.405000001-410000000,

chromosome 2Hashiguchi and Nishida (2007),

Hussain et al. (2009), Vallenderet al. (2010), Eyun et al. (2016)

22 322 019 0

Tammar wallaby 18 3 1 224424132,2070LUS300020N02.g,189272895, 2070LUS280371J18.b,142869977, 222941615,2070LUS280640F03.b,2070LUS330332G15.g,scaffold386681:211:1254,scaffold101726:3050:4087,scaffold5372:18568:19389,scaffold5372:31430:32469,scaffold23666:6728:7759,scaffold25613:18010:19041,scaffold369370:5:625,scaffold361402:565:1491,scaffold64233:11843:12526,scaffold328269:964:1878,scaffold11217:21401:22444

Eyun et al. (2016)

PrototheriaPlatypus 4 0 Nw_001794460.1 Vallender et al. (2010),

Eyun et al. (2016)4 1Sauropsida

American alligator 8 Notreported

Tessarolo et al. (2014)

Carolina anole 3 0 Contig_5904 Tessarolo et al. (2014), Eyun et al.(2016), Gao et al. (2017)3 Not

reported3 Not

reportedChicken 4 1 Chromosome 3 Hashiguchi and Nishida (2007),

Mueller et al. (2008), Hussain et al.(2009), Tessarolo et al. (2014),Eyun et al. (2016), Gao et al. (2017)

3 23 03 Not

reportedZebra finch 1 0 Chromosome 3 Eyun et al. (2016)

AmphibiaWestern clawed frog 7 0 Scaffold_200, scaffold_172,

scaffold_153, scaffold_62Hashiguchi and Nishida (2007),

Hussain et al. (2009), Tessaroloet al. (2014), Syed et al. (2015),Eyun et al. (2016)

6 13 05 Not

reported6Not

reportedAfrican clawed frog 3 Not

reportedSyed et al. (2015)

TeleosteiAtlantic salmon 27 25 Ssa21, Ssa13, Ssa06, Ssa02,

Ssa15, Ssa14, Ssa04, Ssa01Tessarolo et al. (2014)

Amazon molly 2 Notreported

Gao et al. (2017)

Burton’smouthbrooder

23 3 Scaffold_612, scaffold_128,scaffold_24, scaffold_2074,scaffold_2674, scaffold_1343,scaffold_2473, scaffold_1542,scaffold_802, scaffold_2902,scaffold_2545, scaffold_2406,scaffold_1575, scaffold_1303,scaffold_1185, scaffold_802,scaffold_479, scaffold_337,scaffold_2104, scaffold_4685

Azzouzi et al. (2015)

Cave fish 6 Notreported

Gao et al. (2017)

Channel catfish 28 8 Chromosome 2, chromosome 16 Gao et al. (2017)Cod 3 Not

reportedGao et al. (2017)

European eel 13 Notreported

Churcher et al. (2015)

Japanese grenadieranchovy

32 Notreported

Zhu et al. (2017)

(continued)


TABLE 5—Continued



Japanese pufferfish(fugu)

18 1 Scaffold_144, scaffold_2286,scaffold_682, scaffold_2971,scaffold_347, scaffold_36,scaffold_3984, scaffold_5473,scaffold_55, scaffold_62,scaffold_7591, scaffold_3049,scaffold_510, scaffold_2618,scaffold_375

Hashiguchi and Nishida (2007),Hashiguchi et al. (2008), Hussainet al. (2009), Tessarolo et al. (2014),Eyun et al. (2016), Gao et al. (2017)

13 618 06 Not

reported2 Not

reported

Japanese rice fish(medaka)

27 7 Chromosome 2, chromosome 21,chromosome 24, scaffold_2442,scaffold_2165, scaffold_2246,scaffold_3620, scaffold_4535,scaffold_691

Hashiguchi and Nishida (2007),Hashiguchi et al. (2008), Hussainet al. (2009), Tessarolo et al. (2014),Gao et al. (2017)

25 725 16 Not

reported2 Not

reportedMakobe Island

cichlid18 3 Scaffold_133, scaffold_193,

scaffold_490, scaffold_295,scaffold_238, scaffold_947,scaffold_857, scaffold_159,scaffold_1502, scaffold_2325,scaffold_1573, scaffold_698


Maylandia zebra 20 5 Scaffold00183, scaffold00006,scaffold00127, scaffold00676,scaffold00347, scaffold00068,scaffold02965, scaffold00108,scaffold02487, scaffold02369,scaffold02115, scaffold01981,scaffold00834, scaffold00561,scaffold00347, scaffold02144


Nile tilapia 44 8 Scaffold_195, scaffold_84,scaffold_005, scaffold_374,scaffold_16, scaffold_242,scaffold_183, scaffold_78

Azzouzi et al. (2015),Gao et al. (2017)2 Not

reported

Platyfish 2 Notreported

Gao et al. (2017)

Princess cichlid 12 2 Scaffold_18, scaffold_25, scaffold_31,scaffold_166, scaffold_1763,scaffold_26, scaffold_160


Spotted gar 3 Notreported

Gao et al. (2017)

Spotted greenpufferfish

34 3 Chromosomes 3–7, chromosomes14–21, chrUN_random

Hashiguchi et al. (2008), Hussainet al. (2009), Tessarolo et al. (2014),Eyun et al. (2016), Gao et al. (2017)

12 418 017 Not

reported11 Not

reported2 Not

reportedThree-spined

stickleback50 15 LG 9, LG 16, LG 18, scaffold_37,

scaffold_160, scaffold_56Hashiguchi and Nishida (2007),

Hashiguchi et al. (2008), Tessaroloet al. (2014), Gao et al. (2017)

49 1548 05 Not

reported2 Not

reportedWest Indian ocean

coelacanth18 Not

reportedTessarolo et al. (2014)

Zebrafish 110 10 Chromosome 1, chromosome 7,chromosome 10, chromosome 12,chromosome 13, chromosome 15,chromosome 20, Zv6_NA2102

Hashiguchi and Nishida (2007),Hashiguchi et al. (2008), Hussainet al. (2009), Tessarolo et al. (2014),Eyun et al. (2016), Gao et al. (2017)

109 10112 4102 Not

reported94 Not

reportedChondrichthyes

Elephant shark 2 3 AAVX01005735.1,AAVX01475249.1,AAVX01045569.1,AAVX01508596.1,AAVX01365441.1

Hussain et al. (2009), Tessarolo et al.(2014), Eyun et al. (2016), Gaoet al. (2017)

2 02 Not

reported

(continued)


cellular distribution and pharmacological profile ofTAAR1 in validated disease models is summarized inTables 4 and 6 and described in detail in the followingsections.Characterization of TAAR1-KO and TAAR1-

overexpressing (OE) mouse and rat lines has greatlycontributed to a better understanding of TAAR1 function(Wolinsky et al., 2007; Lindemann et al., 2008; Di Caraet al., 2011; Revel et al., 2011, 2012a; Espinoza et al.,2015a; Harmeier et al., 2015; Black et al., 2017; Schwartzet al., 2017). TAAR1-KO mice exhibit an enhancedresponse to amphetamine, resulting in an increasedhyperlocomotion that is correlated with more pronouncedincreases of dopamine, norepinephrine, and serotonin(Wolinsky et al., 2007; Lindemann et al., 2008).TAAR1-OE mice show phenotypically opposite effectswith lowered sensitivity to amphetamine, whereas theirwild-type littermatesmaintain a normal hyperlocomotionresponse (Revel et al., 2012a). Furthermore, the sponta-neous firing rate of dopaminergic neurons in the VTA andserotonergic neurons in the DRN is increased inTAAR1-KO mice (Bradaia et al., 2009; Revel et al.,2011). Thus, TAAR1 plays an active role in the expressionof behaviors traditionally associated with classic neuro-transmitters, supporting its potentially important role inthe modulation of neurotransmitter functions and, hence,in mental illness.1. Pharmacology of Trace Amine–Associated Receptor 1.a. Trace Amine–Associated Receptor 1 Gene

Conservation. The intronless TAAR1 gene is conservedacross mammals, avians, and amphibians and, based onthe rates of amino acid fixation compared with neutralmutations, is under strong purifying selection (Hussainet al., 2009). TAAR1 gene homology between humans andcynomolgus monkeys and between rats and mice is 97%and 77%, respectively. Interestingly, in dogs (Vallenderet al., 2010) and dolphins (Eyun et al., 2016), TAAR1 hasbecome a pseudogene, resulting in an uncompensated lossof function. Comparative genetics analysis in dogs revealedthat this pseudogenization event predated the emergenceof the Canini tribe and is not coincident with caninedomestication (Vallender et al., 2010). All other vertebrateshave maintained a functional TAAR1.b. Expression of Trace Amine–Associated Receptor 1.

Compared with most other GPCRs, one of the definingfeatures of TAAR1 is its generally low expression levels,

and this has contributed to a number of initial reports ofTAAR1 expression failing to be validated with morerobust reagents. Overall advancements in the field havebeen further hampered by a slowdevelopment of suitablyselective pharmacological tools for the systematic prob-ing of tissue and cellular expression profiles (Berry et al.,2017). Inmice, replacement of theTaar1 coding sequencewith a LacZ reporter construct under the control ofthe endogenous Taar1 promoter has allowed demonstra-tion of a heterogenous distribution of TAAR1 withinthe central nervous system (Lindemann et al., 2008;Table 4). This distribution generally mirrors that of theknown dopaminergic, glutamatergic, and serotonergicsystems, with both mRNA and protein confirmed in theamygdala, basal ganglia, limbic areas, prefrontal cortex,raphe nuclei, substantia nigra pars compacta, and VTA(Borowsky et al., 2001; Lindemann et al., 2008). Such adistribution is generally seen in both rodents andprimates and is consistent with the effects of TAAR1-directed ligands described in other sections. At least inhumans, expression may not be limited to neurons withone report of astrocytic TAAR1 (Cisneros and Ghorpade,2014). Spinal cord expression may also occur, at least inrats (Gozal et al., 2014).

One of the other defining features of TAAR1 expres-sion is its absence from the olfactory epithelium, instark contrast to all other TAAR family members(Liberles and Buck, 2006; Carnicelli et al., 2010). Inthe periphery, TAAR1 is prominently located through-out the gastrointestinal system in cells involved inhormone secretion in response to the presence ofnutrients (Table 4). This includes the D cells of thestomach (Chiellini et al., 2012; Revel et al., 2013;Adriaenssens et al., 2015; Raab et al., 2015), pancreaticb cells (Regard et al., 2007; Revel et al., 2013; Raabet al., 2015), and intestinal enterochromaffin mucosalcells (Kidd et al., 2008; Ito et al., 2009; Revel et al., 2013;Raab et al., 2015) of both rodents and humans. Multiplegroups have also reported the presence of TAAR1mRNA and protein in various populations of mouseand human leukocytes (D’Andrea et al., 2003; Nelsonet al., 2007; Wasik et al., 2012; Babusyte et al., 2013),and the presence of TAAR1 protein in breast tissue wassuggested very recently (Vattai et al., 2017), although inthis latter instance no details on the cell type(s) in whichstaining was observed were provided. Furthermore,

TABLE 5—Continued



AgnathaSea lampreya 25 3 GL477485, GL477912, GL478144,

GL478886, GL478986, GL478891,GL479321, GL479771, GL480074,GL480910, GL483319, GL483927,GL484453, GL486051, GL486090,GL490750

Hashiguchi and Nishida (2007),Libants et al. (2009), Eyunet al. (2016)

21 1728 1727 Not

reported

aThere is still some debate about whether lamprey contain TAAR, TAAR-like, or a completely separate family of receptors.


TABLE 6In vivo pharmacological effects of TAAR1-selective agonists

Study Type Species/Test System Pretreatment/Duration

Animals perGroup (M/F)

RO5166017 Dose/Concentration Key Results

min n mg/kgCocaine-induced

hyperlocomotionaC57Bl6 mice 30/30 8 M 0.03–3 p.o. Attenuation at 0.3, 1, and

3 mg/kg p.o.Cocaine-induced

hyperlocomotionaC57Bl6 TAAR1-KO and WT

littermate mice30/30 10 M 0.3–1 p.o. Attenuation at 0.3 and 1 mg/kg p.o.

in WT mice, no effect inTAAR1-KO mice

Spontaneoushyperlocomotion inDAT-KO micea

C57Bl6 DAT-KO and WTlittermate mice

None/90 8 M 0.2–1 p.o. Attenuation at 0.5 and 1 mg/kg p.o.

L-687,414–inducedhyperlocomotiona

NMRI mice 30/30 8 M 0.01–0.1 p.o. Attenuation at 0.1 mg/kg p.o.

L-687,414–inducedhyperlocomotiona

C57Bl6 TAAR1-KO and WTlittermate mice

60/30 8 M 1 p.o. Attenuation at 1 mg/kg p.o. in WTmice, no effect in TAAR1-KOmice

SIHa NMRI mice 45/15 8–16 M 0.01–1 p.o. Reversal of SIH (dT) at doses0.1 and 0.3 mg/kg withoutaffecting Tb

SIHa C57Bl6 TAAR1-KO and WTlittermate mice

45/15 8–10 M 0.1 p.o. Reversal of SIH (dT) at0.1 mg/kg in WT mice, no effectin TAAR1-KO mice

Cocaine-induced CPPb Sprague-Dawley rats 10 9 to 10 M 10 i.p. Inhibition of expression, but notretention of cocaine rewardmemory at 10 mg/kg i.p.

Abuse-related effects ofnicotinec

Sprague-Dawley rats 10 6–9 M 3.2–10 i.p. Reduction of nicotine self-administration (at 5.6 and10 mg/kg i.p.) and attenuation ofcue- and drug-inducedreinstatement of nicotine-seeking (at 10 mg/kg i.p.)

oGTTd C57Bl6 TAAR1-KO and WTlittermate mice

45/120 5–8 M 0.3 mg/kg p.o. Glucose-lowering effect at0.3 mg/kg p.o. in WT mice andincreased PYY and GLP-1levels, no effect in TAAR1-KOmice

oGTTd Diabetic db/db C57Bl6 mice,DIO mice, DIO Glp1R-KOmice

45/120 8 M 0.3 mg/kg p.o. Glucose-lowering effect at0.3 mg/kg p.o. in diabeticdb/db mice

ivGTTd C57Bl6 mice 30/10 10 M 3 mg/kg s.c. Lower amount of meal emptied at0.3 mg/kg p.o.

Gastric emptyingd C57Bl6 mice 45/30 8 M 0.3 mg/kg p.o. Lower amount of meal emptied at0.3 mg/kg p.o.

Food intake and bodyweightd

C57Bl6 mice, DIO mice, 45/60 6 M 0.3 mg/kg s.c. Reduction in food intake and bodyweight, reduced triglyceridelevels, increased insulinsensitivity at0.3 mg/kg p.o.





hyperlocomotioneC57Bl6 mice 30/30 8–16 M 0.3–10 p.o. Attenuation at 1 and 3 mg/kg p.o.

Cocaine-inducedhyperlocomotione

Wistar rats 60/30 7 to 8 M 1–10 p.o. Attenuation at 10 mg/kg p.o.

Spontaneoushyperlocomotion inDAT-KO micee

C57Bl6 DAT-KO and WTlittermate mice

None/60 5–9 M 1 p.o. Attenuation at 1 mg/kg p.o. inDAT-KO mice, but no effect inDAT-KO/TAAR1-KO mice

Spontaneoushyperlocomotion inDAT-KO micee

C57Bl6 DAT-KO, TAAR1-KO,and WT littermate mice

None/60 8–11 M 0.1–1 p.o. Attenuation at 0.1, 0.3, and1 mg/kg p.o.

L-687,414–inducedhyperlocomotione

NMRI mice 15/30 8 M 0.01–0.1 p.o. Attenuation at 0.1 mg/kg p.o.

Spontaneoushyperlocomotion in NR1KD micee

C57Bl6 NR1 KD and WTlittermate mice

None/60 6 to 7 M 0.3–1 p.o. Attenuation at 0.3 and 1 mg/kg p.o.in WT mice, no effect in NR1-KDmice

SIHe NMRI mice 45/15 6–8 M 0.1–1 p.o. Reversal of SIH (dT) at0.3 mg/kg without affecting Tb

Forced swim stresse Wistar rats 24 h, 18 h,1 h/60

8 F 3–30 p.o. Significant decrease in immobilitytime at 10 and 30 mg/kg p.o.

Cocaine self-administratione

Long-Evans rats 60/60 7 to 8 M 3–10 i.p. Reduction of cocaine intake in ratswith a stable history ofintravenous cocaine self-administration at 3 and10 mg/kg i.p.

(continued)


TABLE 6—Continued




min n mg/kgContext-induced cocaine

relapsefLong-Evans rats 15/90 6–8 M 3–10 i.p. Suppression of cocaine seeking

after a 2-wk period ofwithdrawal from chronic cocaineself-administration at 3 and10 mg/kg i.p.

Methamphetamine self-administrationg

Long-Evans rats 10/60 6–10 M 3–10 i.p. Reduction of methamphetamineintake in rats with a stablehistory of intravenousmethamphetamine self-administration at 3 and10 mg/kg i.p.

Methamphetamine-stimulatedhyperactivityg

Long-Evans rats 70/60 5 to 6 M 1.67–5 i.p. Attenuation of methamphetamine-induced hyperactivity andprevention of development ofmethamphetamine sensitizationat 1.67 and 5 mg/kg i.p.

Differential reinforcementof low-rate behaviore

Cynomolgus macaques 4-h injectiontest interval

5 to 6 M 1–30 p.o. Reduced response rate at 10 and30 mg/kg p.o. and increasedinter-response time at30 mg/kg p.o.

Object retrievale Cynomolgus macaques 90/90 12 M 1–10 p.o. Procognitive effect at 10 mg/kg p.o.Haloperidol-induced

catalepsyeWistar rats 60/10 12 M 0.3–10 p.o. Reduction of catalepsy at 0.3, 3,

and 10 mg/kg p.o.Sleep/wake parameterse Sprague-Dawley rats 6 h 8 M 1–10 p.o. Wake-promoting activity at

10 mg/kg p.o.





hyperlocomotionhC57Bl6 mice 60/30 6–8 M 0.3–3 p.o. Attenuation at 1 and 3 mg/kg p.o.

PCP-inducedhyperlocomotionh

C57Bl6 mice 30/60 7–16 M 0.003–1 p.o. Attenuation at 0.01, 0.03, 0.1, and0.3 mg/kg p.o.

L-687,414–inducedhyperlocomotionh

NMRI mice 15/30 8–24 M 0.0003–1 p.o. Attenuation at 0.003, 0.01, 0.03,0.1, 0.3, and 1 mg/kg p.o.

Olanzapine-induced weightgainh

Sprague-Dawley rats None/14 d 8 M 1 p.o. Reduction of fat mass changeinduced by olanzapine at 1 mg/kg p.o.

ICSSi Wistar rats 60/20 8 M 1–10 i.p. Reversal of cocaine (1 mg/kg i.p.)–induced facilitation of changesin ICSS threshold at 1, 3, and10 mg/kg i.p.; no significantchange by RO5263397 alone andtherefore no reinforcing effectsand no abuse potentialanticipated

Abuse-related effects ofcocainej

Sprague-Dawley rats 10/15 6–18 M 1–10 i.p. Reduction of expression of cocainebehavioral sensitization (at3.2 mg/kg i.p.), cue- and cocaineprime-induced reinstatement ofcocaine seeking (at 3.2 and5.6 mg/kg i.p.), and expressionbut not development of cocaine-induced place preference (at10 mg/kg i.p.)

Abuse-related effects ofmethamphetaminek

Sprague-Dawley rats 10/15 7 to 8 M 3.2–10 i.p. Reduction of expression ofbehavioral sensitization (at10 mg/kg i.p.),methamphetamine self-administration (at 3.2 mg/kgi.p.), and both cue- and apriming dose ofmethamphetamine-inducedreinstatement of drug-seekingbehaviors (at 3.2 and10 mg/kg i.p.)

Chronic methamphetamine-treatment 5-CSRTTl

Sprague-Dawley rats 3 d/60 8 M 5.6 i.p. Attenuation of forced abstinence-induced impulsivity(at 5.6 mg/kg)

(continued)


TABLE 6—Continued




min n mg/kgAbuse-related effects of

nicotinecSprague-Dawley rats 10–20 8–10 M 3.2–17.8 i.p Reduction of expression and

development of behavioralsensitization (at 10 mg/kg i.p.),nicotine self-administration (at3.2 and 5.6 mg/kg i.p.) anddiscriminative stimulus effect ofnicotine (at 10 mg/kg i.p.),attenuation of the subjectiveeffects of nicotine(at 10 mg/kg i.p.)

Abuse-related effects ofmorphinem

Sprague-Dawley rats 10 3.2–5.6 i.p Reduction of morphine self-administration (at 3.2 and 5.6mg/kgi.p.) and attenuation of cue- anddrug-induced reinstatement ofmorphine-seeking) at 5.6 mg/kg i.p.);no effect on the expression ofmorphine-induced CPP, naltrexoneprecipitated withdrawal-inducedjumping of CPA, or antinociceptiveeffect of morphine

phMRIh Sprague-Dawley rats 90/12 9 M 1–30 p.o. Activity profile comparable tomarketed antipsychotics in adose-dependent manner.

Forced swim stressh Wistar rats 24 h, 16 h,2 h/60

8 F 3–30 p.o. Significant decrease in immobilitytime at 10 and 30 mg/kg p.o.

Differential reinforcement oflow-rate behaviorh

Cynomolgus macaques 90/90 10 M 1–10 p.o. Number of reinforcers increased at10 mg/kg p.o.

Object retrievalh Cynomolgus macaques 90/90 11 M 0.3–10 p.o Procognitive effect at 1, 3, and10 mg/kg p.o.

Haloperidol-inducedcatalepsyh

Wistar rats 60/10 6–12 M 0.3–10 p.o Reduction of catalepsy at 0.3, 1, 3,and 10 mg/kg p.o.

Sleep/wake parametersn C57Bl6 TAAR1-KO, OE, andWT littermate mice

6 h 8 M 0.1–1 p.o. Wake-promoting activity anddecreased REM and NREM in WTmice at 0.3 and 1 mg/kg p.o., noeffect in TAAR1-KO and potentiatedeffect in TAAR1-OE mice

Sleep/wake parametersh Sprague-Dawley rats 6 h 8 M 0.3–30 p.o. Wake-promoting activity anddecreased REM at 3, 10, and30 mg/kg p.o.

Study Type Species/TestSystem

Pretreatment/Duration



min n mg/kgCocaine-induced hyperlocomotionh C57Bl6 mice 30/60 6–8 M 0.3–3 p.o. Attenuation at 0.3, 1, and

3 mg/kg p.o.PCP-induced hyperlocomotionh C57Bl6 mice 30/60 7–16 M 0.03–3 p.o. Attenuation at 0.03, 0.1, 0.3, 1,

and 3 mg/kg p.o.L-687,414–induced

hyperlocomotionhNMRI mice 15/30 8–24 M 0.01–1 p.o. Attenuation at 0.01, 0.03, 0.1, 0.3,

and 1 mg/kg p.o.ICSSi Wistar rats 60/20 8 M 0.3–3 i.p. Reversal of the cocaine (1 mg/kg

i.p.)–induced facilitation ofchanges in ICSS threshold at0.3, 1, and 3 mg/kg i.p.; nosignificant change byRO5256390 alone and thereforeno reinforcing effects and noabuse potential anticipated

phMRIh Sprague-Dawleyrats

90/12 9 M 1–30 p.o. Activity profile comparable tomarketed antipsychotics in adose-dependent manner

Attentional set-shiftingh Long-Evans rats 60/30 52 M 1–10 p.o. Procognitive effect: improvementof set-shifting performance at1 and 3 mg/kg p.o.

Object retrievalh Cynomolgusmacaques

60/5 11 M 0.3–3 i.m. Procognitive effect: improvementof performance during difficulttrials at 3 mg/kg i.m.

Differential reinforcement of low-rate behaviorh

Cynomolgusmacaques

60/90 10 M 0.3–3 i.m. Increased number of reinforcers at1 mg/kg i.m.

Context-induced cocaine relapsef Long-Evans rats 15/90 6–8 M 310 i.p. Suppression of cocaine seekingafter a 2-wk period ofwithdrawal from chronic cocaineself-administration at 3 and10 mg/kg i.p.

(continued)


details of the antibody used were not provided, and thisputative localization in one or more populations of cellsin breast tissue should be regarded with caution untilvalidation with well defined reagents has been pro-vided. TAAR1 presence in thyroid gland epithelial cellshas also been suggested (Szumska et al., 2015), al-though, again, validation of these findings is required.In contrast to earlier reports of TAAR1 presence inadipose tissue, blood vessels, heart, kidney, liver, lung,and testes (Borowsky et al., 2001; Chiellini et al., 2007,2012; Regard et al., 2008; Fehler et al., 2010), morerecent studies with more robust protocols and reagentshave failed to provide replication of the presence ofTAAR1 (Revel et al., 2013; Raab et al., 2015).Somewhat surprisingly for a GPCR, when TAAR1

protein has been identified, it has almost always beenreported to exhibit a predominantly intracellular local-ization (Bunzow et al., 2001; Lindemann and Hoener,2005; Miller et al., 2005; Revel et al., 2013; Raab et al.,2015; Pei et al., 2016), possibly due to the absence of oneor more N-terminal glycosylation sites (Barak et al.,2008). Intracellular TAAR1 may be associated withmembrane structures (Xie et al., 2008a; Szumskaet al., 2015), although the identity of these is unknown.Although reagents appropriate for ultrastructural stud-ies await development, there is good evidence thatTAAR1 is present both pre- (Bradaia et al., 2009;Revel et al., 2011; Leo et al., 2014) and postsynapticallywithin neurons (Espinoza et al., 2015a).c. Trace Amine–Associated Receptor 1 Ligands.

The archetypal trace amines PEA, TYR, TRP, and OCT,as well as the dopamine metabolite 3-MT and thethyroid hormone metabolite 3IT, are high-affinity en-dogenous agonists at TAAR1 (see Table 7 and referencestherein). In addition, dopamine and serotonin showpartial agonismat physiologically relevant concentrations

(Lindemann et al., 2005). Studies with synthetic com-pounds have revealed a quite remarkably broad ligandtuning of the receptor (Fig. 2; Table 7), with a number ofpsychotropic agents also showing high-affinity agonism atTAAR1 (Simmler et al., 2016). It should be stressed,however, that such effects often show pronounced speciesdependence, and this is perhaps most notable withthe endogenous hallucinogen DMT and lysergic aciddiethylamide, both of which show limited, if any, activityat the human isoform (Table 7). In addition to psychotropicagents, a variety of other synthetic compounds have beenreported to exhibit agonistic activity at TAAR1, includingapomorphine (Sukhanov et al., 2014), ractopamine (Liuet al., 2014), and the imidazoline ligands clonidine,guanabenz, and idazoxan (Hu et al., 2009) (Fig. 2;Table 7). Given this broad substrate tuning of TAAR1, itis interesting to note that OCT2, often described as apolyspecific (i.e., broad substrate tuning) transporter,carries TYR across biologic membranes with a nanomolaraffinity (Berry et al., 2016).

All of the above-mentioned compounds are agonists atTAAR1, and only a single antagonist has been described(which is discussed in detail in section IV.B.1.c.ii). Theapparent resistance of TAAR1 to standard medicinalchemistry approaches toward the development of an-tagonist compounds is one of the unique aspects of thereceptor. Recently, two in silico quantitative structure-activity relationship models were developed to furtherprobe the basis of the broad substrate tuning, speciesspecificity, and lack of antagonist binding (Guarientoet al., 2018). On the basis of the two quantitativestructure-activity relationship models developed,biguanides were identified as a useful chemical skeletonfrom which novel TAAR1 ligands could be developed. It isexpected that further development of suchmodels will aidin the development of much needed TAAR1 antagonists.

TABLE 6—Continued

Study Type Species/TestSystem

Pretreatment/Duration



min n mg/kgCompulsive binge-like eatingo Wistar rats 30/60 12 M 1–10 i.p. Inhibition of binge-like eating

behavior at 3 and 10 mg/kg i.p.Haloperidol-induced catalepsyh Wistar rats 60/10 6–12 M 0.3–3 p.o Reduction of catalepsy at

0.3 mg/kg p.o.Sleep/wake parametersh Sprague-Dawley

rats6 h 8 M 1–10 p.o. No significant effect at 1, 3, and

10 mg/kg p.o. on sleep/wakecycle and core body temperature

5-CSRTT, five-choice serial reaction time task; F, female; ICSS, intracranial self-stimulation; ivGTT, intravenous glucose tolerance test; KD, knockdown; M, male; NMRI,Naval Medical Research Institute; oGTT, oral glucose tolerance test; REM, rapid eye movement; SIH, stress-induced hyperthermia; Tb, basal temperature; WT, wild type.

aRevel et al. (2011).bLiu et al. (2016).cLiu et al. (2018).dRaab et al. (2015).eRevel et al. (2012b).fPei et al. (2014).gCotter et al. (2015).hRevel et al. (2013).iPei et al. (2015).jThorn et al. (2014b).kJing and Li (2015).lXue et al. (2018).mLiu et al. (2017a).nSchwartz et al. (2017).oFerragud et al. (2017).


i. Development of Selective Agonists and PartialAgonists. TAAR1 functionally couples to Gs proteins,and activation of the receptor by trace amines results inan intracellular increase in cAMP (Grandy, 2007). SinceTAAR1-independent effects of endogenous trace aminesare possible through other targets, including monoam-inergic transporters and receptors, as well as the sreceptor (Revel et al., 2011), the development of selec-tive TAAR1 ligands was important for the identificationof specific TAAR1-mediated biologic functions.Starting from the known adrenergic ligand S18616

[(S)-spiro[(1-oxa-2-amino-3azacyclopent-2-ene)-4,29-(89-chloro-19,29,39,49-tetrahydronaphthalene)]], modifyingthe linker region and exploring additional structure-activity relationships, 2-aminooxazolines were discov-ered as a chemical series of novel, highly potent,selective, and orally active TAAR1 full and partialagonists (Galley et al., 2015). Besides functional activityat human TAAR1 and selectivity versus the adrener-gic a2A receptor, metabolic stability as measured inhepatocytes was used as a key parameter to select thefourmolecules that have been extensively characterizedin the literature: RO5166017 [(S)-4-((ethyl(phenyl)-amino)methyl)-4,5-dihydrooxazol-2-amine)], RO5203648[(S)-4-(3,4-dichlorophenyl)-4,5-dihydrooxazol-2-amine],RO5263397 [(S)-4-(3-fluoro-2-methylphenyl)-4,5-dihydrooxazol-2-amine], and RO5256390 [(S)-4-((S)-2-phenylbutyl)-4,5-dihydrooxazol-2-ylamine] (Fig. 2)(Galley et al., 2015). The safety, tolerability, pharmacoki-netics, and pharmacodynamics after oral administrationof one of these molecules, RO5263397, a TAAR1 partialagonist that is well tolerated in rat and cynomolgusmonkey toxicity studies, was determined in a single-ascending dose, randomized, double-blind, placebo-controlled study in healthy male volunteers. Althoughthe compound was found to be generally safe, a 136-foldabove-average systemic exposure to the parent compoundwas found in one participant (Fowler et al., 2015). Anadditional two poor metabolizers were subsequentlyidentified, and all three were of African origin. Additionalin vitro studies with recombinant uridine diphosphateglucuronosyltransferases (UGTs) showed that the contri-bution of UGT2B10 to RO5263397 glucuronidation wasmuch higher at clinically relevant concentrations than theUGT1A4 that had beenpredicted to be themainmetabolicenzyme in preclinical enzyme studies. DNA sequencingidentified a previously uncharacterized splice site muta-tion that prevented assembly of full-length UGT2B10mRNA and thus functional UGT2B10 protein expressionin all of the poor metabolizers (Fowler et al., 2015).Subsequent DNA database analyses revealed theUGT2B10 splice site mutation was highly frequent inindividuals of African origin (45%), compared with onlymoderate frequency in Asians (8%) and an almost com-plete absence in Caucasians (,1%). A prospective studyusing hepatocytes from 20 individual African donorsdemonstrated a greater than 100-fold lower intrinsic

clearance of RO5263397 in cells homozygous for the splicesite variant allele (Fowler et al., 2015). Based on thisunexpected finding in the phase I study, a novel chemicalseries of potent and selective TAAR1 agonists withimproved pharmacokinetic properties will be required toallow further clinical development. Notwithstanding thissetback, the four compounds that are extensively used inthe literature remain excellent tools for the characteriza-tion ofTAAR1-mediated effects in animals andnonhumanprimates.

These TAAR1 partial and full agonists have beenextensively profiled preclinically. A summary of thepreclinical efficacy of the individual compounds inbehavioral in vivo animal models is shown in Tables 6and 8. Notably, the compounds show activity in fivedistinct paradigms based on the modulation of dopami-nergic and/or glutamatergic pathways that are sugges-tive of antipsychotic activity. Furthermore, TAAR1agonists also show antidepressant/stress-reducing ac-tivity; procognitive, wake-promoting, antinarcolepsy,and anticataleptic effects; glucose and weight gain–controlling responses; as well as antiaddiction effects ina wide-range of rodent and nonhuman primate para-digms (see Tables 6 and 8 and references therein). Thus,TAAR1 agonists may constitute a completely new drugclass with a fundamentally new mechanism of actionbased on themodulation of dopaminergic, glutamatergic,and serotonergic neurotransmission for the treatment ofschizophrenia, mood disorders, narcolepsy, addiction, ordiabetes. Importantly, these compounds appear to targetsymptoms in these diseases that currently are not treat-able, including cognitive and negative symptoms inschizophrenia and substance abuse with no concomitantinherent abuse potential. Full details of these effects ofTAAR1 agonists in preclinical models are provided insections IV.B.2 and IV.B.3.

ii. Development of N-(3-Ethoxyphenyl)-4-(1-Pyrrolidinyl)-3-(Trifluoromethyl)Benzamide, the FirstSelective Antagonist. Unlike for most GPCRs, ithas been particularly challenging for standard me-dicinal chemistry approaches to identify selectiveTAAR1 antagonists. Thus far, only one such com-pound, EPPTB [N-(3-ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl)benzamide (RO5212773)] (Fig. 2),has been identified and characterized (Bradaia et al.,2009; Stalder et al., 2011). EPPTB is a highly potentmouse TAAR1-selective antagonist that is largely inac-tive at the rat and human isoforms (Berry et al., 2017);unfortunately, even in mice, the pharmacokinetic prop-erties prevent the in vivo use of EPPTB (Bradaia et al.,2009). This limitation on the use of EPPTB for researchand drug discovery has considerably slowed progress inelucidating TAAR1-dependent mechanisms. Ex vivo ithas been shown that EPPTB prevents the reduction ofthe firing frequency of dopaminergic neurons inducedby TYR (Bradaia et al., 2009). When applied alone,EPPTB increases the firing frequency of dopaminergic


neurons, suggesting that TAAR1 either exhibits con-stitutive activity or is tonically activated by ambientlevels of an endogenous agonist(s). Clear differentia-tion between these two possible scenarios has not beenpossible. Further confusing the situation are sugges-tions that EPPTB may in fact be an inverse agonist(Bradaia et al., 2009; Stalder et al., 2011). EPPTB alsoblocks the TAAR1-mediated activation of an inwardlyrectifying K+ current in VTA slices (Bradaia et al.,2009). When applied alone, EPPTB induced an appar-ent inward current, suggesting the closure of tonicallyactivated K+ channels. Importantly, all of theseEPPTB effects were absent in TAAR1-KO mice, rulingout off-target effects. Together, the above studiessuggest that tonic activation of inwardly rectify-ing K+-channels by TAAR1 leads to a reduction inthe basal firing frequency of dopaminergic neurons inthe VTA.d. Signal Transduction and Molecular Interactions.i. Adenylyl Cyclase. As a Gs-coupled receptor,

TAAR1 promotes cAMP production via stimulation ofadenylyl cyclase (Borowsky et al., 2001; Bunzow et al.,2001; Lindemann and Hoener, 2005). This has beenconfirmed after expression of TAAR1 in a variety of celltypes and with various approaches to analyze cAMPconcentrations used (Reese et al., 2007; Wainscott et al.,2007; Barak et al., 2008; Hu et al., 2009; Espinoza et al.,2011; Revel et al., 2011; Liu et al., 2014). In fact, cAMPassays are now a central component of TAAR1 ligandscreening programs (Bradaia et al., 2009; Revel et al.,2011, 2012a, 2013; Stalder et al., 2011; Galley et al.,2012, 2015).ii. G Protein–Coupled Inwardly Rectifying Potassium

Channels. TAAR1-mediated reduction in the firingrate of VTA dopaminergic neurons appears to occursubsequent to activation of G protein–coupled inwardlyrectifying potassium channels (Bradaia et al., 2009).TYR activation of TAAR1 in VTA slices induces a Gprotein–dependent, inwardly rectifying K+ current,consistent with an activation of G protein–coupledinwardly rectifying potassium channels. Furthermore,in TAAR1 transfected Xenopus oocytes, TAAR1 directlyactivated Kir3 channels via pertussis toxin–insensitiveG proteins in Xenopus oocytes (Bradaia et al., 2009),most likely the aforementioned Gs proteins. As de-scribed above (section IV.B.1.c.ii) inwardly rectifyingK+ currents are also modified by the TAAR1-selectiveantagonist EPPTB.In the samemanner as TYR, the synthetic full TAAR1

agonist RO5166017 also inhibited neuron firing fre-quency by activating a K+-mediated outward current inboth VTA and DRN slices. Such effects are seen atcomparable agonist potencies to those obtained forTAAR1 activation in cAMP assays (Revel et al., 2011).These effects of RO5166017 and TYR on the firing rateof dopamine and serotonin neurons were absent inTAAR1-KO mice and were completely prevented by

EPPTB in wild-type animals (Revel et al., 2011). In-deed, EPPTB reduced the current beyond the pretreat-ment baseline values, providing further evidence forTAAR1 being either constitutively active or under tonicstimulation from an endogenous agonist.

iii. Heterodimerization with the D2-Like DopamineReceptor. A functional physical interaction betweenD2R and TAAR1 has been shown in a number of studiesboth in vitro and in vivo. Cellular studies have revealedthat TAAR1 forms heteromers with the postsynapticD2R-long isoform when coexpressed in human embry-onic kidney 293 (HEK-293) cells, and that the applica-tion of D2R antagonists enhances TAAR1 signaling inthese cells (Espinoza et al., 2011). Although normallyTAAR1 is mainly located intracellularly, coexpressionwith D2R causes TAAR1 appearance at the plasmamembrane (Espinoza et al., 2011; Harmeier et al.,2015). Furthermore, coimmunoprecipitation of TAAR1and D2R, directly reflecting heterodimerization, wasconvincingly demonstrated not only in vitro in HEK-293cells (Espinoza et al., 2011) but also in vivo in midbrainand cortex membrane preparations from TAAR1-OErats (Harmeier et al., 2015).

Such heterodimerization provides an attractive mo-lecular basis for TAAR1-mediated regulation of D2Rfunctioning. In the striatum of TAAR1-KO mice, anincreased expression of D2Rs, but not D1Rs, has beenobserved at both the mRNA and protein levels(Espinoza et al., 2015a). This is in addition to previousobservations of an increased proportion of striatal D2Rsin the high-affinity state after TAAR1-KO (Wolinskyet al., 2007). Furthermore, the locomotor activationinduced by high doses of the D2R agonist quinpirole,but not by the D1R agonist SKF-82958 (6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine), are significantly increased in TAAR1-KOmice (Espinoza et al., 2015a). Recently, quinpirole-induced yawning, a behavioral response thought toreflect the D3 isoform of D2Rs, was reported to bedecreased in the presence of selective TAAR1 agonists(Siemian et al., 2017). In contrast, the purportedly D2

isoform–mediated hypothermic effect of quinpirole wasnot affected by TAAR1 activation (Siemian et al., 2017),raising the possibility that TAAR1 may differentiatebetween D2R isoforms. The effects of D2R antagonistsare also modified by TAAR1, with haloperidol treat-ment causing significantly less catalepsy and striatalc-fos expression in TAAR1-KO animals (Espinozaet al., 2011). An altered function of presynaptic D2

autoreceptors has also been observed in TAAR1-KOmice, as reflected by an increase in the firing rate ofVTA neurons (Bradaia et al., 2009) and deficits inautoinhibitory control of dopamine release (Leo et al.,2014). Taken together, these data clearly demonstratethat the close interaction of TAAR1 and D2R hassignificant functional consequences at both the pre-and postsynaptic levels.


TABLE 7Functional activity (EC50) and binding potency (Ki) of endogenous, selected psychoactive, nonselective, and selective TAAR1 agonists in rats, mice,

and humansValues for EC50 and Ki are given in micromolar, whereas values for efficacy are given in percentages and indicate maximal cAMP levels reached compared with 10 mM

2-phenylethylamine.

TAAR1 AgonistRat TAAR1 Mouse TAAR1 Human TAAR1

EC50 Efficacy Ki EC50 Efficacy Ki EC50 Efficacy Ki

Endogenous ligandsPEAa 0.11 100 0.24 0.20 102 0.31 0.26 104TYRa 0.030 94 0.059 0.28 88 0.38 0.99 91TRPa 0.41 91 0.13 2.7 117 1.4 21 73Octopamineb 2.1 100 20 100 10 100Dopamineb 5.1 50 12 50 16 50Serotoninb 5.2 50 .50 .50 1003-MTc 0.70

Amphetamine likea

Amphetamine 0.66 91 0.23 0.53 90 0.089 2.8 914-fluoroamphetamine 0.069 78 0.081 0.13 77 0.32 3.5 674-methylamphetamine 0.11 93 0.10 0.071 94 0.15 .30Methamphetamine 0.85 73 0.35 0.73 78 0.55 5.3 704-fluoromethamphetamine 0.16 76 0.24 0.46 69 1.7 6.2 44MDMA 1.0 56 0.37 4.0 71 2.4 35 26MDA 0.74 86 0.25 0.58 102 0.16 3.6 11Cathinone 1.2 28 2.2 1.2 66 2.1 6.9 53Methcathinone 8.2 41 4.1 6.8 64 .10 .30

Benzofuransa

4-(2-aminopropyl)benzofuran (4-APB) 0.16 75 0.11 0.85 72 2.1 4.1 505-(2-aminopropyl)benzofuran (5-APB) 0.067 88 0.042 0.13 67 0.11 6.1 436-(2-aminopropyl)benzofuran (6-APB) 0.042 90 0.052 0.067 93 0.056 7.2 477-(2-aminopropyl)benzofuran (7-APB) 0.058 109 0.066 0.11 95 0.13 0.63 896-aminopropyl-2,3-dihydrobenzofuran

(6-APDB)1.0 83 1.0 0.51 95 0.21 .10

Phenethylaminesa

2,5-dimethoxy-4-bromo-phenethylamine(2C-B)

0.24 57 0.079 2.3 69 2.2 3.3 10

8-bromo-2,3,6,7-benzodihydro-difuran-ethylamine (2C-B-Fly)

0.27 48 0.029 1.8 49 0.71 .30

4-ethyl-2,5-dimethoxyphenethylamine(2C-E)

0.18 72 0.066 1.1 64 1.2 .10

2,5-dimethoxyphenethylamine(2C-H)

1.5 80 0.90 7.5 56 11 6.5 53

2,5-dimethoxy-4-propyl-phenethylamine(2C-P)

0.030 84 0.020 0.56 91 0.28 4.2 72

Mescaline 3.7 37 3.3 4.8 25 11 .10(2)-Ephedrine 2.5 42 3.7 14 31 .15 .10

4-thio-substituted phenethylaminesd

2C-T-3 0.0080 0.47 .302C-T-7 (“blue mystic”) 0.010 0.31 .302C-T-19 0.0048 0.096 .302C-T-31 0.0052 0.055 .30

TRPsa

Psilocin 0.92 85 1.4 2.7 80 17 .30DMT 1.5 81 2.2 1.2 73 3.3 .10

ErgolinesLysergic acid diethylamide (LSD)a 1.4 29 0.45 9.7 13 10 .20

Piperazinesa

m-Chlorophenylpiperazine (m-CPP) 0.15 60 0.054 3.2 40 6.6 .30Trifluoromethylphenylpiperazine (TFMPP) 0.75 59 0.38 3.8 44 2.3 .30

Aminoindanesa

2-aminoindane (2-AI) 0.11 90 0.31 0.33 54 2.1 1.5 110N-methyl-2-AI 0.37 63 0.53 0.94 108 2.6 3.3 545-iodo-2-aminoindane (5-IAI) 0.033 96 0.030 0.41 36 1.1 3.2 335,6-methylenedioxy-2-aminoindane (MDAI) 0.22 95 0.57 0.52 99 1.8 4.1 30

Miscellaneous nonselective compoundsApomorphinee 0.99 79 0.37 2.5 59 0.37 .20 0.70Ractopaminef 0.016 1003ITg,h 0.014 0.090 61 1.7 56Clonidinei 0.21 0.97Guanabenzi 0.007 0.025Idazoxani 0.11 6.7

Selective compoundsRO5073012j 0.025 24 0.0011 0.023 26 0.0032 0.025 34 0.0058RO5166017k 0.014 90 0.0027 0.0033 65 0.0019 0.055 95 0.031RO5203648l 0.0068 59 0.0010 0.0040 48 0.0005 0.030 73 0.0068

(continued)


iv. b-Arrestin 2 and Biased Signaling. In additionto the well established cAMP cascade, TAAR1 is alsoable to signal via b-arrestin 2 (Harmeier et al., 2015), amultifunctional scaffolding protein classically associ-ated with GPCR desensitization (Gainetdinov et al.,2004). It is well established that D2R signals through Gi

proteins to reduce cAMP levels. At the same time,however, D2R can also recruit b-arrestin 2 in a Gprotein–independent manner, a pathway that leads tothe dephosphorylation of protein kinase B (AKT)and the subsequent activation of glycogen synthasekinase 3b (GSK3b) (Beaulieu et al., 2009). Both ofthese pathways have been shown to be important fordopamine-mediated behaviors (Beaulieu and Gainetdi-nov, 2011). By using specific b-arrestin 2 complementa-tion assays, it has been demonstrated that whereasTAAR1 normally interacts poorly with b-arrestin 2, theheterodimerization of TAAR1 with D2R significantlyenhances the TAAR1–b-arrestin 2 interaction thatresults in reduced GSK3b activation (Harmeier et al.,2015). Furthermore, both TAAR1-induced cAMP accu-mulation and b-arrestin 2 recruitment to D2R werereduced in the presence of the TAAR1-D2R complex.Further confirmation of heterodimerization of

TAAR1 and D2Rmodulating the AKT/GSK3b pathwaywas obtained in striatal tissues from TAAR1-KO mice(Espinoza et al., 2015a). As measured by Western blot,a basal decrease in the phosphorylation of AKT andGSK3b was found in the striatum of TAAR1-KO mice.Furthermore, the levels of b-catenin, a target of GSK3bthat is degraded in response to increased GSK3bactivity, were also decreased in mutant mice. Incontrast, no alterations were observed in the phos-phorylated forms of extracellular signal–regulatedkinase 1/2, cAMP response element binding protein,or dopamine and cAMP-regulated phosphoprotein(32 kDa) (DARPP32), all of which are regulated by bothD1R and D2R in a G protein–dependent manner(Espinoza et al., 2015a).

Taken together, these results demonstrate thatthe activation of the TAAR1-D2R complex negativelymodulates GSK3b signaling, a situation with impor-tant clinical and pharmacological implications. TheAKT/GSK3b pathway is increasingly implicated inthe pathology of schizophrenia, bipolar disorder, anddepression, with growing evidence that antipsychoticsand antidepressants influence this pathway (Beaulieuet al., 2009). An opportunity to affect D2R signalingmediated by the b-arrestin 2–dependent AKT/GSK3bcascade via targeting the endogenous TAAR1 pathwayprovides an exciting new avenue in the pharmacology ofthese disorders, which is expected to garner consider-able interest over the next few years.

v. Other Signaling Cascades. Different downstreamtargets of TAAR1 signaling were described in activatedrhesus monkey lymphocytes, in which activation of thetranscription factors, cAMP response element bindingprotein and nuclear factor of activated T cells (both ofwhich are associated with immune activation), wasobserved (Panas et al., 2012). TAAR1-dependent phos-phorylation of PKA and PKC after treatment withmethamphetamine has been reported in transfectedHEK-293 cells, immortalized rhesus monkey B cells,and activated rhesus monkey lymphocytes, suggestingthat the high levels of inducible TAAR1 observed inactivated lymphocytes are functionally active (Panaset al., 2012). Whether the PKC activation was down-stream of PKA activation due to TAAR1 coupling to theGs protein was not determined, although this is perhapsthe most likely scenario.

2. Central Nervous System Effects

a. Cellular Effects.i. Dopaminergic Systems. From the first studies on

TAAR1-KO mice, it was clear that TAAR1 is a potentmodulator of dopaminergic neurotransmission(Wolinsky et al., 2007; Lindemann et al., 2008), consis-tent with its expression in the major dopaminergic cell

TABLE 7—Continued

TAAR1 AgonistRat TAAR1 Mouse TAAR1 Human TAAR1

EC50 Efficacy Ki EC50 Efficacy Ki EC50 Efficacy Ki

RO5256390m 0.0051 107 0.0029 0.0020 79 0.0044 0.016 98 0.024RO5263397m 0.047 76 0.0091 0.0013 59 0.0009 0.017 81 0.0041

Selective inverse agonistRO5212773 (EPPTB)n 4.5 0 0.94 0.028 212 0.0009 7.5 0 .5.0

aSimmler et al. (2016).bLindemann et al. (2005).cSotnikova et al. (2010).dLuethi et al. (2017).eSukhanov et al. (2014).fLiu et al. (2014).gScanlan et al. (2004).hCoster et al. (2015).iHu et al. (2009).jRevel et al. (2012a).kRevel et al. (2011).lRevel et al. (2012b).mRevel et al. (2013).nStalder et al. (2011). IC50 values are presented in lieu of EC50 values.


groups of the VTA and to lesser extent the substantianigra (Lindemann et al., 2008; Di Cara et al., 2011;Berry et al., 2017). An enhanced stimulation of locomo-tor activity with concomitant increased dopamine effluxafter amphetamine administration was observed afterTAAR1-KO (Wolinsky et al., 2007; Lindemann et al.,2008). Electrophysiological investigations on brain slicepreparations revealed that the lack of TAAR1 causes anincrease in the firing rate of dopaminergic neuronsof the VTA while simultaneously increasing D2R

agonist potency (Bradaia et al., 2009). Consistent withthis, the firing rate of these neurons in wild-type butnot TAAR1-KO mice is potently enhanced by theTAAR1 antagonist EPPTB and decreased by a varietyof agonists (Bradaia et al., 2009). In vivo microdialysiscoupled with fast-scan cyclic voltammetry revealedthat these changes in TAAR1-KO mice were associatedwith an elevated extracellular level of dopamine inthe nucleus accumbens, whereas synthetic TAAR1ligands (both antagonist and agonists) produced the

TABLE 8Summary of in vivo pharmacological effects of TAAR1-selective agonists

Model/Paradigm Mouse Rat Cynomolgus Monkey

Schizophrenia positive symptomsCocaine-induced LMAa–c RO6017, RO3648, RO6390,

RO3397RO3648

L-687,414–induced LMAa–c RO6017, RO3648, RO6390,RO3397

PCP-induced LMAc RO6390, RO3397DAT-KO (reduction of LMA)a,b RO6017, RO3648NR1-KD (reduction of LMA)a,b RO3648

Schizophrenia cognitive symptomsObject retrievalb,c RO3648, RO6390,

RO3397Attentional set-shiftingc RO6390

Antidepression/stress-reducingForced swim testb RO3648, RO3397Differential reinforcement of low-rate behaviorb,c RO3648, RO6390,

RO3397Stress-induced hyperthermiaa,b RO6017, RO3648

Wake-promotingc,d RO3397 RO3680, RO3397Antiaddiction

Cocaine self-administrationb,e RO3648, RO6390Cocaine-ICSSe RO6390, RO3397Context-induced cocaine relapsef RO3648, RO6390Cocaine reward memoryg RO6017Abuse-related effects of cocaineh RO3397Abuse-related effects of methamphetaminei RO3397Methamphetamine self-administrationj RO3648Methamphetamine-induced impulsivityk RO3397Nicotine self-administration, nicotine-inducedsensitization, reinstatement of nicotine seekingl

RO6017, RO3397

Morphine-induced sensitization, morphine self-administrationm

RO3397

Compulsive binge-like eatingn RO6390Antidiabetic

oGTTo RO6017oGTT in diabetic db/db mice, DIO mice, and diet-induced obese Glp1R-KO miceo

RO6017

Gastric emptyingo RO6017 RO3397Reduction of weight gain and body weightc,o RO6017 RO3397Reduction of weight gain induced by antipsychoticolanzapinec

RO3397

No EPS liability (no effect on catalepsy)b,c RO3648, RO6390,RO3397

Reduction of haloperidol-induced catalepsyb,c RO3648, RO6390,RO3397

DIO, diet-induced obese; EPS, extrapyramidal side-effects; ICSS, intracranial self-stimulation; LMA, locomotor activity; oGTT, oral glucose tolerance test; RO3397,RO5263397; RO3648, RO5203648; RO6017, RO5166017; RO6390, RO5256390;.

aRevel et al. (2011).bRevel et al. (2012b).cRevel et al. (2013).dSchwartz et al. (2017).ePei et al. (2015).fPei et al. (2014).gLiu et al. (2016).hThorn et al. (2014a).iJing and Li (2015).jCotter et al. (2015).kXue et al. (2018).lLiu et al. (2018).mLiu et al. (2017a).nFerragud et al. (2017).oRaab et al. (2015).


corresponding changes in evoked dopamine releasefrom slices of both the nucleus accumbens and striatumof wild-type but not TAAR1-KO animals (Leo et al.,2014). Direct evidence for decreased D2/D3 autoreceptorfunctionality was also demonstrated using paired-pulsefast-scan cyclic voltammetry (Leo et al., 2014).In transfected cells and brain synaptosome prepara-

tions, evidence for PEA (Xie and Miller, 2008) anddopamine (Xie et al., 2008b) modulation of DAT(Slc6A3) function via both TAAR1 and D2 autoreceptorshas been presented. With respect to potential TAAR1-mediated regulation of DAT, it should be noted thatin other studies, no difference in dopamine uptakerate was observed in either the nucleus accumbens orstriatum of TAAR1-KO mice or after TAAR1 agonist/antagonist treatment of wild-type animals (Leo et al.,2014). Furthermore, the partial TAAR1 agonistRO5203648 prevents cocaine-induced dopamine over-flow in the nucleus accumbens without altering dopa-mine uptake (Pei et al., 2014), and TAAR1 agonists areeffective in inhibiting dopamine-dependent hyperac-tivity in DAT-KO mice (Revel et al., 2011, 2012b; Leoet al., 2018) and when DAT has been inhibited byadministration of cocaine (Revel et al., 2013). As such,it now appears unlikely that TAAR1-mediated effectsoccur after a regulation of DAT function. Indeed,rather than modulation of DAT, the full TAAR1agonist RO5256390 blocks cocaine-induced inhibitionof dopamine clearance in nucleus accumbens slices viaa mechanism that requires simultaneous D2R activa-tion and GSK3b signaling, likely originating fromTAAR1-D2R heterodimerization (Asif-Malik et al.,2017), as observed in other systems (see sections IV.B.1.d.iii and IV.B.1.d.iv).When functionality of postsynaptic striatal dopamine

receptors was assessed in TAAR1-KO animals, anincreased expression and pronounced supersensitivityof D2Rs was observed (Wolinsky et al., 2007; Espinozaet al., 2015a), indicating that TAAR1 modulation ofdopaminergic systems also occurs postsynaptically.Furthermore, this postsynaptic supersensitivity wasmediated via the G protein–independent, b-arrestin2–dependent, AKT/GSK3b pathway, and not cAMP/PKA/DARPP32 signaling (Espinoza et al., 2015a). ThatTAAR1-mediated effects can be directed at both pre-and postsynaptic D2R considerably complicates theinterpretation of in vivo findings.ii. Serotonergic Systems. TAAR1 is expressed

within the DRN, the key area where cell bodies ofserotonin neurons are located (Lindemann et al., 2008),suggesting that TAAR1 may modulate brain sero-toninergic activity. Indeed, the full TAAR1 agonistRO5166017 inhibits the firing rate of DRN serotonergicneurons while modulation of TAAR1 activity altersthe desensitization rate and agonist potency at 5-HT1A

receptors (Revel et al., 2011). In brain slices of TAAR1-KOmice, the spontaneous firing frequency of serotonin

neurons was markedly increased compared with wild-type controls, and this increased firing rate was notaffected by RO5166017 (Revel et al., 2011). In wild-typeanimals, however, the TAAR1 partial agonist RO5203648increases the firing frequency of serotonergic neurons inthe DRN (Revel et al., 2012b). Furthermore, in a trans-genic TAAR1-OEmouse line, an augmented extracellularlevel of serotonin in the medial prefrontal cortex wasfound along with an elevated spontaneous firing rate ofDRN neurons (Revel et al., 2012a). Themolecular basis ofthese effects has not been studied in the same detail asthose mediated at dopaminergic systems, and furtherdetailed investigations of the mechanisms of influence ofTAAR1 on brain serotonin functions arewarranted.Usingtransfected cells and brain synaptosome preparations,evidence was presented that PEA (Xie and Miller, 2008)and serotonin can modulate serotonin transporter func-tion via both TAAR1 and 5-HT1A/5-HT1B autoreceptors(Xie et al., 2008b). Notwithstanding the lack of a molec-ularmechanism, the evidence suggests that TAAR1 likelyfunctions as an endogenous mechanism to maintain abalance in serotonergic neuronal activity, similar to itseffects in gating dopaminergic systems.

iii. Glutamatergic Systems. As previously described,TAAR1 agonists can overcome both the hyperlocomotionand cognitive impairments induced by administration ofNMDA receptor antagonists (Revel et al., 2011, 2013).That TAAR1 can regulate glutamatergic transmission isfurther suggested by the significant alterations in gluta-mate transmission found in the prefrontal cortex ofTAAR1-KO mice (Espinoza et al., 2015b), a locationwhere TAAR1 mRNA is found in wild-type mice. Appli-cation of a patch-clamp electrophysiological approach tocortical slices revealed deficient glutamate NMDA re-ceptor function in prefrontal cortex layer V pyramidalneurons of TAAR1-KOmice (Espinoza et al., 2015b). Thiswas associated with a decrease in the expression of theGluN1 andGluN2B subunits, aswell as a decrease in thephosphorylation of the Ser896 residue of GluN1. Incontrast, the levels of GluN2A, the AMPA receptorsubunit GluA1, and the phosphorylation status of post-synaptic density 95 (a postsynaptic protein critical to theproper organization and integrity of postsynaptic struc-tures) were not altered in TAAR1-KO animals. ThatTAAR1 activation acutely regulates the glutamatergicsystem was supported by the observation that culturedcortical neurons treated with the TAAR1 agonistRO5166017 showed a modest increase in GluN1 expres-sion and a significant increase in the phosphorylation ofthe Ser896 residue (Espinoza et al., 2015b). Furthermore,TAAR1 expression was mapped to layer V corticalneurons in TAAR1-KO/dsRed knock-in rats, a recentlydeveloped transgenic rat model in which the fluorescentmarker dsRed is expressed under the control of theTAAR1 promoter (Espinoza et al., 2015b). Since manycortical neurons in layer V project to the striatum, thesedata suggest that TAAR1 may modulate corticostriatal


glutamatergic transmission. Consistent with this hy-pothesis, significant alterations in the total levels andphosphorylation of GluN1 were found in the striatum ofTAAR1-KO mice (Sukhanov et al., 2016). Together, theabove-described studies provide amolecular basis for theability of TAAR1 to prevent hypoglutamatergic states aswell as the pronounced increase in prefrontal cortexactivity seen in pharmacological magnetic resonanceimaging (phMRI) studies with selective TAAR1 agonists(Revel et al., 2013).The TAAR1 agonist RO5166017 has also been report-

ed to counteract L-DOPA–induced phosphorylation ofAMPA receptors resulting in an inhibition of evokedcorticostriatal glutamate release in animals with nigro-striatal lesions (Alvarsson et al., 2015), effects that werereversed by the TAAR1 antagonist EPPTB. Such aneffect has potentially significant functional conse-quences for Parkinson disease–related abnormalities.Interestingly, methamphetamine-induced activation ofTAAR1 leads to intracellular cAMP accumulation inhuman astrocytes, an effect that decreased glutamateclearance abilities due to a downregulation of excit-atory amino acid transporter 2 (EAAT-2) (Cisnerosand Ghorpade, 2014). Although methamphetaminehas a promiscuous pharmacology, molecular alter-ations in astrocyte TAAR1 levels also induced changesin EAAT-2 levels and function, consistent with themethamphetamine response being TAAR1 mediated(Cisneros and Ghorpade, 2014). More recently, it hasbeen shown that high concentrations of dopamineupregulate TAAR1, consistent with TAAR1 acting toprevent dopamine hyperactivity, and this also leads toreduced EAAT-2 expression and glutamate clearance inprimary cortical astrocytes (Ding et al., 2017). In total,the above studies indicate that TAAR1 activity canmodulate glutamatergic responses by selective regula-tion of both receptors and transporter-mediated clear-ance mechanisms.

b. Behavior.i. Schizophrenia and Bipolar Disorder. TAAR1

agonists have the potential to be effective in thetreatment of psychiatric disorders, both directly, as wellas indirectly, through acting as a cellular rheostat ofneurotransmitter pathways. Activation of TAAR1 hasbeen shown to downregulate dopaminergic neurotrans-mission, whereas inhibition of TAAR1 enhances it(Lindemann et al., 2008). Data from studies in severalrodent models—including cocaine-, phencyclidine(PCP)-, and L-687,414 [(3S,4S)-3-amino-1-hydroxy-4-methylpyrrolidin-2-one]–induced hyperlocomotor activ-ity tests; cocaine-induced facilitation of intracranialself-stimulation, and phMRI—indicate that TAAR1agonists exhibit antipsychotic-like activity (Revel et al.,2013; Table 8). At doses that only causedmodest effects onbaseline locomotor activity, TAAR1 agonists significantlyantagonized cocaine-induced hyperlocomotor activity inmice as well as in rats (Revel et al., 2011, 2012b, 2013;

Table 6). In addition, partially active doses of TAAR1agonists and olanzapine, when combined, fully reversedthe hyperlocomotion induced by cocaine (Revel et al.,2013), indicating that TAAR1 agonists can also have anadditive effect on those of the marketed antipsychoticolanzapine. In mice, TAAR1 agonists also inhibitedhyperactivity induced by the noncompetitive NMDA re-ceptor antagonist PCP, as well as that induced byL-687,414, an NMDA glycine-site inhibitor (Revel et al.,2013; Table 6). These are both mechanistic assays thatmimic the NMDA hypofunction seen in schizophrenicpatients. Moreover, modulation of VTA activity in theintracranial self-stimulation test was observed in ratsafter TAAR1 agonist administration (Table 6), furtherdemonstrating regulation of key circuitry known to beassociated with the negative symptoms of schizophrenia(Revel et al., 2013). A series of mixed 5-HT1a/TAAR1agonists fromSunovionPharmaceutical Inc. (Marlborough,MA) (Nazimek et al., 2016) (see Fig. 2 for a representativeexample) have also been shown to be beneficial in prepulseinhibition andPCP-induced hyperactivitymodels of schizo-phrenia (Shao et al., 2011).

In summary, preclinical testing indicates that TAAR1agonists have the potential to treat patients with schizo-phrenia with better efficacy and improved tolerability,due to their lack of polypharmacology and unique abilityto normalize both dopamine hyper-reactive and gluta-mate hyporesponsive circuitry. Furthermore, given thebeneficial metabolic and antidiabetic effects (see sectionIV.B.3.a), they may also provide an additional benefit bynot increasing themetabolic syndrome, amajor side effectof current antipsychotic drugs.

ii. Cognitive Effects. Cognitive deficits are one of thecore symptoms of schizophrenia, symptoms that are onlypoorly controlled by current treatment approaches(Miyamoto et al., 2012; Citrome, 2014). In the primateobject retrieval test, TAAR1 agonists improved the accu-racy of object retrieval in difficult trials, such as thosewhen the object to be retrieved was placed at a distancefrom the test subject (Revel et al., 2013), indicative ofenhanced cognition (Tables 6 and 8). Similar effects werealso observed in a rat attentional set-shifting paradigm.Here, deficits induced by 7 days of PCP treatment in theability to discriminate between a variety of tactile andolfactory cues were reversed by TAAR1 agonists (Revelet al., 2013), indicating a normalization of cognitivedeficits caused by hypoglutamatergic signaling. Sucheffects are consistent with the enhanced prefrontal corti-cal activity seen in phMRI studies after TAAR1 agonisttreatment (Revel et al., 2013).

iii. Depression. TAAR1 agonists have been assessedfor antidepressant-like activity in two validated para-digms: the forced swim stress test in rats and thedifferential reinforcement of low response rate (DRL)behavior schedule in cynomolgus monkeys (Tables 6 and8). The forced swim stress test relies on the principle thatwhen rodents are placed in water in an inescapable


environment, they adopt a characteristic immobile pos-ture after an initial period of vigorous activity and makeonly the minimal movements necessary to stay afloat. Areduction in the time of immobility is considered indicativeof potential antidepressant-like properties, and such aneffect was seen in a dose-dependent manner after admin-istration of TAAR1 partial agonists (Revel et al., 2012b,2013). DRL is a schedule-controlled behavioral test thatinvolves prefrontal cortex and hippocampus activity, inwhich subjects are reinforced to withhold a response over aspecified unsignaled delay interval. Antidepressants froma number of different pharmacological classes, includingselective serotonin reuptake inhibitors, tricyclic antide-pressants, and MAO inhibitors, are active in the DRLschedule by increasing the number of reinforcers obtainedand decreasing intertrial response rates (McGuire andSeiden, 1980; O’Donnell and Seiden, 1983). Consistentwith potential antidepressant activity, TAAR1 agonistssignificantly increased the number of reinforcers obtained(Revel et al., 2012b, 2013). Together, the above studiessuggest that TAAR1 agonists warrant further investiga-tion of their putative antidepressant-like properties. Incombination with the antipsychotic activity profile,these results suggest TAAR1 agonists may also beuseful in bipolar disorder, being able to address bothmanic phases and acute depressive episodes.iv. Sleep, Wake, and Narcolepsy. Although the above

studies have indicated the potential utility of TAAR1agonists and partial agonists, a different spectrum is seenwhen examining sleep. Here, only partial agonists showeffects (Revel et al., 2012b, 2013; Table 6), which isthought to possibly reflect antagonism of endogenousTAAR1 tone. Unfortunately, the lack of suitable TAAR1antagonists prevents the most direct testing of thishypothesis, and the putative benefit of antagonists hasto be inferred from the combinatorial effects of agonists inthe presence of partial agonists or from the effects ofTAAR1-KO where secondary compensatory effects couldalso be involved. TAAR1partial agonists have been shownto cause a generalized increase in wakefulness, decreasedlatency to sleep, and both decreased and lighter NREMsleep in both rats and mice (Revel et al., 2012b, 2013;Black et al., 2017; Schwartz et al., 2017; Table 6).Furthermore, manipulation of TAAR1 levels througheither OE or KO also alters sleep architecture andelectroencephalogram spectral activity, consistent withputative TAAR1-mediated changes in cortical neuronactivity (Schwartz et al., 2017). Such effects were not,however, associated with the hyperactive phenotypeseen with general central nervous system stimulants(Schwartz et al., 2017). In fact, lack of TAAR1 attenuatesthe locomotor activation and electroencephalogram spec-tral changes induced by two unrelated central nervoussystemstimulants, caffeine andmodafinil, neither ofwhichis a TAAR1 ligand (Schwartz et al., 2018). TAAR1-OEmeanwhile produced opposite effects, exacerbating theinduced hyperactivity.

That TAAR1-mediated changes in sleep architecturehave clinical relevance is strengthened by the subse-quent demonstration that TAAR1 partial and fullagonists are effective in two mouse models of narco-lepsy, both involving hypocretin neuronal degeneration(Black et al., 2017). Again, effects appeared to bedependent on the strength of agonism, suggesting thata centrally active, TAAR1 antagonist with appropriatepharmacokinetic properties might be the optimal treat-ment. In the two models used (Atax and DTA mice),beneficial effects of a TAAR1 partial agonist wereobserved in decreasing the cataplexy that is the centralcharacteristic symptom seen in narcolepsy. In total,there is now good evidence for a role of TAAR1 inregulating sleep architecture, and a putative therapeu-tic utility for partial TAAR1 agonists and TAAR1antagonists in sleep-related disorders such as narco-lepsy. It is expected that this will reinvigorate thesearch for TAAR1 antagonists that show suitablepharmacokinetic properties for use in vivo, the identi-fication of which would also be of considerable benefit tothe further pharmacological probing of TAAR1.

v. Addiction and Compulsive Behaviors. There isnow considerable evidence that TAAR1 is a new targetfor the pharmacotherapy of addiction disorders (Tables6 and 8). Intriguingly, this does not appear to be limitedto drugs of abuse that directly interact with TAAR1;rather, this is a general phenomenon for any agent thatmediates its effects through the dopaminergic rewardsystem, and it likely has its cellular andmolecular basisin the prevention of dopaminergic hyperactivity de-scribed elsewhere (see sections IV.B.1.d.iii, IV.B.1.d.iv,and IV.B.2.a.i). Indeed, TAAR1 regulation of cocaine-mediated effects was very recently shown to be afunction of the ability of TAAR1 to form heterodimerswith D2R, and of those heterodimers to recruit theb-arrestin 2 pathway (Asif-Malik et al., 2017). Ingeneral, TAAR1 agonism appears to prevent the re-warding, pleasurable effects of compounds thatmediatethese responses via stimulation of the dopaminergicsystem; as such, TAAR1 agonists themselves do notappear to support self-administration (Cotter et al.,2015; Jing and Li, 2015; Pei et al., 2016; Table 6). Thedopamine reward system has been reported to be atarget of SEP-363856 (a mixed 5-HT1a/TAAR1 agonist)in healthy human volunteers (Nazimek et al., 2016).

Given their demonstrated efficacy at TAAR1, much ofthe initial work focused on amphetamine-type drugs ofabuse. In animals with decreased TAAR1 levels eitherdue to KO (Di Cara et al., 2011; Achat-Mendes et al.,2012; Sukhanov et al., 2016) or an endogenous defunc-tionalizing mutation (Harkness et al., 2015; Reed et al.,2018), increases in acquisition and retention of condi-tioned place preference (CPP) (Achat-Mendes et al.,2012), hyperlocomotion (Achat-Mendes et al., 2012;Sukhanov et al., 2016), reinstatement (Sukhanovet al., 2016), self-administration (Harkness et al.,


2015; Reed et al., 2018), and toxicity (Miner et al., 2017),along with decreased autoinhibitory effects (Di Caraet al., 2011), were seen in response to either amphet-amine, methamphetamine, or 3,4-methylenedioxyme-thamphetamine (MDMA). Such effects suggestedthat TAAR1 agonists may be beneficial in reducingthe abuse potential of amphetamines and TAAR1agonists have now been confirmed to decrease thebehavioral sensitization, self-administration, rein-statement, drug-seeking behavior, and withdrawal-induced impulsivity observed in response to meth-amphetamine administration (Jing et al., 2014; Cotteret al., 2015; Pei et al., 2017; Xue et al., 2018). Consistentwith the TAAR1-mediated effects being due to aninteraction with D2R, the above-described effects areassociated with a decrease in nucleus accumbensdopamine overflow (Cotter et al., 2015; Pei et al., 2017).In addition to amphetamines, a number of other

addictive/abused agents are known to involve thedopaminergic reward systems, including cocaine, etha-nol, food, and nicotine. Intriguingly, TAAR1 agonistsappear to have a similar beneficial anticraving effect inall such instances (Tables 6 and 8). Responses to cocaine(which itself is not a ligand for TAAR1), as well as drug-taking behaviors induced by its administration, aremodified by TAAR1 agonists such that agonist-induceddecreases in cocaine CPP (Thorn et al., 2014a), both cue-and drug-primed reinstatement (Pei et al., 2014; Thornet al., 2014a), hyperactivity (Revel et al., 2011, 2013),reward memory (Liu et al., 2016), self-administration(Pei et al., 2015), sensitization (Thorn et al., 2014a,b),and withdrawal-induced drug seeking (Pei et al., 2014)have all been reported. Furthermore, TAAR1 activationcauses a downward shift in dose-response curves forcocaine reward efficacy, confirming lesser rewardingproperties. Again, effects appear to be focused onTAAR1 activation within the nucleus accumbens aswell as the VTA and prelimbic cortex (Liu et al., 2017b).A similar spectrum of beneficial effects after TAAR1

agonism has recently been reported for a secondaddictive compound that is not a TAAR1 ligand,nicotine (Tables 6 and 8). Administration of TAAR1agonists was able to decrease nicotine-induced hyper-activity (Liu et al., 2018; Sukhanov et al., 2018),sensitization (Liu et al., 2018; Sukhanov et al., 2018),self-administration (Liu et al., 2018), cue- and drug-primed reinstatement (Liu et al., 2018), and discrimi-native stimulus effects (Liu et al., 2018), while simul-taneously increasing the elasticity of the nicotinedemand curve (Liu et al., 2018). These beneficial effectsof TAAR1 activation were associated with a decrease inthe nicotine-induced dopamine release and c-fos expres-sion in the nucleus accumbens, confirming preventionof hyperactivity of the dopamine reward centers (Liuet al., 2018). Furthermore, direct infusion of a TAAR1agonist into the nucleus accumbens also preventeddrug-seeking behaviors, whereas TAAR1-KO enhanced

nicotine-seeking behaviors. Intriguingly, chronic nico-tine administration selectively decreased nucleusaccumbens TAAR1 expression (Liu et al., 2018). In thisway, chronic nicotine administrationmay be removing acellular brake on dopaminergic activity in the centralreward centers, thereby promoting its rewarding andaddicting properties.

With respect to other addictive agents, TAAR1 ago-nists also decrease compulsive binge eating of highlypalatable diets (Ferragud et al., 2017), whereas in-creased ethanol consumption and reward responsesare seen after TAAR1-KO (Lynch et al., 2013) or inanimals now known to contain a defunctionalizingTAAR1 mutation (Fish et al., 2010). Although there isa lack of effect of TAAR1-KO (Achat-Mendes et al., 2012)and TAAR1 agonists (Liu et al., 2017a) on morphine-induced CPP, morphine self-administration and cue-and drug-induced reinstatement are decreased byTAAR1 agonism (Liu et al., 2017a). Importantly, thesebeneficial effects of decreasing the reinforcing prop-erties of morphine are obtained without any changein morphine-induced analgesia (Liu et al., 2017a).In contrast, addictive agents that do not stronglyinvoke the dopamine reward pathways are not affectedby manipulation of TAAR1 activity. TAAR1 agonists donot affect the propensity of rodents to self-administersucrose (Revel et al., 2012b; Jing et al., 2014; Pei et al.,2014, 2017; Cotter et al., 2015). Likewise, TAAR1-KOalso does not affect sucrose self-administration (Lynchet al., 2013).

vi. Feeding Behavior. As described above, withinthe central nervous system TAAR1 is found in brainareas known to be associated with the regulation offeeding behavior, including the area postrema, cortex,hypothalamus, limbic system, and nucleus tractussolitarii (Table 4). This distribution, along with the easewith which TAAR1 ligands are produced from dietaryamino acids, makes TAAR1 an attractive putativemolecular target for involvement in the control of energymetabolism and nutrient intake. Furthermore, the welldocumented beneficial effects of TAAR1 agonism in reduc-ing addiction-associated compulsive behaviors mediatedby the dopaminergic reward system raise the possibilitythat TAAR1 agonists may also be beneficial in control-ling compulsive overeating, since food rewards are wellestablished to be dopaminergic and/or glutamatergic inorigin (Michaelides et al., 2012;Moore et al., 2018). Indeed,TAAR1 agonists have been reported to decrease foodintake in diet-induced obese mice (Raab et al., 2015),resulting in weight loss and improved insulin sensitivity(Table 6). Furthermore, aTAAR1agonist has recently beenshown to prevent rodent binge eating of highly palatablefood by preventing the conditioned rewarding properties ofthe food, preventing compulsive consumption behavior,and decreasing food-seeking behavior during a reinforce-ment paradigm (Ferragud et al., 2017; Table 6). Simi-lar to the situation seen with nicotine exposure, chronic


availability of palatable food resulted in a decrease inTAAR1 expression (Ferragud et al., 2017), again providinga link between the development of an addiction phenotypeand TAAR1. In contrast, standard chow overeating afterfood restriction was not affected. Together, the resultssuggest that TAAR1may uniquely act to normalize centraldrives underlying overeating of palatable food while alsonormalizing hormonal disruptions in the periphery associ-ated with metabolic disorders (see section IV.B.3.a).

3. Effects in the Periphery

a. Diabetes and Obesity. TAAR1 is expressed in themain tissues of the gut-brain axis in addition to specificareas of the brain involved in the control of energymetabolism (Lindemann et al., 2008; Table 4). In bothrodents and humans, the highest peripheral expressionof TAAR1 is seen in the stomach, neuroendocrine cells ofthe intestine, and b cells of the pancreas (Regard et al.,2007; Revel et al., 2013; Raab et al., 2015). In contrast toearlier studies (Borowsky et al., 2001; Fehler et al.,2010; Chiellini et al., 2012; Gozal et al., 2014), noexpression of TAAR1 was observed in the liver, kidney,or skeletal muscle when higher-quality, fully validatedreagents were used (Raab et al., 2015). This expressionpattern of TAAR1 is quite specific and partially overlapswith that of the glucagon-like peptide 1 (GLP-1) recep-tor, consistent with the colocalization of TAAR1 withGLP-1 and PYY in the intestines (Raab et al., 2015).GLP-1 analogs represent a recently developed class of

novel type 2 diabetes mellitus drugs, which improveglucose homeostasis by stimulating insulin secretionfrom pancreatic b cells and inhibiting gastrointestinalmotility and secretion (Baggio and Drucker, 2007).Combined with the at least partially overlapping ex-pression pattern of TAAR1 and GLP-1, this promptedinvestigation into the role of TAAR1 as a potential noveltherapeutic target for type 2 diabetes mellitus. Usingselective small-molecule TAAR1 agonists, in vitro stud-ies demonstrated that activation of TAAR1 results inincreases in glucose-stimulated insulin secretion frompancreatic b-cell lines and isolated human islets (Raabet al., 2015). Importantly, the effect on insulin secretionwas only seen at elevated glucose concentrations,thereby reducing the risk for induction of hypoglycemia.Very recently, signal transduction–altering single nu-cleotide polymorphisms of TAAR1 were reported insome patients with impaired insulin secretion and weresuggested to be a possible predisposing factor fordysfunctional glucose homeostasis (Mühlhaus et al.,2017). The potential utility of TAAR1 agonists wasfurther supported by studies in rodent models of type2 diabetes mellitus (Table 6). Here, TAAR1 agonistswere shown to reduce fasting blood glucose levels andimprove glucose tolerance acutely after either oral orintraperitoneal glucose challenge (Raab et al., 2015). Noeffect of TAAR1 agonists on glucose control was

observed in TAAR1-KOmice, verifying that effects wereindeed TAAR1-mediated.

TAAR1 agonism may also be useful in promotingweight loss in obese individuals. Not only do agonistsprevent the pronounced weight gain associated witholanzapine treatment (Revel et al., 2013), but they alsobring about a significant loss of excess body weight indiet-induced obese mice (Raab et al., 2015). Mechanis-tically, this likely relates to the demonstration thatTAAR1 activation induces a delay in gastric emptyingand decreased food intake (Raab et al., 2015). Further-more, TAAR1 regulates nutrient-induced hormone se-cretion seen as increases in plasma levels of GLP-1 andPYY (Raab et al., 2015) and somatostatin release fromstomach D cells (Adriaenssens et al., 2015).

b. Immunomodulatory Effects. Not only is TAAR1differentially expressed between leukocyte populations(Babusyte et al., 2013), but its expression, along withthat of TAAR2, is increased at both mRNA and proteinlevels after leukocyte activation (Nelson et al., 2007;Wasik et al., 2012; Table 4). As previously noted, PEAand TYR may be released from activated platelets(D’Andrea et al., 2003) and have been reported to bepositive chemotactic agents for leukocytes (Babusyteet al., 2013). The joint regulation of TAAR1 and TAAR2is important from this aspect, as the chemoattractantresponse of leukocytes toward TAAR1-selective agonistsappears to be dependent on the presence of both TAAR1and TAAR2, possibly due to the need for heterodimeriza-tion of the two (Babusyte et al., 2013). From this perspec-tive, the TAAR1/TAAR2 axis may provide a molecularexplanation for the well known immune dysfunction thatis associated with amphetamine-like drugs of abuse(Boyle and Connor, 2010; Sriram et al., 2015), many ofwhich are TAAR1 agonists. Indeed, methamphetaminehas been reported to increaseT-cell TAAR1 expression, aneffect that resulted in a decrease in T-cell interleukin2 levels (Sriram et al., 2016). In addition to thesedecreases, TAAR1 agonists have also been reported toaffect T helper cell differentiation, decreasing the levels ofsecreted phosphoprotein 1 while simultaneously increas-ing interleukin 4 secretion (Babusyte et al., 2013). To-gether, the effects on these cytokines suggest that TAAR1activation may preferentially promote differentiation intothe T helper 2 phenotype, an effect that would be expectedto lead to B-cell activation. Consistent with this, PEA andTYR have been shown to increase immunoglobulin Esecretion from B cells (Babusyte et al., 2013).

Although many of the above effects require indepen-dent verification, in combination with TAAR1 ligandready availability from various environmental sources,they raise the possibility of TAAR1, or indeed otherTAARs, being novel therapeutic targets for environ-mental hyper-reactivity disorders of the immune sys-tem. Consistent with this, TAAR1 has been proposed toplay a role in the susceptibility to fibromyalgia (Smithet al., 2012), TAAR6 implicated in treatment outcomes


in asthma (Chang et al., 2015), and TAAR2, TAAR5, andTAAR9 among the most highly upregulated genes in theinflamed zones of patients with Crohn disease (Taquetet al., 2012), an effect that allowed discrimination of thesepatients from those with irritable bowel syndrome. Aspreviously indicated, two recent unbiased metabolomicstudies have also identified elevated endogenous TAAR1ligands inCrohndisease fecal samples (Jacobs et al., 2016;Santoru et al., 2017). Together, these studies indicate thata careful, systematic investigation of the ability of TAARisoforms to regulate the immune system is warranted.c. Cancer. A recent study reported that an increase in

TAAR1 expression in breast cancer cells is associatedwithlonger survival times for patients (Vattai et al., 2017).TAAR1 expression was further suggested to correlatewith that of the human epidermal growth factor receptorHer2 and to be related to tumor cell differentiation.Unfortunately, no details were provided on the TAAR1antibody used; given the generally poor quality andselectivity of commercial anti-TAAR1 antibodies (Berryet al., 2017), although this study is of interest it should beregarded with considerable caution until such time thatreplication has occurred with fully validated reagents.d. Pregnancy. Very recently, TAAR1 has been

suggested as a novel target for the treatment of miscar-riages. An increased expression of TAAR1 was seen insyncytiotrophoblasts, cytotrophoblasts, decidua, and glandsof placentas obtained from patients suffering spontaneousor recurrent miscarriages (Stavrou et al., 2018). Further-more, this increased expression was associated withelevated levels of phosphorylated GSK3b, suggestingelevated b-arrestin 2 signaling. Whether this requiresheterodimerization of TAAR1, and if so with what, is anarea for further investigation. The authors also providedevidence of TAAR1 agonist-induced increased expressionof TAAR1 (Stavrou et al., 2018) suggestive of a positivefeedback loop. This is not too dissimilar to the reportedelevations of TAAR1 expression in T leukocytes aftertreatment with methamphetamine (Sriram et al., 2016),itself a TAAR1 agonist. Agonist-induced expressionwouldadd a new level of complexity to TAAR1 pharmacologyand may be an area for future systematic study.Although the antibodies used to identify TAAR1 have

previously been reported to be problematic with respect totheir selectivity (Berry et al., 2017), these studies do suggesta newarea for future studies. Intriguingly, the placenta andumbilical cord are well known to be abundant sources ofSSAO/VAP-1/AOC3 (Sikkema et al., 2002), an enzyme thatincludes TAAR1 ligands in its substrate profile, which inlight of these new studies may be indicative of a need totightly control the endogenous TAAR1 agonist levels.

C. Other Tetrapod Trace Amine–Associated Receptors

TAARs are found in all vertebrate species examined:mammalia (including marsupiala), aves, amphibia, reptilia,

osteichthyes (bony fish; teleost), and chondrichthyes (carti-laginous fish) (Fig. 6; Table 5) (Gloriam et al., 2005;Lindemann et al., 2005; Hashiguchi and Nishida, 2007;Grus and Zhang, 2008; Mueller et al., 2008; Eyun et al.,2016). TAAR (or TAAR-like) family members are alsoreported to be present in petromyzontida (jawless fish;lamprey) (Hashiguchi and Nishida, 2007; Eyun et al.,2016), and they likely represent the evolutionary origin ofthe family, although this has been debated (Hussain et al.,2009; Tessarolo et al., 2014). At least in mammals andchickens, TAAR genes are clustered on a single chromo-some, whereas teleost and amphibian TAARs are spreadacross multiple chromosomes/scaffolds (Lindemannet al., 2005; Hashiguchi and Nishida, 2007; Eyun et al.,2016).

In addition to TAAR1, five other functional TAARisoforms are expressed in humans, with single variantsof TAAR2, TAAR5, TAAR6, TAAR8, and TAAR9 pre-sent, all of which contain seven putative transmem-brane domains, as expected for GPCRs (Lee et al., 2000).The individual genes for TAAR3, TAAR4, and TAAR7have undergone defunctionalizing, pseudogenizationevents (Lindemann et al., 2005). Comparison of thehuman TAAR complement to those of non–humanprimates suggests that the pseudogenization of TAAR3likely occurred before the divergence of humans andorangutans, with TAAR4 pseudogenization occurringbetween humans and gorillas (Stäubert et al., 2010). Bycomparison, the marmoset genome was reported tocontain only two functional TAAR genes (TAAR1 andTAAR5) (Eyun et al., 2016), although a functionalmarmoset TAAR2 gene has been reported elsewhere(Vallender et al., 2010), whereas the chimpanzee ge-nome contains three functional genes (TAAR1, TAAR5,and TAAR6) and six pseudogenes (Lindemann andHoener, 2005; Eyun et al., 2016).

1. Trace Amine–Associated Receptor 2

Whereas all other TAAR genes have a single exon,TAAR2 (previously known as GPR58 or G protein–coupled receptor 58; Table 3) contains two exons,encoding a functional protein of 351 amino acids inhumans (Lindemann et al., 2005). TAAR2 shares closesthomology with TAAR5 (previously known as the puta-tive neurotransmitter receptor, PNR; Table 3) at 42%identity, and with the 5-HT4 serotonin receptor at 34%identity (Lindemann et al., 2005). Ligands for TAAR2have not been identified thus far, either among knownendogenous trace amines or other volatile amines.Evolutionary mapping, however, suggests that the re-ceptor will be tuned toward activation by primaryamines (Ferrero et al., 2012). Like all TAARs, with theexception of TAAR1, TAAR2 is found within the olfac-tory epitheliumwhere it is coupled to Golf stimulation ofcAMP accumulation (Liberles and Buck, 2006). The roleof TAAR2 and other TAARs in olfactory sensory func-tion is discussed in detail in section IV.E.


Beyond the olfactory system, TAAR2 mRNA is foundin various leukocyte populations in humans and mice,including B cells, granulocytes, monocytes, naturalkiller cells, and T cells, a pattern that mirrors theexpression of TAAR1 (Nelson et al., 2007; Babusyteet al., 2013; Table 4). The presence of TAAR2 at theprotein level has been confirmed in granulocytes(Babusyte et al., 2013), although it should be noted thatvalidation of the selectivity of the antibody used was notprovided. It has been noted previously that leukocytestimulation results in an increase in both TAAR1 andTAAR2 mRNA levels (Nelson et al., 2007). Further-more, it has been shown that PEA and TYR can bereleased from activated platelets (D’Andrea et al., 2003).Babusyte et al. (2013) also showed that these TAAR1ligands, as well as 3IT, can act as chemoattractants forneutrophils, with subnanomolar EC50 values. Such achemoattractant action requires the presence of bothTAAR1 and TAAR2, as evidenced by the selective smallinterfering RNA knockdown of individual receptors elim-inating the chemotactic responses (Babusyte et al., 2013).Since neither PEA nor TYR is an agonist at TAAR2, aheterodimerization betweenTAAR1andTAAR2 to initiateneutrophil migration has been hypothesized (Babusyteet al., 2013). Indeed, direct coimmunoprecipitation exper-iments indicated that such a heterodimerization is in-volved in the regulation of chemotactic responses to PEA(Babusyte et al., 2013). Taken together, the above studiessuggest that TAAR2 is required for the expression ofTAAR1-mediated recruitment of leukocytes to the sites ofinjury, and that this involves heterodimerization withTAAR1.TAAR2 mRNA transcripts have also been reported in

nonsensory cells in other mammalian species, in duode-nal mucosal cells of the gastrointestinal system in mice(Ito et al., 2009), and in the rat heart and testis (Chielliniet al., 2012). Whether a similar pattern of expression isobserved in human tissues requires further study. In astudy with a small sample size, defunctionalizing singlenucleotide polymorphisms of TAAR2 were reported inup to 10% of humans, with a further minor increase inpatients with schizophrenia (Bly, 2005). These studieshave not been replicated thus far and their clinicalrelevance remains unclear, especially given the lack ofreports of TAAR2 presence in the brain.Recent attempts have been made to apply structure-

based in silico computational protocols to the developmentof TAAR2 homology models for the prediction of TAAR2ligands (Cichero and Tonelli, 2017). With a consensusTAAR defining motif now identified, further developmentof such models is expected to provide new leads and theidentification of TAAR2-selective ligands, which will be amajor boost to elucidating its physiologic roles.


TAAR3 (previously known as GPR57 or G protein–coupled receptor 57; Table 3) is a pseudogene in humans

(Lindemann et al., 2005), although a functional proteinwith defined physiologic roles in olfaction is encoded inother species (discussed in detail in section IV.E.2).


TAAR4 (previously known as trace amine receptor2 or TA2, 5-HT4P; Table 3) is also a pseudogene inhumans (Lindemann et al., 2005) but encodes a func-tional protein in other species, where an overlappingligand selectivity with TAAR1 is present (discussed insection IV.E.3).


The TAAR5 gene encodes a functional protein of337 amino acids in humans. The most prominentexpression of TAAR5 is in the olfactory epithelium,where it plays a role in the detection of socially relevantodor cues (Li et al., 2013; Wallrabenstein et al., 2013;Zhang et al., 2013) (discussed in detail in section IV.E.4). In the olfactory system, TAAR5 appears to becoexpressed with Golf and stimulates cAMP accumula-tion (Liberles and Buck, 2006). It has also been shownthat human TAAR5 can couple to the Gs cascade(Wallrabenstein et al., 2013), the Gq/11 cascade (Dinteret al., 2015c), and G12/13-dependent mitogen-activatedprotein kinase pathways (Dinter et al., 2015c), suggestingthat in different cell groups TAAR5 might demonstratefunctional selectivity by coupling to different signalingmodalities. Whether such an effect is due to receptorheterodimerization, as seen with other TAAR isoforms,with different partners present in different cell types, ordue to ligand bias, requires systematic study.

Beyond the olfactory system, low levels ofTAAR5mRNAhave been reported in various leukocyte populations, withthe greatest expression seen in B cells (Babusyte et al.,2013). Others, however, have reported that TAAR5 is onlyfound in mouse, and not in human, leukocytes (Nelsonet al., 2007). Expression of TAAR5 mRNA has also beenreported in several mouse brain regions such as theamygdala, arcuate nucleus, and ventromedial hypothala-mus, with an overlapping localization of TAAR1 andTAAR5 in the amygdala and ventromedial hypothalamus(Dinter et al., 2015c; Table 4). Spinal cord (Gozal et al.,2014), testis (Chiellini et al., 2012), and intestinal (Kuboet al., 2015) expression has also been reported in rats,although it should benoted that, at leastwithTAAR1, suchexpression patterns could not be validated with higher-quality reagents. Whether similar patterns of TAAR5expression occur in humans requires further detailedstudies.

As previously described, TAAR5 is predicted to betuned to activation by tertiary amines (Ferrero et al.,2012), and trimethylamine has consistently been foundto be the most active and selective agonist at humanTAAR5, with dimethylethylamine a less potent partialagonist (Liberles and Buck, 2006; Wallrabenstein et al.,2013; Zhang et al., 2013). One group, however, found


activity of trimethylamine only at rodent, and nothuman, TAAR5 (Stäubert et al., 2010). Several syn-thetic ligands and one additional natural ligand forTAAR5 have also been identified. Interestingly, theputative thyroid hormone metabolite and TAAR1 ago-nist 3IT has been reported to be a TAAR5 inverseagonist (Dinter et al., 2015c). The activity of 3IT,trimethylamine, and dimethylethylamine at TAAR5has been confirmed by recent docking studies usinghomology models (Cichero et al., 2016). Furthermore,these studies identified two TAAR5 antagonists (Fig. 7),each of which was active at micromolar concentrations.The synthetic chemical 1-(2,2,6-trimethylcyclohexyl)-hexan-3-ol (Timberol; Symrise AG, Holzminden,Germany) (Fig. 7) has also been identified as anantagonist of TAAR5 (Wallrabenstein et al., 2015).Although none of these antagonists have to date beentested in animals to better evaluate TAAR5 functional-ity, this is a considerable advance given the lack of good-quality antagonists at all other TAAR isoforms, makingthis is an exciting area for future development. Asynthetic TAAR5-selective agonist, 2-(a-naphthoyl)-ethyltrimethylammonium iodide (alpha-NETA) (Fig. 7),has also recently been described (Aleksandrov et al.,2018a; Aleksandrov et al., 2018b). Using the brainevent-related potentials in the paired-click paradigm,a model that directly recapitulates sensory gatingdeficits seen in schizophrenia, alpha-NETA was shownto decrease sensory gating in rats (Aleksandrov et al.,2018a), suggesting that TAAR5 may be a novel molec-ular locus for sensory gating deficits seen in schizophre-nia. Furthermore, alpha-NETA affected mismatchnegativity-like response in rats, a cognitive paradigmknown to be reflective of schizophrenia-related cognitivedeficits described in experimental animal models andhumans (Aleksandrov et al., 2018b). It is alsoworthnotingthat a protocol for the generation of large (milligram)quantities of human TAAR5 has been described (Wanget al., 2011), providing an additional approach to furtherunderstanding TAAR5 structure and functions.


The TAAR6 gene (previously known as TRAR4, TA4, ortrace amine receptor 4; Table 3) encodes a functionalhuman protein of 345 amino acids. Similar to TAAR2, noligands for TAAR6have been found thus far, although it ispredicted to be activated by tertiary amines (Ferrero et al.,2012) and/or diamines (Li et al., 2015). The signal trans-duction events occurring at TAAR6 have also not beeninvestigated but it is expected that like other olfactoryTAARs, TAAR6 will be coupled to Golf and subsequentcAMPaccumulation.Beyond the olfactory system,TAAR6mRNA has been found in mouse duodenal mucosal cells(Ito et al., 2009), in the rat spinal cord (Gozal et al., 2014)but not in the human spinal cord (Duan et al., 2004), andin the rat testis (Chiellini et al., 2012) (Table 4). TAAR6transcripts have been reported in several human brain

regions, including the amygdala and hippocampus(Borowsky et al., 2001; Duan et al., 2004), basal ganglia,and frontal cortex and substantia nigra (Duan et al.,2004), and these levels may exceed those of the mostthoroughly investigated member of the TAAR family,TAAR1 (Duan et al., 2004). In the periphery, TAAR6mRNA has been reported to be present in the humankidney (Borowsky et al., 2001) and in various humanleukocyte populations (D’Andrea et al., 2003; Babusyteet al., 2013). In all cases, the functional consequences ofTAAR6 activation are unknown. The flying fox TAARgene family expansion (26 genes and 10 pseudogenes) is inpart due to an expansion of its TAAR6 (four functional, sixpseudogenes) complement (Eyun et al., 2016).

Despite minimal knowledge on the biology and physio-logic role(s) of TAAR6, several studies have identifiedsingle nucleotide polymorphisms (Duan et al., 2004;Chang et al., 2015) and other genetic variants (Pae et al.,2010) in patients with schizophrenia (Duan et al., 2004;Pae et al., 2008a) and affective disorders (Abou Jamraet al., 2005; Pae et al., 2008b, 2010). Particularly interest-ing are the investigations of the potential role of TAAR6single nucleotide polymorphisms in schizophrenia etiologyand treatment, even though results of these studiesare somewhat conflicting. Although several studies in-dicated a link in patients of Korean, European, and Afro-American origin (Duan et al., 2004; Vladimirov et al., 2007;Pae et al., 2008a,b), later studies failed to confirm associ-ations in Japanese, Chinese, and European populations(Ikeda et al., 2005; Duan et al., 2006; Ludewick et al.,2008; Sanders et al., 2008; Vladimirov et al., 2009).TAAR6 polymorphisms have also been reported to beconnected with therapeutic responses to the antipsychoticaripiprazole (Serretti et al., 2009), whereas amore complexepistatic relationship between TAAR6 and heat shockprotein 70 polymorphisms has been associated with boththe development of schizophrenia and treatment outcomes(Pae et al., 2009). TAAR6 variants have also beensuggested to influence asthma patient responsivity tocorticosterone (Chang et al., 2015).


TAAR7 is a pseudogene in humans (Lindemann et al.,2005) but isamajor site ofmammalian species variation inother species. The increased TAAR repertoire in standardlaboratory rodents—the mouse genome contains 15 func-tional receptors and one pseudogene, whereas the ratgenome contains 17 functional TAAR genes and twopseudogenes (Borowsky et al., 2001; Lindemann et al.,2005; Lindemann and Hoener, 2005; Eyun et al., 2016)—is primarily due to an expansion of the TAAR7 subfamily.Likewise, the flying fox possesses a pronounced TAAR7expansion with 16 functional variants (Eyun et al., 2016).


The TAAR8 gene (previously known as GPR102,TRAR5, TAR5, TA5, or trace amine receptor 5; Table 3)


encodes a functional protein of 342 amino acids inhumans. At present, only tertiary volatile amine ligands,N-methylpiperidine and N,N-dimethylcyclohexylamine,for nonhuman TAAR8 isoforms have been identified(Ferrero et al., 2012; Li et al., 2015). TAAR8, includingthe human isoform, does contain a putative diaminebinding domain (Li et al., 2015), suggesting the existenceof diamine ligands as well and this is described in moredetail in IV.E. Trace Amine–Associated Receptors inOlfaction.Like other olfactory TAARs, TAAR8 is coupled to Golf,

stimulating cAMP accumulation (Li et al., 2015), buthas also been reported to be coupled to Gi (Mühlhauset al., 2014). Beyond the olfactory system, TAAR8mRNA has been reported in the human amygdala(Borowsky et al., 2001) and leukocytes (D’Andrea

et al., 2003; Babusyte et al., 2013; Table 4). Multipleisoforms of TAAR8 were found in various tissues fromother species, including the rat cortex and cerebellum(Chiellini et al., 2012) and spinal cord (Gozal et al.,2014). In astroglia, expression was increased by lipo-polysaccharide activation (D’Andrea et al., 2012). Low-level expression of the TAAR8b isoform was reported invarious mouse brain regions (Mühlhaus et al., 2014),leukocytes (Nelson et al., 2007), kidney (Borowsky et al.,2001; Chiellini et al., 2012), heart, intestines, lung,skeletal muscle, spleen, stomach, and testis (Chielliniet al., 2012).


The TAAR9 gene (previously known as TRAR3, TAR3,or trace amine receptor 3; Table 3) encodes a functionalhumanprotein of 348 amino acids. TAAR9 is thought to betuned toward activation by tertiary amines, and like thecase of TAAR8c, rat TAAR9 can be activated by N-methylpiperidine and N,N-dimethylcyclohexylamine, al-though only at micromolar concentrations (Liberles andBuck, 2006; Ferrero et al., 2012). It is notable that ratTAAR9 can also be activated by a currently unknowncomponent(s) of both carnivore and noncarnivore urine(Ferrero et al., 2011), and this provides a good startingpoint for identifying putative endogenous ligands. Aswithother olfactory TAARs, TAAR9 is coupled toGolf-mediatedcAMP accumulation (Ferrero et al., 2011). Beyond its rolein the mammalian olfactory system, TAAR9 mRNA hasbeen found in the full range of human leukocytes(D’Andrea et al., 2003; Babusyte et al., 2013), in additionto the pituitary gland and skeletal muscle (Vanti et al.,2003) (Table 4). TAAR9 mRNA has also been reported tobe present in the mouse gastrointestinal tract, where it ispreferentially localized to duodenal mucosal cells (Itoet al., 2009), as well as the spleen (Regard et al., 2008)and spinal cord (Gozal et al., 2014). A loss-of-functionmutation in the TAAR9 gene with unknown clinicalrelevance has been reported to be present in up to 20%of the human population (Vanti et al., 2003; Müller et al.,2010).

D. Trace Amine–Associated Receptors in Teleostand Other Fish

Like in mammals, the number of functional TAARgenes varies significantly among teleost fish but isgenerally higher than in tetrapod genomes, rangingfrom approximately 18 in fugu to 112 in zebrafish(Hussain et al., 2009), with only minor differencesgenerally observed between independent studies ofindividual species (Fig. 6; Table 5). Although somezebrafish TAARs (27 of 112), like all human TAARs(six of six) and most mouse TAARs (13 of 15), share theability with other biogenic amine receptors to recognizemonoamines via a specific, conserved binding site, themajority of zebrafish TAARs (85 of 112) evolved to a newFig. 7. Recently described synthetic TAAR5 ligands.


way to recognize amines, losing the classic monoaminebinding pocket and acquiring an inverted bindingpocket, thus giving a rise to a distinct, third clade ofTAARs that is unique to teleosts and Xenopus (Hussainet al., 2009, 2013; Li et al., 2015). In total, teleost TAARsspan at least three separate phylogenetic groups, two ofwhich have not been fully characterized (Hashiguchiand Nishida, 2007; Eyun et al., 2016). By comparison,cartilaginous fish (e.g., elephant shark) are evolution-arily the earliest representatives of jawed vertebratesand have two TAAR genes, both of which maintain theTAAR signature motif (Eyun et al., 2016). Sea lampreymay be the evolutionary origin of TAARs and have beenreported to contain 25 TAAR-like proteins that form aseparate family (Hashiguchi and Nishida, 2007; Eyunet al., 2016). Whether these lamprey receptors areindeed true TAAR family members has been debated(Hussain et al., 2009; Tessarolo et al., 2014) largely dueto the TAAR signature motif that is well represented inthe TAAR1–TAAR9 family being only weakly conservedin the sea lamprey TAAR-like genes.Ligands for 12 teleost TAARs have been identified

(Table 2), including members of the classic amine-sensingclade I receptors (TAAR10a, TAAR10b, TAAR12h, andTAAR12i) and the clade III representatives TAAR16c,TAAR16e, and TAAR16f, as well as TAAR13a, TAAR13c,TAAR13d, TAAR13e, and TAAR14d, which contain bothclassic and inverted binding pockets that combine to createa potential diamine binding site (Hussain et al., 2013; Liet al., 2015; Gao et al., 2017).

E. Trace Amine–Associated Receptorsin Olfaction

Although TAARs are only distantly related to biogenicamine receptors and they also have no phylogeneticrelationship with classic chemosensory receptors, it ap-pears that all subfamilies of TAARs, with the exception ofTAAR1, serve chemosensory functions in detecting sociallyor ecologically relevant olfactory cues (Liberles and Buck,2006; Hussain et al., 2009; Horowitz et al., 2014; Liberles,2015). Indeed, mammalian TAAR2–TAAR9 subfamilies,as well as teleost-specific TAARs, have distinct expressionpatterns in the olfactory system, being present in theolfactory epithelium, and neonatal Grueneberg ganglionbut not in the vomeronasal organ (Liberles andBuck, 2006;Fleischer et al., 2007; Hussain et al., 2009). Like olfactoryreceptors, each TAAR is expressed in sparsely distrib-uted sensory neurons (,0.1% of the total), with aspecific spatial distribution (Liberles and Buck, 2006).Neurons expressing a functional TAAR do not expressother TAARs or olfactory receptors, whereas neuronsexpressing a TAAR pseudogene may express a secondTAAR (Liberles and Buck, 2006; Johnson et al., 2012;Pacifico et al., 2012). Expressed TAAR proteins arefound in the olfactory cilia, the site of odor detection, andaxons project to distinct glomeruli within the dorsomedial

domain of the olfactory bulb (Pacifico et al., 2012; Dieriset al., 2017), although this may be developmentallyregulated in some species (Gliem et al., 2009; Shaoet al., 2017). Like classic olfactory receptor neurons, TAARneurons express Golf and related signaling proteins, andinhibition of adenylyl cyclase blocks the odor-relatedresponses of TAAR neurons (Liberles and Buck, 2006;Ferrero et al., 2012; Zhang et al., 2013). It has, however,been reported that innate behavioral responses to theTAAR4 ligand PEA are not attenuated after conditionalGolf knockdown (Pérez-Gómez et al., 2015), raising thepossibility of currently unknown coupling mechanismsin vivo.

In vitro screening for odorant TAAR ligands using cAMPreporter assays in HEK-293 cells transfected with individ-ual TAARs has identified a number of small, volatilemolecules that activate individual TAARs in severalspecies, with affinities comparable to those of knownligands for olfactory receptors (EC50 , 10 mM). Thus far,such agonists have been identified for one human, onemacaque, six mouse, three rat, and 12 zebrafish TAARs(Table 2 and Table 9) (Liberles and Buck, 2006; Ferreroet al., 2012; Wallrabenstein et al., 2013; Horowitz et al.,2014; Liberles, 2015; Saraiva et al., 2016). Inmany of thesecases, however, further investigation of receptor andspecies specificity, as well as the physiologic functionsmediated by receptor activation, is required to fullyvalidate the identified chemical cues as the true ligandsof individual TAAR (Liberles, 2015).

Notwithstanding the need for further validation, anemerging picture is developing of TAARs playing a role indetecting socially relevant odors from diverse ecologicalsources, in particular those originating from urine or themicrobial metabolism of decomposing flesh, resulting inthe induction of innate behavioral responses (Liberles,2015). As such, individual TAARs have been implicated inthe detection and avoidance of predators, avoidance (ormore rarely attraction) to rotting food sources, and eventhe detection of putative pheromones. For example, urinehas been shown to activate mouse TAAR4, TAAR5,TAAR7f, TAAR8c, and TAAR9 (Liberles and Buck, 2006;Ferrero et al., 2011; Li et al., 2013), although the specificcomponent responsible has not always been identified.Recent comprehensive screening for olfactory TAARligands and analysis of their attractive and avoidanceproperties in mice has revealed a complex organization ofTAAR-mediated innate behavioral effects. These innatebehavioral responses are not only both context andconcentration dependent, but they are also subject tomodification by other odorants (Saraiva et al., 2016).Thus, it is likely that TAAR-mediated hard-wired behav-ioral responses to socially relevant odorant cues arefurther modulated in brain circuitry due to interactionswith signals derived from other odorant receptors orenvironmental cues.

Two notes of caution are necessary, however, withrespect to the interpretation of olfaction-mediated


behaviors. First, in particular with respect to dosedependence, none of the studies in terrestrial verte-brates have measured the ligand concentration presentin the vapor phase, a major confound with respect totrying to compare the potency of effects across indepen-dent studies. Even with a defined liquid concentrationused, the vapor phase concentration can vary markedlyas a result of variations in the surface area of the liquidphase, temperature of the testing environment, andvolume of the testing chamber, as well as the nature ofany device used to prevent physical contact of experi-mental animals with the liquid phase. Although thisdoes not diminish the importance of the observedbehavioral changes, it does necessitate that caution beused in making dose-dependent comparisons acrossstudies. Second, studies have so far assumed that allresponses are downstream of olfactory epitheliumTAAR activation. At least some of the ligands (e.g.,PEA) have been shown to readily cross lipid bilayers(Berry et al., 2013), and individual TAARs are presentin peripheral cell populations (see previous sections). Assuch, it is possible that the physiologic responses tosome olfactory delivered trace amines may occur due toa combination of downstream central nervous systemsignaling after olfactory epithelium TAAR activation,and systemic responses after ligand entry in to thebloodstream after passage across pulmonarymembranes.The current state of knowledge of olfaction-mediated

responses of individual TAARs is presented in thefollowing sections.


Potent ligands for TAAR2 are yet to be identified,although several compounds that can activate thisreceptor at high concentrations were recently reported(Saraiva et al., 2016).


Isoamylamine, a biogenic amine produced by leucinedecarboxylation and known to be innately aversive tomice, was identified as an agonist of mouse TAAR3(Liberles and Buck, 2006; Liberles, 2015), a receptorthat is a pseudogene in humans (Lindemann andHoener, 2005). Isoamylamine has been proposed to actas a pheromone in mice, inducing puberty in females(Nishimura et al., 1989), although this has also beenquestioned (Price and Vandenbergh, 1992). Intrigu-ingly, strong class I major histocompatibility complex(MHC)–dependent female choice for genetically diverseand dissimilar males in the greater sac-winged bat(Saccopteryx bilineata) was recently correlated with thefemale TAAR3 genotype (Santos et al., 2016), indicatingthat TAARs and olfactory cues may be key mediators inmammalian MHC and immune system-based matechoice. The MHC-based ligand responsible for thisresponse is currently unknown.


TAAR4, which is a functional protein in many mam-malian species but not in humans, has an overlappingpharmacological profile with TAAR1, being potentlyactivated by the archetypal trace amine PEA (Liberlesand Buck, 2006; Dewan et al., 2013). PEA is a componentof urine, and analysis of 38 mammalian species indicatedsignificantly higher PEA concentrations in carnivoreurine, with some producing in excess of 1000-fold morethan herbivores (Dewan et al., 2013).Mice, as well as rats,are known to innately avoid a PEA odor source (Ferreroet al., 2011) and enzymatic depletion of PEA fromcarnivore urine reduced the repellant properties of theurine (Dewan et al., 2013). Furthermore, TAAR4-KOmiceshow no avoidance response to either PEA or carnivoreurine (Dewan et al., 2013). Thus, at least in mice, PEAactivation of TAAR4 underlies innate avoidance re-sponses to predator urine. Consistent with this, PEAolfactory exposure sufficient to induce an innate avoid-ance response has been reported to result in activation(as measured by increased c-fos expression) of theposteroventral region of the medial amygdala and thedorsomedial region of the ventromedial hypothalamus(Pérez-Gómez et al., 2015), brain areas implicated in fear,anxiety, panic, and defensive behaviors in rodents. Inter-estingly, PEA urinary levels have also been reported to beincreased in response to stress (Grimsby et al., 1997;Paulos and Tessel, 1982); as such, PEA may serve asboth a conspecific and heterospecific urinary warningcue. In tigers, PEA has also been proposed to serve apheromone function (Brahmachary and Dutta, 1979),although the TAAR4 expression profile of tigers iscurrently unknown.


Human, macaque, rat, and mouse TAAR5 are po-tently activated by trimethylamine (Liberles and Buck,2006; Horowitz et al., 2014), which is most oftenregarded as a product of choline and/or L-carnitinemetabolism in the body by the microflora of thegastrointestinal tract, oral cavity, and vagina, as de-scribed above (Fennema et al., 2016; Zhang and Davies,2016). TAAR5 is also activated by spoiled fish, likely dueto the presence of trimethylamine (Horowitz et al.,2014), and might serve as a mechanism for the innateavoidance of foods that could harbor pathogenic micro-organisms and thus pose a danger to health.

In mice, trimethylamine is a sexually dimorphic odorthat can evoke sex-specific, concentration-dependent be-haviors (Liberles and Buck, 2006; Li et al., 2013), and thishas led to suggestions that trimethylamine is a murinepheromone (Liberles, 2014). Trimethylamine is present inmale mouse urine from where it is detected by TAAR5(Liberles and Buck, 2006); enzymatic elimination of uri-nary trimethylamine disrupts behavioral responses tomale mouse urine (Li et al., 2013). Male mice have


particularlyhigh concentrations of trimethylamine inurine(up to 5mM;.1000-fold higher than in femalemice or rats)due to a genetic deficiency in FMO3 (Li et al., 2013;Liberles, 2015) preventing its metabolism. Whereas rats(like humans) find trimethylamine innately aversive, micedemonstrate attraction to lower (urinary) concentrations,with aversion occurring to higher levels (Li et al., 2013;Liberles, 2015). Mice lacking TAAR5 demonstrate noattraction to trimethylamine but still avoid high concen-trations (Li et al., 2013), suggesting that TAAR5 mediatesthe attractive responses due to high-affinity detection oftrimethylamine, whereas a second, low-affinity, unknownreceptor is responsible for the aversive behavior. Interest-ingly, specific anosmia for trimethylamine in humans was

reported to be unrelated to polymorphisms in the codingsequence of the TAAR5 gene (Wallrabenstein et al., 2013).

5. Trace Amine–Associated Receptors 7, 8, and 9

It has been shown that unknown components of carni-vore and noncarnivore urine can activate rat TAAR7f,TAAR8c, and TAAR9 (Ferrero et al., 2011). TAAR8c andTAAR9 canalso be activated byN-methylpiperidine andN,N-dimethylcyclohexylamine, respectively (Liberles andBuck, 2006; Ferrero et al., 2012).

6. Teleost Olfactory Responses

The archetypal diamine cadaverine is a major compo-nent of rotting flesh that potently activates zebrafishTAAR13c present in olfactory sensory neurons that

TABLE 9Functional activity (EC50) of endogenous and synthetic agonists of rat, mouse, human, and zebrafish olfactory TAARs

Mammalian TAAR Species Ligand EC50

mMTAAR3 Rat, mouse Cyclohexylamine 7 (mouse),b 20 (mouse)c

Isoamylamine 10 (mouse)b,c

Octylamine 50 (mouse)b

PEA 40 (mouse)b

TAAR4 Rat, mouse PEA 0.7 (mouse),b 1 (mouse)c

TAAR5 Human, rat, mouse Trimethylamine 0.7 (mouse),b 0.3 (mouse),d

1 (rat),d 0.12 (human)e

N,N-dimethylethylamine 1 (mouse),b 0.17 (human)e

N,N-dimethyloctylamine 1 (mouse)b

N-methylpiperidine 3 (mouse),b 20 (mouse),c

.10 (human)e

Pyrrolidine 17 (mouse)b

Triethylamine 22 (mouse)b

3-pyrroline 42 (mouse)b

2-methy-1-pyrroline 46 (mouse)b

3ITa 4.4 (human)f

TAAR7b Rat, mouse N,N-dimethyloctylamine 3 (mouse)b

N,N-dimethylbutylamine 30 (mouse)b

TAAR7e Rat, mouse Octylamine 28 (mouse)b

Heptylamine 34 (mouse)b


TAAR7d Rat, mouse N,N-dimethylcyclohexylamine 0.5 (mouse)b

N-methylpiperidine 16 (mouse)b

N,N-dimethylbutylamine 30 (mouse)b

1-(2-aminoethyl)piperidine 30 (mouse)b

Heptylamine 33 (mouse)b


TAAR8c Human, rat, mouse N-methylpiperidine 0.5 (rat)g

N,N-dimethylcyclohexylamine 13 (rat)g

TAAR9 Human, rat, mouse N-methylpiperidine 18 (rat)g

N,N-dimethylcyclohexylamine 27 (rat)g

Zebrafish TAARsTAAR10a Zebrafish Serotonin 0.5h

5-methoxytryptamine 20h

TAAR10b Zebrafish TRP 50h

TAAR12h Zebrafish b-phenyethylamine 0.3h

TAAR13a Zebrafish Histamine 20h

TAAR13c Zebrafish Cadaverine 20,i 10h

TAAR13d Zebrafish Putrescine 1h

TAAR16c Zebrafish N-methylpiperidine 10h

TAAR16e Zebrafish N,N-dimethylcyclohexylamine 30h

Only compounds with ,50 mM activity are listed.aInverse agonist.bSaraiva et al. (2016).cLiberles and Buck (2006).dLi et al. (2015).eWallrabenstein et al. (2013).fDinter et al. (2015c).gFerrero et al. (2012).hLi et al. (2015).iHussain et al. (2013).


project to dorsolateral glomeruli of the olfactory bulb,evoking innate avoidance behavior (Hussain et al., 2013;Dieris et al., 2017). Structure-activity analysis indicatedthat TAAR13c has a preference for medium-sized di-amines containing an odd number of carbons and containsa nonclassic monoamine recognition pocket with twodistinct cation recognition sites (Hussain et al., 2013).Intriguingly, cadaverine also induces avoidance responsesin mice, and this behavior is lost in mice with a clusterTAAR2-TAAR9-KO, suggesting that at least one of thesereceptors is responsible for the detection (Dewan et al.,2013). Interestingly, human TAAR6 and TAAR8, mouseTAAR6 and TAAR8b, and rat TAAR6 and TAAR8a alsohave TAAR13c-like diamine binding pockets and thus arelikely candidates for cadaverine-detectingTAARs (Li et al.,2015). Ecologically relevant ligands for a number of otherteleost TAARs have been identified (Table 2), although thephysiologic responses to these ligands of other teleostTAARs have not yet been investigated (Li et al., 2015). Aspreviously described, lamprey have also been reported toinnately avoid a PEA source (Imre et al., 2014), althoughwhether this is TAAR mediated is unknown.

V. Future Directions

A. Better Understanding of the Physiologic Role(s) ofTrace Amine–Associated Receptors and TheirEndogenous Ligands

1. Trace Amine–Associated Receptor 1. Althoughgreat advances have occurred in understanding thephysiologic roles of TAAR1 in the central nervous system,its functions in peripheral tissues remain largely un-known. Advances in these areas have begun, in particularin establishing TAAR1 as playing a role in the control ofnutrient-induced hormone secretion throughout the gas-trointestinal system, with particularly robust effectsdemonstrated in the pancreas. Unfortunately, furtherprogress has been hindered by a lack of suitable pharma-cological tools (particularly useable receptor antagonists).In addition, it is only in the last 2 years thatwell validatedantibodies have been described, one suitable for immuno-histochemistry of human tissue and one for Western blotof rat tissues. Although progress has been made throughthe use of transgenic and gene silencing technologies,again the development of missing high-quality pharma-cological tools such as receptor antagonists and validatedselective antibodies will be crucial to furthering theseendeavors and this was discussed more fully recently(Berry et al., 2017). Application ofmodern optogenetic andDREADD (designer receptors exclusively activated bydesigner drugs) technologies will also be of considerablebenefit to further understanding the homeostatic physio-logic properties of TAAR1 aswell as other TAAR isoforms.Outside of the central nervous system and tissues

controlling energy metabolism, the role of TAAR1 inmodulating the immune system is a notable area forfuture study. Although this area is currently not as well

developed as others, there is slowly accumulatingevidence that TAAR1 and its ligands exert immuno-modulatory effects. With endogenous TAAR1 ligandsappearing to readily cross cell membranes and TAAR1seemingly present in a variety of leukocyte populationswhere it may regulate cytokine and immunoglobulinsecretion, a particularly intriguing possibility is thatTAAR1 activation may play a role in environmentalhypersensitivity reactions, particularly in the pulmo-nary and gastrointestinal systems.

There also remains a surprising sparsity of knowl-edge about the basic homeostatic mechanisms thatcontrol trace aminergic functioning. Although endoge-nous ligands for TAAR1 are now well established,questions remain about their synthesis and how cellularlevels and access to receptors are controlled. Thepossibility that the TAAR1 system serves as an endog-enous mechanism for promoting biased agonism at oneor more neurotransmitter receptors is intriguing andsomething that, should it be firmly established, isexpected to generate considerable interest within thepharmaceutical industry.

2. Other Trace Amine–Associated Receptors. Theestablishment of TAARs as a new class of receptor forolfaction has allowed the first steps to be taken indetermining the function of the wider TAAR family,with the identification of ligands for several TAARs,including putative endogenous ligands for TAAR3,TAAR4, and TAAR5. As progress continues to be madein establishing physiologic roles for olfactory TAARs indetecting environmental cues that induce innate be-havioral responses related to survival and speciespropagation, it will be important not to lose sight ofthe presence of these receptors in other tissues as well.Furthermore, identification of the neural pathwaysactivated downstream of individual olfactory TAARisoforms will allow a greater understanding of conspe-cific and heterospecific signaling mechanisms.

Clarifying the cellulardistributionof eachTAARthrough-out the body will be an important early step in futurestudies, alongwithdevelopmentofhigh-selectivity syntheticligands. In this latter aspect, the identification of a consen-sus TAAR binding pocket and development of in silicomolecular docking programs, allowing for high-throughputvirtual screening of vast chemical libraries, is expected to beof considerable utility. As endogenous ligands for individualTAARs are identified, the identification of synthetic anddegradative routes will be required. A number of pharma-cological tools have been developed for the study of olfactoryTAARs, and application of these tools to nonolfactorysystemswill beuseful inallowingamore rapid identificationof physiologic responses than was possible with TAAR1.

B. Development of Selective Trace Amine–AssociatedReceptor 1 Ligands as Therapeutics

1. Schizophrenia and Bipolar Disorder. Schizophre-nia and bipolar disorders are world-leading causes of


disability, with a combined lifetime prevalence of approx-imately 3% in the general population (Perälä et al., 2007).Symptoms and signs of schizophrenia are clustered intothree major domains: positive (e.g., delusions or halluci-nations), negative (e.g., blunted affect, amotivation), andcognitive (e.g., executive dysfunction, poor verbal mem-ory), with frequent occurrences of comorbid depressivesymptoms. The primary symptom domain of bipolardisorder is the presence of a sustained abnormality ofmood state, including abnormal mood elevation, depres-sion, irritability ofmood, and so-called “mixed states” of allthree symptom clusters. In cases of psychotic bipolardisorder, co-occurring psychotic symptoms also appearduring severe phases.Antipsychotic treatment of schizophrenia and bipolar

disorders mainly targets the symptoms found duringacute exacerbations, in particular acute positive symp-toms and mania. These drugs act as antagonists atD2Rs, and they act on serotonergic (e.g., 5-HT2A),cholinergic (e.g., muscarinic M1), and histaminergic(e.g., H1) receptors as well (Kim et al., 2009). Theseinclude first-generation drugs such as the phenothia-zines and butyrophenones, which are associated withfrequent and severe extrapyramidal symptoms such asdrug-induced parkinsonism and the neuroleptic malig-nant syndrome despite having great potency asantipsychotics. Second-generation (atypical) antipsy-chotics are currently the mainstay of therapy for bothdisorders and include compounds such as clozapine,olanzapine, quetiapine, risperidone, and aripiprazole.These second-generation compounds are associatedwith less severe extrapyramidal symptoms but areassociated with a metabolic syndrome (dyslipidemia,insulin resistance, pronounced weight gain, and eleva-tions in blood pressure), which can unfavorably alter thebenefit-risk ratio (Gründer et al., 2009; Kim et al., 2009;Meyer and Stahl, 2009; Thomas et al., 2009). Themetabolic syndrome and obesity are estimated to occurin 33% of patients with schizophrenia and bipolardisorders. In particular, patients who require treatmentwith olanzapine, clozapine, risperidone, or quetiapinehave high rates of these conditions. Even before theadvent of modern pharmacotherapy, however, patientswith schizophrenia and related-disorders were knownto suffer disproportionately from chronic disorders suchas cardiovascular disease and diabetes (Reynolds andKirk, 2010), suggesting a potential molecular common-ality. Until recently, the excess dopamine hypothesiswas the major pathophysiological theory of psychosis,based largely on the effectiveness of D2R antagonists.More recently, dysfunctions in other neurotransmitters,in particular glutamate, have been identified and arebeing explored as new therapeutic targets (Moghaddamand Javitt, 2012).As described in previous sections, abnormalities in

trace amine physiology have long been associated withschizophrenia and bipolar disorders. In schizophrenia,

increased urinary levels of PEA and alterations in themetabolism of TRP and TYR have been proposed, includ-ing enzymes involved in the synthetic and catabolicpathways of these molecules. TAAR1 is expressed in brainstructures associated with psychiatric disorders, in partic-ular inkeyareaswheremodulation of dopamine (VTA) andserotonin (DRN) occurs, but also in the amygdala, hypo-thalamus, rhinal cortices, and subiculum (Lindemannet al., 2008) where it modulates dopaminergic, serotoner-gic, and glutamatergic neurotransmission. As such, TAAR1may be a novel target for development of novel antipsy-chotic, potentially mood-stabilizing, and antidepressantdrugs with high potential for differentiation, by exploitingthe fundamentally new mechanism and target of action.Furthermore, given their positive energy metabolism pro-file, TAAR1 agonists may provide additional benefit forpatients with schizophrenia or bipolar disorder and thecomorbidities of associated metabolic syndrome. Indeed,the first TAAR1-directed agent (SEP-363856) is currentlyin clinical trials for schizophrenia (Berry et al., 2017).

Given that TAAR1 is the only validated receptor targetof the amphetamines and there is an abundant literaturelinking amphetamine-like effects to schizophrenia, it mayappear counterintuitive that TAAR1 agonists exert anti-psychotic actions. As described, however, TAAR1 agonismonly recapitulates a small portion of the physiologicresponses to amphetamine and none of the pleasurable,reinforcing properties. Indeed, TAAR1 agonism counter-acts these reinforcing properties. As such, it is incorrect toregard TAAR1 agonists as amphetamine like. Rather,TAAR1 agonism is just one of the many molecularmechanisms by which the promiscuous amphetaminesexert their effects.

2. Addiction and Compulsive Behaviors. Since theidentification that a number of psychotropic drugs ofabuse were agonists at TAAR1, there has been consid-erable interest in determining the role(s) that TAAR1might play in drug abuse and compulsive behaviors ingeneral. These studies have firmly established TAAR1as a novel target for the treatment of addiction, an effectthat relates to TAAR1 localization in the centraldopamine reward centers and regulation of D2R signal-ing. Not only does this realize control of cravings andrelapse toward psychotropic agents that are agonists atTAAR1 (amphetamine, methamphetamine, MDMA),but also to other addictive agents (cocaine, morphine,nicotine, and ethanol) and behaviors (binge eating)where TAAR1 is not a direct target of the addictiveagent. Importantly, the effects of TAAR1 agonists do notmerely substitute for the agent of abuse, with TAAR1agonists alone not supporting self-administration;rather, they address the underlying overactivity ofdopaminergic neural drives of drug-seeking behavior.Although no known clinical trials have yet occurred,dopamine reward pathways were identified as a TAAR1target in healthy human volunteers and it is anticipatedthat TAAR1 agonists will be the subject of future


development activities for the control of drug-takingbehavior. Such development activities may also includeenvironmental stress disorders such as post-traumaticstress disorder, in which an increased susceptibility todrug abuse is prevalent. In this regard, it is interestingto note that the possible beneficial effects of thepsychotropic TAAR1 ligand MDMA in post-traumaticstress disorder are being actively investigated (Amorosoand Workman, 2016; Sessa, 2017).3. Metabolic Syndromes. Diabetes is one of the

fastest growing health concerns worldwide. The totalnumber of people with all types of diabetes is projectedto rise from approximately 285 million in 2010 to439million in 2030, primarily due to population growth,aging, urbanization, and an increasing prevalence ofobesity and physical inactivity (Shaw et al., 2010). Type2 diabetes mellitus accounts for 90%–95% of all cases ofdiabetes in patients older than 20 years of age. It occursmore frequently in adults but is also being increasinglydiagnosed in adolescents (Aye and Levitsky, 2003).Type 2 diabetes mellitus is a chronic metabolic diseasecharacterized by hyperglycemia resulting from pancre-atic b-cell dysfunction, deficiency in insulin secretion,insulin resistance, and/or increased hepatic glucoseproduction. Current antidiabetic medications are ofteninefficient at achieving glycemic control because theypredominantly address only a single underlying defect(namely, the insulin secretion deficits). In addition, theytend to be associated with undesirable side effects suchas weight gain and episodes of hypoglycemia, conges-tive heart failure, and impaired renal function. Newand more effective drugs for the prevention and treat-ment of type 2 diabetes mellitus are therefore urgentlyrequired. The emerging profile of TAAR1 agonistspossessing incretin-like effects, increasing insulin se-cretion at elevated, but not basal, glucose levels, makesthem an attractive prospect for further development.Furthermore, the unique ability of TAAR1 agonists toalso regulate hormone secretion from the stomach andgastrointestinal tract in response to nutrients, decreasediabetes-associated obesity, as well as decrease thepropensity for binge eating and addictive behaviors ingeneral suggests that TAAR1 agonists may be uniquelyable to address both central and peripheral aspects ofmetabolic disorders.

C. Trace Amine–Associated Receptors in Ecology

The demonstration that olfactory TAARs are in-volved in the initiation of innate behavioral responsesto diverse environmental cues has generated interestin TAARs within the olfaction research community.Interest is also beginning to be shown by thosestudying ecology and there have been the first sugges-tions that TAARs may be involved in migration andmate choice of individual species. It is expected thatthese initial results will generate further interest thatwill allow further advances to be made in identifying

innate behaviors that are controlled by olfactoryTAARs. Such advances will almost certainly also leadto the identification of new TAAR ligands, which it ishoped will allow for the deorphaning of additionalTAAR family members. With a select number ofcompounds that are associated with diverse ecologicalcontexts and selective toward individual TAARs al-ready identified, the olfactory TAARs offer an amena-ble system to probing the neural basis of survivalbehaviors associated with different ecological contexts.Currently, only one neural pathway involved in theinnate behaviors initiated in response to olfactoryTAAR activation has begun to be mapped. It isexpected that this will be resolved in the coming years,providing a major breakthrough in understanding thecellular basis of the drives for various vertebratebehaviors.

With large variations in the complement of TAARsbetween species and activation of individual TAARsdemonstrated in response to both conspecific andheterospecific urines, it seems clear that TAARs areinvolved in species-specific adaptation responses. Assuch, the evolution of TAARs appears to be a goodoption for studying the evolution of olfactory systems,at least with respect to innate survival mechanisms.From this perspective, amphibians are of particularinterest because their olfactory systems have beensuggested to represent an evolutionary midpoint be-tween teleosts and mammals. There are few completegenome sequences available for amphibia and theTAAR complement of two Xenopus species are the onlyones to have been reported. Unfortunately, Xenopusare evolutionarily rather distant from the true frogs(Ranitidae sp.). Determining the TAAR complement ofrepresentative true frog species would be advanta-geous and provide a starting point for addressingevolutionary questions about the origins of vertebrateolfactory systems.

One interesting aspect that the TAAR systemmay offeris the ability of a single cue to directly activate bothcentrally and peripherallymediated responses. Certainly,centrally mediated behaviors are induced by activation ofTAARs in the olfactory passages. In addition, however, atleast some TAAR ligands can readily cross cell mem-branes; with TAARs present throughout the body, directactivation of systemic responses may also be possible. Inthis regard, it is particularly interesting that the strongestevidence for peripheral TAAR responses so far is in cellscontrolling energy metabolism and those of the immunesystem. These are two systems that depending on theprecise context would, on the surface, appear to beadvantageous to activate in response to the possiblepresence of predators, pathogenic microbes (spoiled food),or in preparation for mating. Such a situation couldprovide an explanation for why the evolutionary originof the entire family (TAAR1) is not involved in thedetection of olfactory cues.


D. Trace Amine–Associated Receptors, Nutrition, theMicrobiome, and Health

The roles of nutrition and the constitutive microbiotain health and disease are a growing area of research.The presence within various foodstuffs of compoundsthat are now known to be selective ligands for individualTAARs has long been known. Within the food sciencecommunity, the presence of these compounds has beenalmost exclusively studied from the point of view of foodspoilage and microbial contamination. With the grow-ing recognition and acceptance of TAAR functionsthroughout the body, but particularly in the gastroin-testinal tract and immune system, it is expectedthat over the next few years there will be a growinginterest in whether there is a role to play for TAARs inboth the beneficial and adverse effects of food in someindividuals.The search for metabolites that can link the consti-

tutive microbiota to health outcomes has become anactive area considerably aided by the development ofpowerful metabolomic techniques. With the levels ofknownTAAR ligands readilymodified by themicrobiotaand a defined family of receptors present, it is expectedthat the trace amine system will develop into a locus forsuch studies. Besides the potential of the trace aminesystem providing a molecular pathway for the putativelink between the microbiota, gastrointestinal tract, andcentral nervous system disorders, particularly intrigu-ing is the molecular link that the trace amine systemcould provide between nutrient intake, the microbiota,and the immune system.

VI. Conclusion

The identification of the TAAR family has resurrectedinterest in a series of compounds that had originallybeen identified as a topic for study at the advent of the20th century by some of the founding fathers of thefields of biochemistry, cellular physiology, and pharma-cology. Some basic knowledge gaps remain; for example,many TAARs remain orphan receptors with no knowndefining ligand, there is sparse information on cellularand tissue distribution of receptors, even the term“trace amine” has become nebulous and poorly defined,and suitably selective pharmacological reagents remainat a premium. Yet an exciting field with broad multi-disciplinary implications has begun to emerge. Al-though the 1960s and 1970s focus of trace amines (andthereby the subsequently identified TAARs) as putativeetiologic factors in drug abuse and psychiatric disorderscontinues to be a driving force, hitherto unsuspectedroles have emerged. Prominent among those are roles inthe peripheral control of energy metabolism as well as agrowing recognition of a putative role in the control ofcellular immune responses. Even with respect to psy-chiatric disorders, the journey of the last few years has

taken a number of unexpected twists and turns. WhereasTAAR1 is a direct target of a number of psychotropic drugsof abuse, activation of TAAR1 unexpectedly shows con-siderable promise as a novel therapeutic target for thepharmacotherapy of schizophrenia and drug abuse, withavailable evidence indicating no abuse potential ofTAAR1-directed agents. This likely reflects the promiscu-ity of molecular action of many of the drugs of abuse, inparticular those that are amphetamine like. AlthoughTAAR1 agonism is one of those sites of action, this is notrelated to their reinforcing properties and, hence, abusepotential. As such, TAAR1 agonists should not be viewedas amphetamine like. Rather, amphetaminergic com-pounds include TAAR1 agonism as one of their manymolecular sites of action.

After the identification of TAARs, the unexpecteddemonstration that they also serve a role as a new classof receptors for olfaction has opened up new vistas.Diverse environmental sources of ligands for individualTAARs have been identified, making the TAAR familyof interest for investigating ecological questions aroundpredator-prey interactions, mate selection, nutrient,and habitat choices. Furthermore, with TAARs beingunique to vertebrates and showing a high degree ofinterspecies variability, the ability to detect traceamines having arisen independently in invertebratesand vertebrates, and different TAAR isoforms undereither positive or negative selection pressures, TAARshave emerged as an excellent system for addressingquestions of the evolutionary origin of vertebrate olfac-tory systems. The independence of evolution betweeninvertebrate and vertebrate trace amine systems hasalso seen the invertebrate trace amine systemsemerge as potential targets for development of novelpest control agents that may have a better safetyprofile with respect to vertebrate exposure.

TAAR ligands are readily produced andmetabolized bymicrobes, and as TAARs become more widely recognizedand their cellular effects more fully established, they arelikely to attract the interest of microbiologists studyinghost-microbe interactions, food scientists studying fer-mentation processes in food production and qualitycontrol, and nutritional biochemists interested in the roleof nutrients in health and disease. Although the journeyfromNencki’s products of putrid flesh is far fromcomplete,the last decade has seen considerable advances and newavenues discovered. With a critical mass of trace amineresearchers originating from diverse scientific back-grounds now emerging, it is expected that an acceleratedphase of advancement of the field will occur over the nextfew years, including an increased understanding of theevolution of environmental adaptation strategies and theclinical testing of the first pharmacotherapies directedtoward individual TAARs.


Acknowledgments

We thank Martin Ebeling and Jitao David Zhang (F. Hoffmann-LaRoche, Basel, Switzerland) for kind help and excellent technicalassistance in preparation of the phylogenetic tree of TAARs.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Gainetdinov,Hoener, Berry.

ReferencesAbou Jamra R, Sircar I, Becker T, Freudenberg-Hua Y, Ohlraun S, Freudenberg J,Brockschmidt F, Schulze TG, Gross M, Spira F, et al. (2005) A family-based andcase-control association study of trace amine receptor genes on chromosome 6q23in bipolar affective disorder. Mol Psychiatry 10:618–620.

Achat-Mendes C, Lynch LJ, Sullivan KA, Vallender EJ, and Miller GM (2012)Augmentation of methamphetamine-induced behaviors in transgenic mice lackingthe trace amine-associated receptor 1. Pharmacol Biochem Behav 101:201–207.

Ackermans MT, Klieverik LP, Ringeling P, Endert E, Kalsbeek A, and Fliers E (2010)An online solid-phase extraction-liquid chromatography-tandem mass spectrome-try method to study the presence of thyronamines in plasma and tissue and theirputative conversion from 13C6-thyroxine. J Endocrinol 206:327–334.

Adriaenssens A, Lam BY, Billing L, Skeffington K, Sewing S, Reimann F,and Gribble F (2015) A transcriptome-led exploration of molecular mechanismsregulating somatostatin-producing D-cells in the gastric epithelium. Endocrinology156:3924–3936.

Aguanno A, Afar R, and Albert VR (1996) Tissue-specific expression of the nonneuronalpromoter of the aromatic L-amino acid decarboxylase gene is regulated by hepatocytenuclear factor 1. J Biol Chem 271:4528–4538.

Aguanno A, Lee MR, Marden CM, Rattray M, Gault A, and Albert VR (1995) Analysisof the neuronal promoter of the rat aromatic L-amino acid decarboxylase gene.J Neurochem 65:1944–1954.

Albert VR, Lee MR, Bolden AH, Wurzburger RJ, and Aguanno A (1992) Distinctpromoters direct neuronal and nonneuronal expression of rat aromatic L-aminoacid decarboxylase. Proc Natl Acad Sci USA 89:12053–12057.

Aleksandrov AA, Dmitrieva ES, Volnova AB, Knyazeva VM, Gerasimov AS,and Gainetdinov RR (2018a) TAAR5 receptor agonist affects sensory gating in rats.Neurosci Lett 666:144–147.

Aleksandrov AA, Knyazeva VM, Volnova AB, Dmitrieva ES, Korenkova O, EspinozaS, Gerasimov A, and Gainetdinov RR (2018b) Identification of TAAR5 agonist ac-tivity of alpha-NETA and its effect on mismatch negativity amplitude in awakerats. Neurotox Res DOI:10.1007/s12640-018-9902-6 [published ahead of print].

Alexander JL, Wilson ID, Teare J, Marchesi JR, Nicholson JK, and Kinross JM(2017) Gut microbiota modulation of chemotherapy efficacy and toxicity. Nat RevGastroenterol Hepatol 14:356–365.

Alexander SPH, Kelly E, Marrion N, Peters JA, Benson HE, Faccenda E, Pawson AJ,Sharman JL, Southan C, Buneman OP, et al.; CGTP Collaborators (2015) Theconcise guide to pharmacology 2015/16: overview. Br J Pharmacol 172:5729–5743.

Alkema MJ, Hunter-Ensor M, Ringstad N, and Horvitz HR (2005) Tyramine func-tions independently of octopamine in the Caenorhabditis elegans nervous system.Neuron 46:247–260.

Altar CA, Wasley AM, and Martin LL (1986) Autoradiographic localization andpharmacology of unique [3H]tryptamine binding sites in rat brain. Neuroscience17:263–273.

Alvarsson A, Zhang X, Stan TL, Schintu N, Kadkhodaei B, Millan MJ, Perlmann T,and Svenningsson P (2015) Modulation by trace amine-associated receptor 1 ofexperimental parkinsonism, L-DOPA responsivity, and glutamatergic neuro-transmission. J Neurosci 35:14057–14069.

Amoroso T and Workman M (2016) Treating posttraumatic stress disorder withMDMA-assisted psychotherapy: a preliminary meta-analysis and comparison toprolonged exposure therapy. J Psychopharmacol 30:595–600.

An D, Chen Z, Zheng J, Chen S, Wang L, Huang Z, and Weng L (2015) Determina-tion of biogenic amines in oysters by capillary electrophoresis coupled withelectrochemiluminescence. Food Chem 168:1–6.

Ando-Yamamoto M, Hayashi H, Sugiyama T, Fukui H, Watanabe T, and Wada H(1987) Purification of L-dopa decarboxylase from rat liver and production of poly-clonal and monoclonal antibodies against it. J Biochem 101:405–414.

Andrés M, Seifert M, Spalthoff C, Warren B, Weiss L, Giraldo D, Winkler M, Pauls S,and Göpfert MC (2016) Auditory efferent system modulates mosquito hearing.Curr Biol 26:2028–2036.

Anton S, Dufour M-C, and Gadenne C (2006) Plasticity of olfactory-guided behaviourand its neurobiological basis. Entomol Experiment Appl 123:1–11.

Aperia A, Hökfelt T, Meister B, Bertorello A, Fryckstedt J, Holtbäck U, and Seri I(1990) The significance of L-amino acid decarboxylase and DARPP-32 in the kid-ney. Am J Hypertens 3:11S–13S.

Arakawa S, Gocayne JD, McCombie WR, Urquhart DA, Hall LM, Fraser CM,and Venter JC (1990) Cloning, localization, and permanent expression of a Dro-sophila octopamine receptor. Neuron 4:343–354.

Ashley-Montagu MF (1938) Trimethylamine in menstruous women. Nature 142:1121–1122.

Asif-Malik A, Hoener MC, and Canales JJ (2017) Interaction between the traceamine-associated receptor 1 and the dopamine D2 receptor controls cocaine’sneurochemical actions. Sci Rep 7:13901.

Axelrod J and Saavedra JM (1977) Octopamine. Nature 265:501–504.Aye T and Levitsky LL (2003) Type 2 diabetes: an epidemic disease in childhood.Curr Opin Pediatr 15:411–415.

Azam B, Wienecke J, Jensen DB, Azam A, and Zhang M (2015) Spinal cord hem-isection facilitates aromatic L-amino acid decarboxylase cells to produce sero-tonin in the subchronic but not the chronic phase. Neural Plast 2015:549671.

Azzouzi N, Barloy-Hubler F, and Galibert F (2015) Identification and characteriza-tion of cichlid TAAR genes and comparison with other teleost TAAR repertoires.BMC Genomics 16:335.

Babusyte A, Kotthoff M, Fiedler J, and Krautwurst D (2013) Biogenic amines acti-vate blood leukocytes via trace amine-associated receptors TAAR1 and TAAR2. JLeukoc Biol 93:387–394.

Bach B, Le Quere S, Vuchot P, Grinbaum M, and Barnavon L (2012) Validation of amethod for the analysis of biogenic amines: histamine instability during winesample storage. Anal Chim Acta 732:114–119.

Bacon JP, Thompson KS, and Stern M (1995) Identified octopaminergic neuronsprovide an arousal mechanism in the locust brain. J Neurophysiol 74:2739–2743.

Baggio LL and Drucker DJ (2007) Biology of incretins: GLP-1 and GIP. Gastroen-terology 132:2131–2157.

Bahado-Singh RO, Akolekar R, Chelliah A, Mandal R, Dong E, Kruger M, WishartDS, and Nicolaides K (2013) Metabolomic analysis for first-trimester trisomy18 detection. Am J Obstet Gynecol 209:65.e1–65.e9.

Bain MA, Faull R, Fornasini G, Milne RW, and Evans AM (2006) Accumulation oftrimethylamine and trimethylamine-N-oxide in end-stage renal disease patientsundergoing haemodialysis. Nephrol Dial Transplant 21:1300–1304.

Baker GB, Bornstein RA, Rouget AC, Ashton SE, van Muyden JC, and Coutts RT(1991) Phenylethylaminergic mechanisms in attention-deficit disorder. Biol Psy-chiatry 29:15–22.

Baker GB, Raiteri M, Bertollini A, and del Carmine R (1976) Interaction of beta-phenethylamine with dopamine and noradrenaline in the central nervous systemof the rat. J Pharm Pharmacol 28:456–457.

Balfanz S, Jordan N, Langenstück T, Breuer J, Bergmeier V, and Baumann A (2014)Molecular, pharmacological, and signaling properties of octopamine receptors fromhoneybee (Apis mellifera) brain. J Neurochem 129:284–296.

Balfanz S, Strünker T, Frings S, and Baumann A (2005) A family of octopamine[corrected] receptors that specifically induce cyclic AMP production or Ca2+ releasein Drosophila melanogaster [published correction appears in J Neurochem (2005)94:1168]. J Neurochem 93:440–451.

Barak LS, Salahpour A, Zhang X, Masri B, Sotnikova TD, Ramsey AJ, Violin JD,Lefkowitz RJ, Caron MG, and Gainetdinov RR (2008) Pharmacological character-ization of membrane-expressed human trace amine-associated receptor 1 (TAAR1)by a bioluminescence resonance energy transfer cAMP biosensor. Mol Pharmacol74:585–594.

Barger G and Dale HH (1910) Chemical structure and sympathomimetic action ofamines. J Physiol 41:19–59.

Barger G and Walpole GS (1909) Isolation of the pressor principles of putrid meat.J Physiol 38:343–352.

Barron AB, Schulz DJ, and Robinson GE (2002) Octopamine modulates responsive-ness to foraging-related stimuli in honey bees (Apis mellifera). J Comp Physiol ANeuroethol Sens Neural Behav Physiol 188:603–610.

Barygin OI, Komarova MS, Tikhonova TB, Korosteleva AS, Nikolaev MV, MagazanikLG, and Tikhonov DB (2017) Complex action of tyramine, tryptamine and hista-mine on native and recombinant ASICs. Channels (Austin) 11:648–659.

Bayliss A, Roselli G, and Evans PD (2013) A comparison of the signalling propertiesof two tyramine receptors from Drosophila. J Neurochem 125:37–48.

Beaulieu JM and Gainetdinov RR (2011) The physiology, signaling, and pharma-cology of dopamine receptors. Pharmacol Rev 63:182–217.

Beaulieu JM, Gainetdinov RR, and Caron MG (2009) Akt/GSK3 signaling in theaction of psychotropic drugs. Annu Rev Pharmacol Toxicol 49:327–347.

Beggs KT, Tyndall JD, and Mercer AR (2011) Honey bee dopamine and octopaminereceptors linked to intracellular calcium signaling have a close phylogenetic andpharmacological relationship. PLoS One 6:e26809.

Bender DA and Coulson WF (1972) Variations in aromatic amino acid decarboxylaseactivity towards DOPA and 5-hydroxytryptophan caused by pH changes and de-naturation. J Neurochem 19:2801–2810.

Bengtsson AA, Trygg J, Wuttge DM, Sturfelt G, Theander E, Donten M, Moritz T,Sennbro CJ, Torell F, Lood C, et al. (2016) Metabolic profiling of systemic lupuserythematosus and comparison with primary Sjögren’s syndrome and systemicsclerosis. PLoS One 11:e0159384.

Berry MD (2004) Mammalian central nervous system trace amines. Pharmacologicamphetamines, physiologic neuromodulators. J Neurochem 90:257–271.

Berry MD (2007) The potential of trace amines and their receptors for treatingneurological and psychiatric diseases. Rev Recent Clin Trials 2:3–19.

Berry MD (2011) The effects of pargyline and 2-phenylethylamine on D1-like dopa-mine receptor binding. J Neural Transm (Vienna) 118:1115–1118.

Berry MD, Gainetdinov RR, Hoener MC, and Shahid M (2017) Pharmacology ofhuman trace amine-associated receptors: therapeutic opportunities and chal-lenges. Pharmacol Ther 180:161–180.

Berry MD, Hart S, Pryor AR, Hunter S, and Gardiner D (2016) Pharmacologicalcharacterization of a high-affinity p-tyramine transporter in rat brain synapto-somes. Sci Rep 6:38006.

Berry MD, Juorio AV, Li XM, and Boulton AA (1996) Aromatic L-amino acid decarboxylase:a neglected and misunderstood enzyme. Neurochem Res 21:1075–1087.

Berry MD, Scarr E, Zhu M-Y, Paterson IA, and Juorio AV (1994) The effects ofadministration of monoamine oxidase-B inhibitors on rat striatal neurone re-sponses to dopamine. Br J Pharmacol 113:1159–1166.

Berry MD, Shitut MR, Almousa A, Alcorn J, and Tomberli B (2013) Membranepermeability of trace amines: evidence for a regulated, activity-dependent, non-exocytotic, synaptic release. Synapse 67:656–667.

Bierut LJ, Rice JP, Edenberg HJ, Goate A, Foroud T, Cloninger CR, Begleiter H,Conneally PM, Crowe RR, Hesselbrock V, et al. (2000) Family-based study of theassociation of the dopamine D2 receptor gene (DRD2) with habitual smoking. Am JMed Genet 90:299–302.


http://dx.doi.org/10.1007/s12640-018-9902-6

Biji KB, Ravishankar CN, Venkateswarlu R, Mohan CO, and Gopal TK (2016) Bio-genic amines in seafood: a review. J Food Sci Technol 53:2210–2218.

Black SW, Schwartz MD, Chen TM, Hoener MC, and Kilduff TS (2017) Trace amine-associated receptor 1 agonists as narcolepsy therapeutics. Biol Psychiatry 82:623–633.

Blakeley AG and Nicol CJ (1978) Accumulation of amines by rabbit erythrocytesin vitro. J Physiol 277:77–90.

Blaschko H, Richter D, and Schlossmann H (1937) The oxidation of adrenaline andother amines. Biochem J 31:2187–2196.

Blenau W, Balfanz S, and Baumann A (2017) PeaTAR1B: characterization of a sec-ond type 1 tyramine receptor of the American cockroach, Periplaneta americana.Int J Mol Sci 18:E2279.

Blenau W and Baumann A (2001) Molecular and pharmacological properties of insectbiogenic amine receptors: lessons from Drosophila melanogaster and Apis mellifera.Arch Insect Biochem Physiol 48:13–38.

Bloch G and Meshi A (2007) Influences of octopamine and juvenile hormone on locomotorbehavior and period gene expression in the honeybee, Apis mellifera. J Comp Physiol ANeuroethol Sens Neural Behav Physiol 193:181–199.

Blum K, Noble EP, Sheridan PJ, Montgomery A, Ritchie T, Jagadeeswaran P,Nogami H, Briggs AH, and Cohn JB (1990) Allelic association of human dopamineD2 receptor gene in alcoholism. JAMA 263:2055–2060.

Bly M (2005) Examination of the trace amine-associated receptor 2 (TAAR2).Schizophr Res 80:367–368.

Bocian R, Posluszny A, Kowalczyk T, Kazmierska P, and Konopacki J (2011) Gapjunction modulation of hippocampal formation theta and local cell discharges inanesthetized rats. Eur J Neurosci 33:471–481.

Børglum AD, Hampson M, Kjeldsen TE, Muir W, Murray V, Ewald H, Mors O,Blackwood D, and Kruse TA (2001) Dopa decarboxylase genotypes may influenceage at onset of schizophrenia. Mol Psychiatry 6:712–717.

Borison RL, Mosnaim AD, and Sabelli HC (1975) Brain 2-phenylethylamine as amajor mediator for the central actions of amphetamine and methylphenidate. LifeSci 17:1331–1343.

Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, DurkinMM, Lakhlani PP, Bonini JA, Pathirana S, et al. (2001) Trace amines: identifica-tion of a family of mammalian G protein-coupled receptors. Proc Natl Acad SciUSA 98:8966–8971.

Bouchard S and Roberge AG (1979) Biochemical properties and kinetic parameters ofdihydroxyphenylalanine–5-hydroxytryptophan decarboxylase in brain, liver, andadrenals of cat. Can J Biochem 57:1014–1018.

Boulton AA (1974) Letter: amines and theories in psychiatry. Lancet 2:52–53.Boulton AA (1976) Identification, distribution, metabolism, and function of meta andpara tyramine, phenylethylamine and tryptamine in brain. Adv Biochem Psycho-pharmacol 15:57–67.

Boulton AA and Wu PH (1972) Biosynthesis of cerebral phenolic amines. I. In vivoformation of p-tyramine, octopamine, and synephrine. Can J Biochem 50:261–267.

Boulton AA and Wu PH (1973) Biosynthesis of cerebral phenolic amines. II. In vivoregional formation of p-tyramine and octopamine from tyrosine and dopamine. CanJ Biochem 51:428–435.

Boyle NT and Connor TJ (2010) Methylenedioxymethamphetamine (‘Ecstasy’)-inducedimmunosuppression: a cause for concern? Br J Pharmacol 161:17–32.

Bradaia A, Trube G, Stalder H, Norcross RD, Ozmen L, Wettstein JG, Pinard A,Buchy D, Gassmann M, Hoener MC, et al. (2009) The selective antagonist EPPTBreveals TAAR1-mediated regulatory mechanisms in dopaminergic neurons of themesolimbic system. Proc Natl Acad Sci USA 106:20081–20086.

Brahmachary RL and Dutta J (1979) Phenylethylamine as a biochemical marker oftiger. Z Naturforsch C 34:632–633.

Braulke LJ, Klingenspor M, DeBarber A, Tobias SC, Grandy DK, Scanlan TS,and Heldmaier G (2008) 3-Iodothyronamine: a novel hormone controlling the bal-ance between glucose and lipid utilisation. J Comp Physiol B 178:167–177.

Braun G and Bicker G (1992) Habituation of an appetitive reflex in the honeybee.J Neurophysiol 67:588–598.

Bromek E, Haduch A, and Daniel WA (2010) The ability of cytochrome P450 2D isoformsto synthesize dopamine in the brain: An in vitro study. Eur J Pharmacol 626:171–178.

Brown JM and Hazen SL (2017) Targeting of microbe-derived metabolites toimprove human health: the next frontier for drug discovery. J Biol Chem 292:8560–8568.

Brunkwall L and Orho-Melander M (2017) The gut microbiome as a target for pre-vention and treatment of hyperglycaemia in type 2 diabetes: from current humanevidence to future possibilities. Diabetologia 60:943–951.

Buckland PR, Marshall R, Watkins P, and McGuffin P (1997) Does phenylethylaminehave a role in schizophrenia?: LSD and PCP up-regulate aromatic L-amino aciddecarboxylase mRNA levels. Brain Res Mol Brain Res 49:266–270.

Buckland PR, O’Donovan MC, and McGuffin P (1992) Changes in dopa decarboxylasemRNA but not tyrosine hydroxylase mRNA levels in rat brain following antipsy-chotic treatment. Psychopharmacology (Berl) 108:98–102.

Buckland PR, Spurlock G, and McGuffin P (1996) Amphetamine and vigabatrin downregulate aromatic L-amino acid decarboxylase mRNA levels. Brain Res Mol BrainRes 35:69–76.

Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI,Darland T, Suchland KL, Pasumamula S, Kennedy JL, et al. (2001) Amphetamine,3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabo-lites of the catecholamine neurotransmitters are agonists of a rat trace aminereceptor. Mol Pharmacol 60:1181–1188.

Burczynski ME, Peterson RL, Twine NC, Zuberek KA, Brodeur BJ, Casciotti L,Maganti V, Reddy PS, Strahs A, Immermann F, et al. (2006) Molecular classifi-cation of Crohn’s disease and ulcerative colitis patients using transcriptional pro-files in peripheral blood mononuclear cells. J Mol Diagn 8:51–61.

Büschges A, Kittmann R, and Ramirez JM (1993) Octopamine effects mimick state-dependent changes in a proprioceptive feedback system. J Neurobiol 24:598–610.

Cao Q, Martinez M, Zhang J, Sanders AR, Badner JA, Cravchik A, Markey CJ,Beshah E, Guroff JJ, Maxwell ME, et al. (1997) Suggestive evidence for a schizo-phrenia susceptibility locus on chromosome 6q and a confirmation in an indepen-dent series of pedigrees. Genomics 43:1–8.

Carbonaro TM and Gatch MB (2016) Neuropharmacology of N,N-dimethyltryptamine.Brain Res Bull 126:74–88.

Carnicelli V, Santoro A, Sellari-Franceschini S, Berrettini S, and Zucchi R (2010)Expression of trace amine-associated receptors in human nasal mucosa. Chemo-sens Percept 3:99–107.

Cassenaer S and Laurent G (2012) Conditional modulation of spike-timing-dependent plasticity for olfactory learning. Nature 482:47–52.

Cazzamali G, Klaerke DA, and Grimmelikhuijzen CJ (2005) A new family of insecttyramine receptors. Biochem Biophys Res Commun 338:1189–1196.

Chang D-J, Li X-C, Lee Y-S, Kim H-K, Kim US, Cho NJ, Lo X, Weiss KR, Kandel ER,and Kaang B-K (2000) Activation of a heterologously expressed octopamine re-ceptor coupled only to adenylyl cyclase produces all the features of presynapticfacilitation in aplysia sensory neurons. Proc Natl Acad Sci USA 97:1829–1834.

Chang HS, Heo JS, Shin SW, Bae DJ, Song HJ, Jun JA, Kim JD, Park JS, Park BL,Shin HD, et al. (2015) Association between TAAR6 polymorphisms and airwayresponsiveness to inhaled corticosteroids in asthmatic patients. PharmacogenetGenomics 25:334–342.

Chang WP, Wu JJ, and Shyu BC (2013) Thalamic modulation of cingulate seizureactivity via the regulation of gap junctions in mice thalamocingulate slice. PLoSOne 8:e62952.

Chang YT, Mues G, and Hyland K (1996) Alternative splicing in the coding region ofhuman aromatic L-amino acid decarboxylase mRNA. Neurosci Lett 202:157–160.

Châtel A, Murillo L, Bourdin CM, Quinchard S, Picard D, and Legros C (2013)Characterization of tyramine b-hydroxylase, an enzyme upregulated by stress inPeriplaneta americana. J Mol Endocrinol 50:91–102.

Chhibber-Goel J, Gaur A, Singhal V, Parakh N, Bhargava B, and Sharma A (2016)The complex metabolism of trimethylamine in humans: endogenous and exogenoussources. Expert Rev Mol Med 18:e8.

Chiellini G, Bellusci L, Sabatini M, and Zucchi R (2017) Thyronamines and analogues- the route from rediscovery to translational research on thyronergic amines. MolCell Endocrinol 458:149–155.

Chiellini G, Erba P, Carnicelli V, Manfredi C, Frascarelli S, Ghelardoni S, Mariani G,and Zucchi R (2012) Distribution of exogenous [125I]-3-iodothyronamine in mousein vivo: relationship with trace amine-associated receptors. J Endocrinol 213:223–230.

Chiellini G, Frascarelli S, Ghelardoni S, Carnicelli V, Tobias SC, DeBarber A, Brogioni S,Ronca-Testoni S, Cerbai E, Grandy DK, et al. (2007) Cardiac effects of3-iodothyronamine: a new aminergic system modulating cardiac function. FASEB J 21:1597–1608.

Chireux M, Raynal JF, Le Van Thai A, Cadas H, Bernard C, Martinou I, MartinouJC, and Weber MJ (1994) Multiple promoters of human choline acetyltransferaseand aromatic L-amino acid decarboxylase genes. J Physiol Paris 88:215–227.

Cho S, Duchemin A-M, Neff NH, and Hadjiconstantinou M (1999) Tyrosine hydrox-ylase, aromatic L-amino acid decarboxylase and dopamine metabolism afterchronic treatment with dopaminergic drugs. Brain Res 830:237–245.

Cho S, Neff NH, and Hadjiconstantinou M (1997) Regulation of tyrosine hydroxylaseand aromatic L-amino acid decarboxylase by dopaminergic drugs. Eur J Pharmacol323:149–157.

Christensen TA, Sherman TG, McCaman RE, and Carlson AD (1983) Presence ofoctopamine in firefly photomotor neurons. Neuroscience 9:183–189.

Christenson JG, Dairman W, and Udenfriend S (1970) Preparation and properties ofa homogeneous aromatic L-amino acid decarboxylase from hog kidney. Arch BiochemBiophys 141:356–367.

Christie AE, Fontanilla TM, Roncalli V, Cieslak MC, and Lenz PH (2014) Identifi-cation and developmental expression of the enzymes responsible for dopamine,histamine, octopamine and serotonin biosynthesis in the copepod crustacean Calanusfinmarchicus. Gen Comp Endocrinol 195:28–39.

Churcher AM, Hubbard PC, Marques JP, Canário AV, and Huertas M (2015) Deep se-quencing of the olfactory epithelium reveals specific chemosensory receptors are expressedat sexual maturity in the European eel Anguilla anguilla. Mol Ecol 24:822–834.

Cichero E, Espinoza S, Tonelli M, Franchini S, Gerasimov AS, Sorbi C, GainetdinovRR, Brasili L, and Fossa P (2016) A homology modelling-driven study leading tothe discovery of the first mouse trace amine-associated receptor 5 (TAAR5) an-tagonists. MedChemComm 7:353–364.

Cichero E and Tonelli M (2017) New insights into the structure of the trace amine-associated receptor 2: homology modelling studies exploring the binding mode of3-iodothyronamine. Chem Biol Drug Des 89:790–796.

Cisneros IE and Ghorpade A (2014) Methamphetamine and HIV-1-induced neuro-toxicity: role of trace amine associated receptor 1 cAMP signaling in astrocytes.Neuropharmacology 85:499–507.

Citrome L (2014) Unmet needs in the treatment of schizophrenia: new targets to helpdifferent symptom domains. J Clin Psychiatry 75 (Suppl 1):21–26.

Clark A (1911) The clinical application of ergotamine (tyramine). Biochem J 5:236–242.

Coecke S, Debast G, Phillips IR, Vercruysse A, Shephard EA, and Rogiers V (1998)Hormonal regulation of microsomal flavin-containing monooxygenase activity bysex steroids and growth hormone in co-cultured adult male rat hepatocytes. BiochemPharmacol 56:1047–1051.

Cole SH, Carney GE, McClung CA, Willard SS, Taylor BJ, and Hirsh J (2005) Twofunctional but noncomplementing Drosophila tyrosine decarboxylase genes: dis-tinct roles for neural tyramine and octopamine in female fertility. J Biol Chem 280:14948–14955.

Comings DE, Comings BG, Muhleman D, Dietz G, Shahbahrami B, Tast D, Knell E,Kocsis P, Baumgarten R, Kovacs BW, et al. (1991) The dopamine D2 receptor locusas a modifying gene in neuropsychiatric disorders. JAMA 266:1793–1800.


Comings DE, Muhleman D, Ahn C, Gysin R, and Flanagan SD (1994) The dopamine D2receptor gene: a genetic risk factor in substance abuse. Drug Alcohol Depend 34:175–180.

Comings DE, Rosenthal RJ, Lesieur HR, Rugle LJ, Muhleman D, Chiu C, Dietz G,and Gade R (1996) A study of the dopamine D2 receptor gene in pathologicalgambling. Pharmacogenetics 6:223–234.

Consoulas C, Johnston RM, Pflüger HJ, and Levine RB (1999) Peripheral distribu-tion of presynaptic sites of abdominal motor and modulatory neurons in Manducasexta larvae. J Comp Neurol 410:4–19.

Copeland J and Robertson HA (1982) Octopamine as the transmitter at the fireflylantern: presence of an octopamine-sensitive and a dopamine-sensitive adenylatecyclase. Comp Biochem Physiol C Toxicol Pharmacol 72:125–127.

Cöster M, Biebermann H, Schöneberg T, and Stäubert C (2015) Evolutionary con-servation of 3-iodothyronamine as an agonist at the trace amine-associated re-ceptor 1. Eur Thyroid J 4 (Suppl 1):9–20.

Coton M, Delbés-Paus C, Irlinger F, Desmasures N, Le Fleche A, Stahl V, MontelMC, and Coton E (2012) Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses. Food Microbiol 29:88–98.

Cotter R, Pei Y, Mus L, Harmeier A, Gainetdinov RR, Hoener MC, and Canales JJ(2015) The trace amine-associated receptor 1 modulates methamphetamine’sneurochemical and behavioral effects. Front Neurosci 9:39.

Couroussé T and Gautron S (2015) Role of organic cation transporters (OCTs) in thebrain. Pharmacol Ther 146:94–103.

Coutts RT, Baker GB, and Pasutto FM (1986) Foodstuffs as sources of psychoactiveamines and their precursors: content, significance and identification. Adv Drug Res15:169–232.

Craciun S and Balskus EP (2012) Microbial conversion of choline to trimethylaminerequires a glycyl radical enzyme. Proc Natl Acad Sci USA 109:21307–21312.

Cumero S, Fogolari F, Domenis R, Zucchi R, Mavelli I, and Contessi S (2012) Mito-chondrial F(0) F(1) -ATP synthase is a molecular target of 3-iodothyronamine, anendogenous metabolite of thyroid hormone. Br J Pharmacol 166:2331–2347.

Cunha SC, Faria MA, and Fernandes JO (2011) Gas chromatography-mass spec-trometry assessment of amines in Port wine and grape juice after fast chloroformateextraction/derivatization. J Agric Food Chem 59:8742–8753.

D’Andrea G, D’Arrigo A, Facchinetti F, Del Giudice E, Colavito D, Bernardini D,and Leon A (2012) Octopamine, unlike other trace amines, inhibits responses ofastroglia-enriched cultures to lipopolysaccharide via a b-adrenoreceptor-mediatedmechanism. Neurosci Lett 517:36–40.

D’Andrea G, Perini F, Terrazzino S, and Nordera GP (2004) Contributions of bio-chemistry to the pathogenesis of primary headaches. Neurol Sci 25 (Suppl 3):S89–S92.

D’Andrea G, Terrazzino S, Fortin D, Farruggio A, Rinaldi L, and Leon A (2003) HPLCelectrochemical detection of trace amines in human plasma and platelets and ex-pression of mRNA transcripts of trace amine receptors in circulating leukocytes.Neurosci Lett 346:89–92.

Danielson TJ, Boulton AA, and Robertson HA (1977) m-Octopamine, p-octopamineand phenylethanolamine in rat brain: a sensitive, specific assay and the effects ofsome drugs. J Neurochem 29:1131–1135.

Davis BA (1989) Biogenic amines and their metabolites in body fluids of normal,psychiatric and neurological subjects. J Chromatogr A 466:89–218.

de la Barca JMC, Huang NT, Jiao H, Tessier L, Gadras C, Simard G, Natoli R,Tcherkez G, Reynier P, and Valter K (2017) Retinal metabolic events in preconditioninglight stress as revealed by wide-spectrum targeted metabolomics. Metabolomics 13:22.

Dewan A, Pacifico R, Zhan R, Rinberg D, and Bozza T (2013) Non-redundant codingof aversive odours in the main olfactory pathway. Nature 497:486–489.

Dewhurst SA, Croker SG, Ikeda K, and McCaman RE (1972) Metabolism ofbiogenic amines in Drosophila nervous tissue. Comp Biochem Physiol B 43:975–981.

Dhillo WS, Bewick GA, White NE, Gardiner JV, Thompson EL, Bataveljic A, MurphyKG, Roy D, Patel NA, Scutt JN, et al. (2009) The thyroid hormone derivative3-iodothyronamine increases food intake in rodents. Diabetes Obes Metab 11:251–260.

Di Cara B, Maggio R, Aloisi G, Rivet JM, Lundius EG, Yoshitake T, Svenningsson P,Brocco M, Gobert A, De Groote L, et al. (2011) Genetic deletion of trace amine1 receptors reveals their role in auto-inhibiting the actions of ecstasy (MDMA).J Neurosci 31:16928–16940.

Dial EJ, Cooper LC, and Lichtenberger LM (1991) Amino acid- and amine-inducedgastrin release from isolated rat endocrine granules. Am J Physiol 260:G175–G181.

Dieris M, Ahuja G, Krishna V, and Korsching SI (2017) A single identified glomerulusin the zebrafish olfactory bulb carries the high-affinity response to death-associated odorcadaverine. Sci Rep 7:40892.

Dinan TG and Cryan JF (2015) The impact of gut microbiota on brain and behaviour:implications for psychiatry. Curr Opin Clin Nutr Metab Care 18:552–558.

Ding S, Wang X, Zhuge W, Yang J, and Zhuge Q (2017) Dopamine induces glutamateaccumulation in astrocytes to disrupt neuronal function leading to pathogenesis ofminimal hepatic encephalopathy. Neuroscience 365:94–113.

Dinter J, Khajavi N, Mühlhaus J, Wienchol CL, Cöster M, Hermsdorf T, Stäubert C,Köhrle J, Schöneberg T, Kleinau G, et al. (2015a) The multitarget ligand3-iodothyronamine modulates b-adrenergic receptor 2 signaling. Eur Thyroid J 4(Suppl 1):21–29.

Dinter J, Mühlhaus J, Jacobi SF, Wienchol CL, Cöster M, Meister J, Hoefig CS,Müller A, Köhrle J, Grüters A, et al. (2015b) 3-iodothyronamine differentiallymodulates a-2A-adrenergic receptor-mediated signaling. J Mol Endocrinol 54:205–216.

Dinter J, Mühlhaus J, Wienchol CL, Yi CX, Nürnberg D, Morin S, Grüters A, Köhrle J,Schöneberg T, Tschöp M, et al. (2015c) Inverse agonistic action of 3-iodothyronamine atthe human trace amine-associated receptor 5. PLoS One 10:e0117774.

Djali PK, Dimaline R, and Dockray GJ (1998) Novel form of L-aromatic amino aciddecarboxylase mRNA in rat antral mucosa. Exp Physiol 83:617–627.

Dominici P, Tancini B, Barra D, and Voltattorni CB (1987) Purification and char-acterization of rat-liver 3,4-dihydroxyphenylalanine decarboxylase. Eur J Biochem169:209–213.

Donly BC and Caveney S (2005) A transporter for phenolamine uptake in the ar-thropod CNS. Arch Insect Biochem Physiol 59:172–183.

Dourish CT (1982) An observational analysis of the behavioural effects of beta-phenylethylamine in isolated and grouped mice. Prog Neuropsychopharmacol BiolPsychiatry 6:143–158.

Downer RG, Hiripi L, and Juhos S (1993) Characterization of the tyraminergic sys-tem in the central nervous system of the locust, Locusta migratoria migratoides.Neurochem Res 18:1245–1248.

Doyle KP, Suchland KL, Ciesielski TM, Lessov NS, Grandy DK, Scanlan TS,and Stenzel-Poore MP (2007) Novel thyroxine derivatives, thyronamine and3-iodothyronamine, induce transient hypothermia and marked neuroprotectionagainst stroke injury. Stroke 38:2569–2576.

Duan J, Martinez M, Sanders AR, Hou C, Saitou N, Kitano T, Mowry BJ, Crowe RR,Silverman JM, Levinson DF, et al. (2004) Polymorphisms in the trace amine re-ceptor 4 (TRAR4) gene on chromosome 6q23.2 are associated with susceptibility toschizophrenia. Am J Hum Genet 75:624–638.

Duan S, Du J, Xu Y, Xing Q, Wang H, Wu S, Chen Q, Li X, Li X, Shen J, et al. (2006)Failure to find association between TRAR4 and schizophrenia in the Chinese Hanpopulation. J Neural Transm (Vienna) 113:381–385.

Duchamp-Viret P and Duchamp A (1997) Odor processing in the frog olfactory sys-tem. Prog Neurobiol 53:561–602.

Duchemin AM, Berry MD, Neff NH, and Hadjiconstantinou M (2000) Phosphoryla-tion and activation of brain aromatic L-amino acid decarboxylase by cyclic AMP-dependent protein kinase. J Neurochem 75:725–731.

Duchemin AM, Neff NH, and Hadjiconstantinou M (2010) Aromatic L-amino aciddecarboxylase phosphorylation and activation by PKGIalpha in vitro. J Neurochem114:542–552.

Dumas ME, Barton RH, Toye A, Cloarec O, Blancher C, Rothwell A, Fearnside J,Tatoud R, Blanc V, Lindon JC, et al. (2006) Metabolic profiling reveals a contri-bution of gut microbiota to fatty liver phenotype in insulin-resistant mice. ProcNatl Acad Sci USA 103:12511–12516.

Durden DA and Philips SR (1980) Kinetic measurements of the turnover rates of phen-ylethylamine and tryptamine in vivo in the rat brain. J Neurochem 34:1725–1732.

Dyck LE (1989) Release of some endogenous trace amines from rat striatal slices inthe presence and absence of a monoamine oxidase inhibitor. Life Sci 44:1149–1156.

Dyck LE, Yang CR, and Boulton AA (1983) The biosynthesis of p-tyramine, m-tyramine, andbeta-phenylethylamine by rat striatal slices. J Neurosci Res 10:211–220.

Eisenberg DP, Kohn PD, Hegarty CE, Ianni AM, Kolachana B, Gregory MD, Masdeu JC,and Berman KF (2016) Common variation in the DOPA decarboxylase (DDC) gene andhuman striatal DDC activity in vivo. Neuropsychopharmacology 41:2303–2308.

El-Kholy S, Stephano F, Li Y, Bhandari A, Fink C, and Roeder T (2015) Expressionanalysis of octopamine and tyramine receptors in Drosophila. Cell Tissue Res 361:669–684.

Elliott J, Callingham BA, and Sharman DF (1989) Semicarbazide-sensitive amineoxidase (SSAO) of the rat aorta. Interactions with some naturally occurring aminesand their structural analogues. Biochem Pharmacol 38:1507–1515.

Elliott P, Posma JM, Chan Q, Garcia-Perez I, Wijeyesekera A, Bictash M, Ebbels TM,Ueshima H, Zhao L, van Horn L, et al. (2015) Urinary metabolic signatures of humanadiposity. Sci Transl Med 7:285ra62.

Erspamer V (1952) Identification of octopamine as l-p-hydroxyphenylethanolamine.Nature 169:375–376.

Espinoza S, Ghisi V, Emanuele M, Leo D, Sukhanov I, Sotnikova TD, Chieregatti E,and Gainetdinov RR (2015a) Postsynaptic D2 dopamine receptor supersensitivityin the striatum of mice lacking TAAR1. Neuropharmacology 93:308–313.

Espinoza S, Lignani G, Caffino L, Maggi S, Sukhanov I, Leo D, Mus L, Emanuele M,Ronzitti G, Harmeier A, et al. (2015b) TAAR1 modulates cortical glutamate NMDAreceptor function. Neuropsychopharmacology 40:2217–2227.

Espinoza S, Manago F, Leo D, Sotnikova TD, and Gainetdinov RR (2012) Role ofcatechol-O-methyltransferase (COMT)-dependent processes in Parkinson’s diseaseand L-DOPA treatment. CNS Neurol Disord Drug Targets 11:251–263.

Espinoza S, Salahpour A, Masri B, Sotnikova TD, Messa M, Barak LS, Caron MG,and Gainetdinov RR (2011) Functional interaction between trace amine-associatedreceptor 1 and dopamine D2 receptor. Mol Pharmacol 80:416–425.

Evans PD and Maqueira B (2005) Insect octopamine receptors: a new classificationscheme based on studies of cloned Drosophila G-protein coupled receptors. InvertNeurosci 5:111–118.

Eyun SI, Moriyama H, Hoffmann FG, and Moriyama EN (2016) Molecular evolution andfunctional divergence of trace amine-associated receptors. PLoS One 11:e0151023.

Farooqui T (2007) Octopamine-mediated neuromodulation of insect senses. NeurochemRes 32:1511–1529.

Farooqui T, Robinson K, Vaessin H, and Smith BH (2003) Modulation of early ol-factory processing by an octopaminergic reinforcement pathway in the honeybee.J Neurosci 23:5370–5380.

Fatsini E, Bautista R, Manchado M, and Duncan NJ (2016) Transcriptomic profiles of theupper olfactory rosette in cultured and wild Senegalese sole (Solea senegalensis) males.Comp Biochem Physiol Part D Genomics Proteomics 20:125–135.

Fayad LM, Wang X, Blakeley JO, Durand DJ, Jacobs MA, Demehri S, SubhawongTK, Soldatos T, and Barker PB (2014) Characterization of peripheral nerve sheathtumors with 3T proton MR spectroscopy. AJNR Am J Neuroradiol 35:1035–1041.

Fehler M, Broadley KJ, Ford WR, and Kidd EJ (2010) Identification of trace-amine-associated receptors (TAAR) in the rat aorta and their role in vasoconstriction byb-phenylethylamine. Naunyn Schmiedebergs Arch Pharmacol 382:385–398.

Fennema D, Phillips IR, and Shephard EA (2016) Trimethylamine and trimethyl-amine N-oxide, a flavin-containing monooxygenase 3 (FMO3)-mediated host-microbiome metabolic axis implicated in health and disease. Drug Metab Dispos44:1839–1850.


Fernández de Palencia P, Fernández M, Mohedano ML, Ladero V, Quevedo C,Alvarez MA, and López P (2011) Role of tyramine synthesis by food-borne Enterococcusdurans in adaptation to the gastrointestinal tract environment. Appl Environ Microbiol77:699–702.

Ferragud A, Howell AD, Moore CF, Ta TL, Hoener MC, Sabino V, and Cottone P (2017)The trace amine-associated receptor 1 agonist RO5256390 blocks compulsive, binge-likeeating in rats. Neuropsychopharmacology 42:1458–1470.

Ferrero DM, Lemon JK, Fluegge D, Pashkovski SL, Korzan WJ, Datta SR, Spehr M,Fendt M, and Liberles SD (2011) Detection and avoidance of a carnivore odor byprey. Proc Natl Acad Sci USA 108:11235–11240.

Ferrero DM, Wacker D, Roque MA, Baldwin MW, Stevens RC, and Liberles SD(2012) Agonists for 13 trace amine-associated receptors provide insight into themolecular basis of odor selectivity. ACS Chem Biol 7:1184–1189.

Fetissov SO, Bensing S, Mulder J, Le Maitre E, Hulting AL, Harkany T, Ekwall O,Sköldberg F, Husebye ES, Perheentupa J, et al. (2009) Autoantibodies in autoimmunepolyglandular syndrome type I patients react with major brain neurotransmitter sys-tems. J Comp Neurol 513:1–20.

Finberg JPM and Gillman K (2011) Selective inhibitors of monoamine oxidase type Band the “cheese effect”. Int Rev Neurobiol 100:169–190.

Fischer W, Neubert RHH, and Brandsch M (2010) Transport of phenylethylamine atintestinal epithelial (Caco-2) cells: mechanism and substrate specificity. EurJ Pharm Biopharm 74:281–289.

Fish EW, Riday TT, McGuiganMM, Faccidomo S, Hodge CW, and Malanga CJ (2010)Alcohol, cocaine, and brain stimulation-reward in C57Bl6/J and DBA2/J mice.Alcohol Clin Exp Res 34:81–89.

Fisher A, Biggs CS, and Starr MS (1998) Differential effects of NMDA and non-NMDA antagonists on the aromatic L-amino acid decarboxylase activity in thenigrostriatal pathway of the rat. Brain Res 792:126–132.

Flecke C and Stengl M (2009) Octopamine and tyramine modulate pheromone-sensitive olfactory sensilla of the hawkmoth Manduca sexta in a time-dependentmanner. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 195:529–545.

Fleischer J, Schwarzenbacher K, and Breer H (2007) Expression of trace amine-associated receptors in the Grueneberg ganglion. Chem Senses 32:623–631.

Fowler S, Kletzl H, Finel M, Manevski N, Schmid P, Tuerck D, Norcross RD, HoenerMC, Spleiss O, and Iglesias VA (2015) A UGT2B10 splicing polymorphism commonin African populations may greatly increase drug exposure. J Pharmacol Exp Ther352:358–367.

Frascarelli S, Ghelardoni S, Chiellini G, Galli E, Ronca F, Scanlan TS, and Zucchi R(2011) Cardioprotective effect of 3-iodothyronamine in perfused rat heart subjectedto ischemia and reperfusion. Cardiovasc Drugs Ther 25:307–313.

Frenken M and Kaumann AJ (1988) Effects of tryptamine mediated through 2 statesof the 5-HT2 receptor in calf coronary artery. Naunyn Schmiedebergs Arch Pharmacol337:484–492.

Fung TC, Olson CA, and Hsiao EY (2017) Interactions between the microbiota, im-mune and nervous systems in health and disease. Nat Neurosci 20:145–155.

Furuzawa Y, Ohmori Y, and Watanabe T (1994) Immunohistochemical colocalizationof serotonin, aromatic L-amino acid decarboxylase and polypeptide hormones inislet A- and PP-cells of the cat pancreas. J Vet Med Sci 56:911–916.

Fussnecker BL, Smith BH, and Mustard JA (2006) Octopamine and tyramine in-fluence the behavioral profile of locomotor activity in the honey bee (Apis melli-fera). J Insect Physiol 52:1083–1092.

Fuxe K, Grobecker H, and Jonsson J (1967) The effect of beta-phenylethylamine oncentral and peripheral monoamine-containing neurons. Eur J Pharmacol 2:202–207.

Gainetdinov RR, Premont RT, Bohn LM, Lefkowitz RJ, and Caron MG (2004) De-sensitization of G protein-coupled receptors and neuronal functions. Annu RevNeurosci 27:107–144.

Gainetdinov RR, Wetsel WC, Jones SR, Levin ED, Jaber M, and Caron MG (1999)Role of serotonin in the paradoxical calming effect of psychostimulants on hyper-activity. Science 283:397–401.

Gajda Z, Gyengési E, Hermesz E, Ali KS, and Szente M (2003) Involvement of gapjunctions in the manifestation and control of the duration of seizures in rats in vivo.Epilepsia 44:1596–1600.

Gallant P, Malutan T, McLean H, Verellen L, Caveney S, and Donly C (2003)Functionally distinct dopamine and octopamine transporters in the CNS of thecabbage looper moth. Eur J Biochem 270:664–674.

Galley G, Beurier A, Décoret G, Goergler A, Hutter R, Mohr S, Pähler A, Schmid P,Türck D, Unger R, et al. (2015) Discovery and characterization of2-aminooxazolines as highly potent, selective, and orally active TAAR1 agonists.ACS Med Chem Lett 7:192–197.

Galley G, Stalder H, Goergler A, Hoener MC, and Norcross RD (2012) Optimisation ofimidazole compounds as selective TAAR1 agonists: discovery of RO5073012. BioorgMed Chem Lett 22:5244–5248.

Gao S, Liu S, Yao J, Li N, Yuan Z, Zhou T, Li Q, and Liu Z (2017) Genomic organi-zation and evolution of olfactory receptors and trace amine-associated receptors inchannel catfish, Ictalurus punctatus. Biochim Biophys Acta 1861:644–651.

Gardini F, Özogul Y, Suzzi G, Tabanelli G, and Özogul F (2016) Technological factorsaffecting biogenic amine content in foods: a review. Front Microbiol 7:1218.

Gavaghan McKee CL, Wilson ID, and Nicholson JK (2006) Metabolic phenotyping ofnude and normal (Alpk:ApfCD, C57BL10J) mice. J Proteome Res 5:378–384.

Ghelardoni S, Suffredini S, Frascarelli S, Brogioni S, Chiellini G, Ronca-Testoni S,Grandy DK, Scanlan TS, Cerbai E, and Zucchi R (2009) Modulation of cardiac ionichomeostasis by 3-iodothyronamine. J Cell Mol Med 13 (9B):3082–3090.

Gjedde A, Léger GC, Cumming P, Yasuhara Y, Evans AC, Guttman M,and Kuwabara H (1993) Striatal L-dopa decarboxylase activity in Parkinson’sdisease in vivo: implications for the regulation of dopamine synthesis. J Neurochem61:1538–1541.

Gliem S, Schild D, and Manzini I (2009) Highly specific responses to amine odorantsof individual olfactory receptor neurons in situ. Eur J Neurosci 29:2315–2326.

Gliem S, Syed AS, Sansone A, Kludt E, Tantalaki E, Hassenklöver T, Korsching SI,and Manzini I (2013) Bimodal processing of olfactory information in an amphibiannose: odor responses segregate into a medial and a lateral stream. Cell Mol Life Sci70:1965–1984.

Gloriam DE, Bjarnadóttir TK, Schiöth HB, and Fredriksson R (2005) High speciesvariation within the repertoire of trace amine receptors. Ann N Y Acad Sci 1040:323–327.

Gozal EA, O’Neill BE, Sawchuk MA, Zhu H, Halder M, Chou CC, and Hochman S(2014) Anatomical and functional evidence for trace amines as unique modulatorsof locomotor function in the mammalian spinal cord. Front Neural Circuits 8:134.

Grandy DK (2007) Trace amine-associated receptor 1-family archetype or iconoclast?Pharmacol Ther 116:355–390.

Graziano C, Wischmeijer A, Pippucci T, Fusco C, Diquigiovanni C, Nõukas M, SaukM, Kurg A, Rivieri F, Blau N, et al. (2015) Syndromic intellectual disability: a newphenotype caused by an aromatic amino acid decarboxylase gene (DDC) variant.Gene 559:144–148.

Greenshaw AJ (1984) beta-Phenylethylamine and reinforcement. Prog Neuro-psychopharmacol Biol Psychiatry 8:615–620.

Greenshaw AJ, Turrkish S, and Davis BA (2002) The enigma of conditioned tasteaversion learning: stimulus properties of 2-phenylethylamine derivatives. PhysiolRes 51 (Suppl 1):S13–S20.

Grillo MA, Battaglia V, Colombatto S, Rossi CA, Simonian AR, Salvi M, KhomutovAR, and Toninello A (2007) Inhibition of agmatine transport in liver mitochondriaby new charge-deficient agmatine analogues. Biochem Soc Trans 35:401–404.

Grimsby J, Toth M, Chen K, Kumazawa T, Klaidman L, Adams JD, Karoum F, Gal J,and Shih JC (1997) Increased stress response and beta-phenylethylamine inMAOB-deficient mice. Nat Genet 17:206–210.

Grohmann L, Blenau W, Erber J, Ebert PR, Strünker T, and Baumann A (2003)Molecular and functional characterization of an octopamine receptor from honey-bee (Apis mellifera) brain. J Neurochem 86:725–735.

Gründemann D, Hahne C, Berkels R, and Schömig E (2003) Agmatine is efficientlytransported by non-neuronal monoamine transporters extraneuronal monoaminetransporter (EMT) and organic cation transporter 2 (OCT2). J Pharmacol Exp Ther304:810–817.

Gründer G, Hippius H, and Carlsson A (2009) The ‘atypicality’ of antipsychotics: aconcept re-examined and re-defined. Nat Rev Drug Discov 8:197–202.

Grus WE and Zhang J (2008) Distinct evolutionary patterns between chemoreceptorsof 2 vertebrate olfactory systems and the differential tuning hypothesis. Mol BiolEvol 25:1593–1601.

Guariento S, Tonelli M, Espinoza S, Gerasimov AS, Gainetdinov RR, and Cichero E(2018) Rational design, chemical synthesis and biological evaluation of novelbiguanides exploring species-specificity responsiveness of TAAR1 agonists. Eur JMed Chem 146:171–184.

Guest I, Cyr DG, and Varma DR (1994) Mechanism of trimethylamine-induced in-hibition of macromolecular synthesis by mouse embryos in culture. Food ChemToxicol 32:365–371.

Guest I and Varma DR (1992) Teratogenic and macromolecular synthesis inhibitoryeffects of trimethylamine on mouse embryos in culture. J Toxicol Environ Health36:27–41.

Guest I and Varma DR (1993) Selective growth inhibition of the male progeny of micetreated with trimethylamine during pregnancy. Can J Physiol Pharmacol 71:185–187.

Ha K, Shin H, Ju H, Chung CM, and Choi I (2017) Behavioral hypothermia of adomesticated lizard under treatment of the hypometabolic agent3-iodothyronamine. Exp Anim 66:99–105.

Hackenmueller SA, Marchini M, Saba A, Zucchi R, and Scanlan TS (2012) Bio-synthesis of 3-iodothyronamine (T1AM) is dependent on the sodium-iodide sym-porter and thyroperoxidase but does not involve extrathyroidal metabolism of T4.Endocrinology 153:5659–5667.

Hackenmueller SA and Scanlan TS (2012) Identification and quantification of3-iodothyronamine metabolites in mouse serum using liquid chromatography-tandem mass spectrometry. J Chromatogr A 1256:89–97.

Hacker K, Maas R, Kornhuber J, FrommMF, and Zolk O (2015) Substrate-dependentinhibition of the human organic cation transporter OCT2: a comparison of met-formin with experimental substrates. PLoS One 10:e0136451.

Hadjiconstantinou M, Duchemin AM, Azad A, and Neff NH (2010) Aromatic L-aminoacid decarboxylase, in Biogenic Amines: Pharmacological, Neurochemical andMolecular Aspects in the CNS (Farooqui T and Farooqui AA eds) pp 25–45, NovaBiomedical, New York.

Hadjiconstantinou M, Rossetti Z, Silvia C, Krajnc D, and Neff NH (1988) AromaticL-amino acid decarboxylase activity of the rat retina is modulated in vivo by en-vironmental light. J Neurochem 51:1560–1564.

Hadjiconstantinou M, Rossetti ZL, Wemlinger TA, and Neff NH (1995) Dizocilpineenhances striatal tyrosine hydroxylase and aromatic L-amino acid decarboxylaseactivity. Eur J Pharmacol 289:97–101.

Hadjiconstantinou M, Wemlinger TA, Sylvia CP, Hubble JP, and Neff NH (1993)Aromatic L-amino acid decarboxylase activity of mouse striatum is modulated viadopamine receptors. J Neurochem 60:2175–2180.

Hahn SL, Hahn M, Kang UJ, and Joh TH (1993) Structure of the rat aromaticL-amino acid decarboxylase gene: evidence for an alternative promoter usage.J Neurochem 60:1058–1064.

Hammer M (1997) The neural basis of associative reward learning in honeybees.Trends Neurosci 20:245–252.

Han KA, Millar NS, and Davis RL (1998) A novel octopamine receptor withpreferential expression in Drosophila mushroom bodies. J Neurosci 18:3650–3658.

Han Q, Ding H, Robinson H, Christensen BM, and Li J (2010) Crystal structure andsubstrate specificity of Drosophila 3,4-dihydroxyphenylalanine decarboxylase.PLoS One 5:e8826.


Hao X, Liu X, Wang W, Ren H, Xie J, Shen P, Lin D, and Chen N (2013) Distinctmetabolic profile of primary focal segmental glomerulosclerosis revealed by NMR-based metabolomics. PLoS One 8:e78531.

Harada K (1985) Feeding attraction activities of amino acids and nitrogenous basesfor oriental weatherfish. Bull Jap Soc Sci Fish 51:461–466.

Harkness JH, Shi X, Janowsky A, and Phillips TJ (2015) Trace amine-associatedreceptor 1 regulation of methamphetamine intake and related traits. Neuro-psychopharmacology 40:2175–2184.

Harmeier A, Obermueller S, Meyer CA, Revel FG, Buchy D, Chaboz S, Dernick G,Wettstein JG, Iglesias A, Rolink A, et al. (2015) Trace amine-associated receptor1 activation silences GSK3b signaling of TAAR1 and D2R heteromers. Eur Neu-ropsychopharmacol 25:2049–2061.

Harris J, Trivedi S, and Ramakrishna BL (1988) A contribution to theneuromodulator/neurotransmitter role of trace amines, in Trace Amines: Com-parative and Clinical Neurobiology (Boulton AA, Juorio AV, and Downer RGH eds)pp 213–221, Humana Press, Totowa, NJ.

Hashemzadeh H, Hollingworth RM, and Voliva A (1985) Receptors for 3H-octopamine in the adult firefly light organ. Life Sci 37:433–440.

Hashiguchi Y, Furuta Y, and Nishida M (2008) Evolutionary patterns and selectivepressures of odorant/pheromone receptor gene families in teleost fishes. PLoS One3:e4083.

Hashiguchi Y and Nishida M (2007) Evolution of trace amine associated receptor(TAAR) gene family in vertebrates: lineage-specific expansions and degradations ofa second class of vertebrate chemosensory receptors expressed in the olfactoryepithelium. Mol Biol Evol 24:2099–2107.

Hauger RL, Skolnick P, and Paul SM (1982) Specific [3H] beta-phenylethylaminebinding sites in rat brain. Eur J Pharmacol 83:147–148.

Haughton BG and King HK (1961) Induced formation of leucine decarboxylase inProteus vulgaris. Biochem J 80:268–277.

Hauser F, Cazzamali G, Williamson M, Blenau W, and Grimmelikhuijzen CJ (2006)A review of neurohormone GPCRs present in the fruitfly Drosophila melanogasterand the honey bee Apis mellifera. Prog Neurobiol 80:1–19.

Hayashi M, Yamaji Y, Kitajima W, and Saruta T (1990) Aromatic L-amino aciddecarboxylase activity along the rat nephron. Am J Physiol 258:F28–F33.

Henry DP, Russell WL, Clemens JA, and Plebus LA (1988) Phenylethylamine andp-tyramine in the extracellular space of the rat brain: quantification using a newradioenzymatic assay and in situ microdialysis, in Trace Amines; Comparative andClinical Neurobiology (Boulton AA, Juorio AV, and Downer RGH eds) pp 239–250,Humana Press, Totowa, NJ.

Hicks TP and McLennan H (1978) Comparison of the actions of octopamine andcatecholamines on single neurones of the rat cerebral cortex. Br J Pharmacol 64:485–491.

Hiripi L, Juhos S, and Downer RG (1994) Characterization of tyramine and octop-amine receptors in the insect (Locusta migratoria migratorioides) brain. Brain Res633:119–126.

Hiroi T, Imaoka S, and Funae Y (1998) Dopamine formation from tyramine byCYP2D6. Biochem Biophys Res Comm 249:838–843.

Hjort AM (1934) Some physiological properties of certain n-methylated-beta-phen-ylethylamines. J Pharmacol Exp Ther 52:101–112.

Hoefig CS, Köhrle J, Brabant G, Dixit K, Yap B, Strasburger CJ, and Wu Z (2011)Evidence for extrathyroidal formation of 3-iodothyronamine in humans as providedby a novel monoclonal antibody-based chemiluminescent serum immunoassay.J Clin Endocrinol Metab 96:1864–1872.

Hoefig CS, Renko K, Piehl S, Scanlan TS, Bertoldi M, Opladen T, Hoffmann GF,Klein J, Blankenstein O, Schweizer U, et al. (2012) Does the aromatic L-amino aciddecarboxylase contribute to thyronamine biosynthesis? Mol Cell Endocrinol 349:195–201.

Hoefig CS, Wuensch T, Rijntjes E, Lehmphul I, Daniel H, Schweizer U, Mittag J,and Kohrle J (2015) Biosynthesis of 3-iodothyronamine from T4 in murine in-testinal tissue. Endocrinology 156:4356–4364.

Hoefig CS, Zucchi R, and Köhrle J (2016) Thyronamines and derivatives: physio-logical relevance, pharmacological actions, and future research directions. Thyroid26:1656–1673.

Homberg U, Seyfarth J, Binkle U, Monastirioti M, and Alkema MJ (2013) Iden-tification of distinct tyraminergic and octopaminergic neurons innervating thecentral complex of the desert locust, Schistocerca gregaria. J Comp Neurol 521:2025–2041.

Honda K and Littman DR (2016) The microbiota in adaptive immune homeostasisand disease. Nature 535:75–84.

Horowitz LF, Saraiva LR, Kuang D, Yoon KH, and Buck LB (2014) Olfactory receptorpatterning in a higher primate. J Neurosci 34:12241–12252.

Hoyer SC, Eckart A, Herrel A, Zars T, Fischer SA, Hardie SL, and Heisenberg M(2008) Octopamine in male aggression of Drosophila. Curr Biol 18:159–167.

Hu LA, Zhou T, Ahn J, Wang S, Zhou J, Hu Y, and Liu Q (2009) Human and mousetrace amine-associated receptor 1 have distinct pharmacology towards endogenousmonoamines and imidazoline receptor ligands. Biochem J 424:39–45.

Huang J, Ohta H, Inoue N, Takao H, Kita T, Ozoe F, and Ozoe Y (2009) Molecularcloning and pharmacological characterization of a Bombyx mori tyramine receptorselectively coupled to intracellular calcium mobilization. Insect Biochem Mol Biol39:842–849.

Huang J, Wu SF, Li XH, Adamo SA, and Ye GY (2012) The characterization of aconcentration-sensitive a-adrenergic-like octopamine receptor found on insect im-mune cells and its possible role in mediating stress hormone effects on immunefunction. Brain Behav Immun 26:942–950.

Hukkanen J, Dempsey D, Jacob P 3rd, and Benowitz NL (2005) Effect of pregnancyon a measure of FMO3 activity. Br J Clin Pharmacol 60:224–226.

Humbert JA, Hammond KB, and Hathaway WE (1970) Trimethylaminuria: the fish-odour syndrome. Lancet 2:770–771.

Hussain A, Saraiva LR, Ferrero DM, Ahuja G, Krishna VS, Liberles SD,and Korsching SI (2013) High-affinity olfactory receptor for the death-associatedodor cadaverine. Proc Natl Acad Sci USA 110:19579–19584.

Hussain A, Saraiva LR, and Korsching SI (2009) Positive Darwinian selection andthe birth of an olfactory receptor clade in teleosts. Proc Natl Acad Sci USA 106:4313–4318.

Ianculescu AG and Scanlan TS (2010) 3-Iodothyronamine (T(1)AM): a new chapter ofthyroid hormone endocrinology? Mol Biosyst 6:1338–1344.

Ichinose H, Sumi-Ichinose C, Ohye T, Hagino Y, Fujita K, and Nagatsu T (1992)Tissue-specific alternative splicing of the first exon generates two types ofmRNAs in human aromatic L-amino acid decarboxylase. Biochemistry 31:11546–11550.

Ikeda M, Iwata N, Suzuki T, Kitajima T, Yamanouchi Y, Kinoshita Y, Inada T,and Ozaki N (2005) No association of haplotype-tagging SNPs in TRAR4 withschizophrenia in Japanese patients. Schizophr Res 78:127–130.

Ikemoto K, Nishimura A, Oda T, Nagatsu I, and Nishi K (2003) Number of striatalD-neurons is reduced in autopsy brains of schizophrenics. Leg Med (Tokyo) 5(Suppl 1):S221–S224.

Imre I, Di Rocco RT, Belanger CF, Brown GE, and Johnson NS (2014) The behav-ioural response of adult Petromyzon marinus to damage-released alarm andpredator cues. J Fish Biol 84:1490–1502.

Irsfeld M, Spadafore M, and Prüß BM (2013) b-phenylethylamine, a small moleculewith a large impact. Webmedcentral 4:4409.

Ishida Y and Ozaki M (2012) Aversive odorant causing appetite decrease down-regulates tyrosine decarboxylase gene expression in the olfactory receptor neuronof the blowfly, Phormia regina. Naturwissenschaften 99:71–75.

Ito J, Ito M, Nambu H, Fujikawa T, Tanaka K, Iwaasa H, and Tokita S (2009)Anatomical and histological profiling of orphan G-protein-coupled receptor ex-pression in gastrointestinal tract of C57BL/6J mice. Cell Tissue Res 338:257–269.

Jacob MS and Presti DE (2005) Endogenous psychoactive tryptamines reconsidered:an anxiolytic role for dimethyltryptamine. Med Hypotheses 64:930–937.

Jacobs JP, Goudarzi M, Singh N, Tong M, McHardy IH, Ruegger P, Asadourian M,Moon BH, Ayson A, Borneman J, et al. (2016) A disease-associated microbial andmetabolomics state in relatives of pediatric inflammatory bowel disease patients.Cell Mol Gastroenterol Hepatol 2:750–766.

Jaeger CB, Ruggiero DA, Albert VR, Park DH, Joh TH, and Reis DJ (1984) AromaticL-amino acid decarboxylase in the rat brain: immunocytochemical localization inneurons of the brain stem. Neuroscience 11:691–713.

Jaeger CB, Teitelman G, Joh TH, Albert VR, Park DH, and Reis DJ (1983) Someneurons of the rat central nervous system contain aromatic-L-amino-acid decar-boxylase but not monoamines. Science 219:1233–1235.

James TD, Moffett SX, Scanlan TS, and Martin JV (2013) Effects of acute microinjectionsof the thyroid hormone derivative 3-iodothyronamine to the preoptic region of adultmale rats on sleep, thermoregulation and motor activity. Horm Behav 64:81–88.

Janssen PA, Leysen JE, Megens AA, and Awouters FH (1999) Does phenylethyl-amine act as an endogenous amphetamine in some patients? Int J Neuro-psychopharmacol 2:229–240.

Jansson J, Willing B, Lucio M, Fekete A, Dicksved J, Halfvarson J, Tysk C,and Schmitt-Kopplin P (2009) Metabolomics reveals metabolic biomarkers ofCrohn’s disease. PLoS One 4:e6386.

Jing L and Li JX (2015) Trace amine-associated receptor 1: a promising target for thetreatment of psychostimulant addiction. Eur J Pharmacol 761:345–352.

Jing L, Zhang Y, and Li JX (2014) Effects of the trace amine associated receptor1 agonist RO5263397 on abuse-related behavioral indices of methamphetamine inrats. Int J Neuropsychopharmacol 18:pyu060.

John J, Kukshal P, Bhatia T, Chowdari KV, Nimgaonkar VL, Deshpande SN,and Thelma BK (2017) Possible role of rare variants in trace amine associatedreceptor 1 in schizophrenia. Schizophr Res 189:190–195.

Johnson MA, Tsai L, Roy DS, Valenzuela DH, Mosley C, Magklara A, Lomvardas S,Liberles SD, and Barnea G (2012) Neurons expressing trace amine-associated re-ceptors project to discrete glomeruli and constitute an olfactory subsystem. ProcNatl Acad Sci USA 109:13410–13415.

Jones RS (1982a) Noradrenaline-octopamine interactions on cortical neurones in therat. Eur J Pharmacol 77:159–162.

Jones RS (1982b) Tryptamine modifies cortical neurone responses evoked by stim-ulation of nucleus raphe medianus. Brain Res Bull 8:435–437.

Jones RS and Boulton AA (1980a) Tryptamine and 5-hydroxytryptamine: actions andinteractions of cortical neurones in the rat. Life Sci 27:1849–1856.

Jones RSG (1983) Trace biogenic-amines - a possible functional-role in the CNS.Trends Pharmacol Sci 4:426–429.

Jones RSG and Boulton AA (1980b) Interactions between p-tyramine, m-tyramine, orbeta-phenylethylamine and dopamine on single neurones in the cortex and caudatenucleus of the rat. Can J Physiol Pharmacol 58:222–227.

Ju H, So H, Ha K, Park K, Lee JW, Chung CM, and Choi I (2011) Sustained torpidityfollowing multi-dose administration of 3-iodothyronamine in mice. J Cell Physiol226:853–858.

Juorio AV (1979) Drug-induced changes in the formation, storage and metabolism oftyramine in the mouse. Br J Pharmacol 66:377–384.

Juorio AV, Greenshaw AJ, and Wishart TB (1988) Reciprocal changes in striatal dopa-mine and beta-phenylethylamine induced by reserpine in the presence of monoamineoxidase inhibitors. Naunyn Schmiedebergs Arch Pharmacol 338:644–648.

Juorio AV, Greenshaw AJ, Zhu MY, and Paterson IA (1991) The effects of someneuroleptics and d-amphetamine on striatal 2-phenylethylamine in the mouse.GenPharmacol 22:407–413.

Juorio AV, Li XM, Walz W, and Paterson IA (1993) Decarboxylation of L-dopa bycultured mouse astrocytes. Brain Res 626:306–309.

Juorio AV and Yu PH (1985a) Effects of benzene and other organic solvents on thedecarboxylation of some brain aromatic-L-amino acids. Biochem Pharmacol 34:1381–1387.


Juorio AV and Yu PH (1985b) Effects of benzene and pyridine on the concentration ofmouse striatal tryptamine and 5-hydroxytryptamine. Biochem Pharmacol 34:3774–3776.

Kalnins G, Kuka J, Grinberga S, Makrecka-Kuka M, Liepinsh E, Dambrova M,and Tars K (2015) Structure and function of cutC choline lyase from humanmicrobiota bacterium Klebsiella pneumoniae. J Biol Chem 290:21732–21740.

Karoum F, Linnoila M, Potter WZ, Chuang LW, Goodwin FK, and Wyatt RJ (1982)Fluctuating high urinary phenylethylamine excretion rates in some bipolar affec-tive disorder patients. Psychiatry Res 6:215–222.

Kastner KW, Shoue DA, Estiu GL, Wolford J, Fuerst MF, Markley LD, Izaguirre JA,and McDowell MA (2014) Characterization of the Anopheles gambiae octopaminereceptor and discovery of potential agonists and antagonists using a combinedcomputational-experimental approach. Malar J 13:434–447.

Kaufmann CA, Suarez B, Malaspina D, Pepple J, Svrakic D, Markel PD, Meyer J,Zambuto CT, Schmitt K, Matise TC, et al. (1998) NIMH Genetics Initiative Mil-lenium Schizophrenia Consortium: linkage analysis of African-American pedi-grees. Am J Med Genet 81:282–289.

Kellar KJ and Cascio CS (1982) [3H]Tryptamine: high affinity binding sites in ratbrain. Eur J Pharmacol 78:475–478.

Khajavi N, Reinach PS, Slavi N, Skrzypski M, Lucius A, Strauß O, Köhrle J,and Mergler S (2015) Thyronamine induces TRPM8 channel activation in humanconjunctival epithelial cells. Cell Signal 27:315–325.

Kidd M, Modlin IM, Gustafsson BI, Drozdov I, Hauso O, and Pfragner R (2008)Luminal regulation of normal and neoplastic human EC cell serotonin release ismediated by bile salts, amines, tastants, and olfactants. Am J Physiol GastrointestLiver Physiol 295:G260–G272.

Kido Y, Matsson P, and Giacomini KM (2011) Profiling of a prescription drug libraryfor potential renal drug-drug interactions mediated by the organic cation trans-porter 2. J Med Chem 54:4548–4558.

Kim DH, Maneen MJ, and Stahl SM (2009) Building a better antipsychotic: receptortargets for the treatment of multiple symptom dimensions of schizophrenia. Neu-rotherapeutics 6:78–85.

Kim SG, Bae HS, and Lee ST (2001) A novel denitrifying bacterial isolate that de-grades trimethylamine both aerobically and anaerobically via two different path-ways. Arch Microbiol 176:271–277.

Kirkwood S and Marion L (1950) The biogenesis of alkaloids I. The isolation ofN-methyltyramine from barley. J Am Chem Soc 72:2522–2524.

Kitahama K, Ikemoto K, Jouvet A, Araneda S, Nagatsu I, Raynaud B, NishimuraA, Nishi K, and Niwa S (2009) Aromatic L-amino acid decarboxylase-immunoreactive structures in human midbrain, pons, and medulla. J ChemNeuroanat 38:130–140.

Kleinau G, Pratzka J, Nürnberg D, Grüters A, Führer-Sakel D, Krude H, Köhrle J,Schöneberg T, and Biebermann H (2011) Differential modulation of beta-adrenergic receptor signaling by trace amine-associated receptor 1 agonists.PLoS One 6:e27073.

Klieverik LP, Foppen E, Ackermans MT, Serlie MJ, Sauerwein HP, Scanlan TS,Grandy DK, Fliers E, and Kalsbeek A (2009) Central effects of thyronamines onglucose metabolism in rats. J Endocrinol 201:377–386.

Knight ME and Harris J (1993) Investigations into the biochemical basis of neuro-modulation by 2-phenylethylamine: effect on microtubule protein. Neurochem Res18:1221–1229.

Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y,Li L, et al. (2013) Intestinal microbiota metabolism of L-carnitine, a nutrient in redmeat, promotes atherosclerosis. Nat Med 19:576–585.

Kojima K, Anzai R, Ohba C, Goto T, Miyauchi A, Thöny B, Saitsu H, Matsumoto N,Osaka H, and Yamagata T (2016) A female case of aromatic l-amino acid decar-boxylase deficiency responsive to MAO-B inhibition. Brain Dev 38:959–963.

Könczöl Á, Rendes K, Dékány M, Müller J, Riethmüller E, and Balogh GT (2016)Blood-brain barrier specific permeability assay reveals N-methylated tyraminederivatives in standardised leaf extracts and herbal products of Ginkgo biloba.J Pharm Biomed Anal 131:167–174.

Kononenko NL, Wolfenberg H, and Pflüger HJ (2009) Tyramine as an independenttransmitter and a precursor of octopamine in the locust central nervous system: animmunocytochemical study. J Comp Neurol 512:433–452.

Kubo H, Shibato J, Saito T, Ogawa T, Rakwal R, and Shioda S (2015) Unraveling therat intestine, spleen and liver genome-wide transcriptome after the oral adminis-tration of lavender oil by a two-color dye-swap DNA microarray approach. PLoSOne 10:e0129951.

Kutsukake M, Komatsu A, Yamamoto D, and Ishiwa-Chigusa S (2000) A tyraminereceptor gene mutation causes a defective olfactory behavior in Drosophila mela-nogaster. Gene 245:31–42.

Laakso A, Pohjalainen T, Bergman J, Kajander J, Haaparanta M, Solin O, SyvälahtiE, and Hietala J (2005) The A1 allele of the human D2 dopamine receptor gene isassociated with increased activity of striatal L-amino acid decarboxylase in healthysubjects. Pharmacogenet Genomics 15:387–391.

Lam VM, Espinoza S, Gerasimov AS, Gainetdinov RR, and Salahpour A (2015) In-vivopharmacology of trace-amine associated receptor 1.Eur J Pharmacol 763 (Pt B):136–142.

Lancaster GA and Sourkes TL (1972) Purification and properties of hog-kidney 3,4-dihydroxyphenylalanine decarboxylase. Can J Biochem 50:791–797.

Lang DH, Yeung CK, Peter RM, Ibarra C, Gasser R, Itagaki K, Philpot RM,and Rettie AE (1998) Isoform specificity of trimethylamine N-oxygenation by hu-man flavin-containing monooxygenase (FMO) and P450 enzymes: selective catal-ysis by FMO3. Biochem Pharmacol 56:1005–1012.

Lange AB (2009) Tyramine: from octopamine precursor to neuroactive chemical ininsects. Gen Comp Endocrinol 162:18–26.

Laube G and Bernstein HG (2017) Agmatine: multifunctional arginine metaboliteand magic bullet in clinical neuroscience? Biochem J 474:2619–2640.

Laurino A, De Siena G, Saba A, Chiellini G, Landucci E, Zucchi R, and Raimondi L(2015) In the brain of mice, 3-iodothyronamine (T1AM) is converted into3-iodothyroacetic acid (TA1) and it is included within the signaling network

connecting thyroid hormone metabolites with histamine. Eur J Pharmacol 761:130–134.

Laurino A, Matucci R, Vistoli G, and Raimondi L (2016) 3-iodothyronamine (T1AM),a novel antagonist of muscarinic receptors. Eur J Pharmacol 793:35–42.

Laurino A and Raimondi L (2017) Commentary: torpor: the rise and fall of3-monoiodothyronamine from brain to gut-from gut to brain? Front Endocrinol(Lausanne) 8:206.

Lauweryns JM and Van Ranst L (1988) Immunocytochemical localization of aromaticL-amino acid decarboxylase in human, rat, and mouse bronchopulmonary andgastrointestinal endocrine cells. J Histochem Cytochem 36:1181–1186.

Lawford BR, Young RM, Noble EP, Sargent J, Rowell J, Shadforth S, Zhang X,and Ritchie T (2000) The D(2) dopamine receptor A(1) allele and opioid de-pendence: association with heroin use and response to methadone treatment. AmJ Med Genet 96:592–598.

Lee DK, Lynch KR, Nguyen T, Im DS, Cheng R, Saldivia VR, Liu Y, Liu IS, Heng HH,Seeman P, et al. (2000) Cloning and characterization of additional members of theG protein-coupled receptor family. Biochim Biophys Acta 1490:311–323.

Lee H-G, Seong C-S, Kim Y-C, Davis RL, and Han K-A (2003) Octopamine receptorOAMB is required for ovulation in Drosophila melanogaster. Dev Biol 264:179–190.

Lehman HK, Schulz DJ, Barron AB, Wraight L, Hardison C, Whitney S, Takeuchi H,Paul RK, and Robinson GE (2006) Division of labor in the honey bee (Apis melli-fera): the role of tyramine beta-hydroxylase. J Exp Biol 209:2774–2784.

Lenders JW, Eisenhofer G, Abeling NG, Berger W, Murphy DL, Konings CH,Wagemakers LM, Kopin IJ, Karoum F, van Gennip AH, et al. (1996) Specific ge-netic deficiencies of the A and B isoenzymes of monoamine oxidase are charac-terized by distinct neurochemical and clinical phenotypes. J Clin Invest 97:1010–1019.

Lenders JWM and Eisenhofer G (2017) Update on modern management of pheo-chromocytoma and paraganglioma. Endocrinol Metab (Seoul) 32:152–161.

Leniger T, Thöne J, Bonnet U, Hufnagel A, Bingmann D, and Wiemann M (2004)Levetiracetam inhibits Na+-dependent Cl-/HCO3- exchange of adult hippocampalCA3 neurons from guinea-pigs. Br J Pharmacol 142:1073–1080.

Leo D, Mus L, Espinoza S, Hoener MC, Sotnikova TD, and Gainetdinov RR (2014)Taar1-mediated modulation of presynaptic dopaminergic neurotransmission: roleof D2 dopamine autoreceptors. Neuropharmacology 81:283–291.

Leo D, Sukhanov I, Zoratto F, Illiano P, Caffino L, Sanna F, Messa G, Emanuele M,Esposito A, Dorofeikova M, et al. (2018) Pronounced hyperactivity, cognitive dys-functions, and BDNF dysregulation in dopamine transporter knock-out rats.J Neurosci 38:1959–1972.

Leonti M and Casu L (2014) Soma, food of the immortals according to the BowerManuscript (Kashmir, 6th century A.D.). J Ethnopharmacol 155:373–386.

Levinson DF, Holmans P, Straub RE, Owen MJ, Wildenauer DB, Gejman PV, PulverAE, Laurent C, Kendler KS, Walsh D, et al. (2000) Multicenter linkage study ofschizophrenia candidate regions on chromosomes 5q, 6q, 10p, and 13q: schizo-phrenia linkage collaborative group III. Am J Hum Genet 67:652–663.

Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R, and Reis DJ (1994) Agmatine:an endogenous clonidine-displacing substance in the brain. Science 263:966–969.

Li J and Meltzer HY (2014) A genetic locus in 7p12.2 associated with treatmentresistant schizophrenia. Schizophr Res 159:333–339.

Li Q, Korzan WJ, Ferrero DM, Chang RB, Roy DS, Buchi M, Lemon JK, Kaur AW,Stowers L, Fendt M, et al. (2013) Synchronous evolution of an odor biosynthesispathway and behavioral response. Curr Biol 23:11–20.

Li Q, Tachie-Baffour Y, Liu Z, Baldwin MW, Kruse AC, and Liberles SD (2015) Non-classical amine recognition evolved in a large clade of olfactory receptors. eLife 4:e10441.

Li W-G, Yu Y, Zhang Z-D, Cao H, and Xu T-L (2010) ASIC3 channels integrateagmatine and multiple inflammatory signals through the nonproton ligand sensingdomain. Mol Pain 6:88–102.

Li XM, Juorio AV, Paterson IA, and Boulton AA (1992a) Absence of2-phenylethylamine binding after monoamine oxidase inhibition in rat brain. EurJ Pharmacol 210:189–193.

Li XM, Juorio AV, Paterson IA, Walz W, Zhu MY, and Boulton AA (1992b) Geneexpression of aromatic L-amino acid decarboxylase in cultured rat glial cells. JNeurochem 59:1172–1175.

Li Y, Li L, Stephens MJ, Zenner D, Murray KC, Winship IR, Vavrek R, Baker GB,Fouad K, and Bennett DJ (2014) Synthesis, transport, and metabolism of serotoninformed from exogenously applied 5-HTP after spinal cord injury in rats. J Neu-rophysiol 111:145–163.

Libants S, Carr K, Wu H, Teeter JH, Chung-Davidson YW, Zhang Z, Wilkerson C,and Li W (2009) The sea lamprey Petromyzon marinus genome reveals the earlyorigin of several chemosensory receptor families in the vertebrate lineage. BMCEvol Biol 9:180.

Liberles SD (2014) Mammalian pheromones. Annu Rev Physiol 76:151–175.Liberles SD (2015) Trace amine-associated receptors: ligands, neural circuits, andbehaviors. Curr Opin Neurobiol 34:1–7.

Liberles SD and Buck LB (2006) A second class of chemosensory receptors in theolfactory epithelium. Nature 442:645–650.

Lichtenberger LM, Delansorne R, and Graziani LA (1982) Importance of amino aciduptake and decarboxylation in gastrin release from isolated G cells. Nature 295:698–700.

Lichtenberger LM, Gardner JW, Barreto JC, and Morriss FH Jr (1991) Evidence for arole of volatile amines in the development of neonatal hypergastrinemia. J PediatrGastroenterol Nutr 13:342–346.

Lim J, Sabandal PR, Fernandez A, Sabandal JM, Lee HG, Evans P, and Han KA(2014) The octopamine receptor Octb2R regulates ovulation in Drosophila mela-nogaster. PLoS One 9:e104441.

Lindemann L, Ebeling M, Kratochwil NA, Bunzow JR, Grandy DK, and Hoener MC(2005) Trace amine-associated receptors form structurally and functionally distinctsubfamilies of novel G protein-coupled receptors. Genomics 85:372–385.


Lindemann L and Hoener MC (2005) A renaissance in trace amines inspired by anovel GPCR family. Trends Pharmacol Sci 26:274–281.

Lindemann L and Hoener MC (2008) Trace amines, in Encyclopedia of MolecularPharmacology (Offermanns S and Rosenthal W eds) pp 1217–1223, Springer,Berlin.

Lindemann L, Meyer CA, Jeanneau K, Bradaia A, Ozmen L, Bluethmann H, BettlerB, Wettstein JG, Borroni E, Moreau JL, et al. (2008) Trace amine-associated re-ceptor 1 modulates dopaminergic activity. J Pharmacol Exp Ther 324:948–956.

Lindström P and Sehlin J (1983) Mechanisms underlying the effects of5-hydroxytryptamine and 5-hydroxytryptophan in pancreatic islets. A proposedrole for L-aromatic amino acid decarboxylase. Endocrinology 112:1524–1529.

Linnoila RI, Gazdar AF, Funa K, and Becker KL (1993) Long-term selective cultureof hamster pulmonary endocrine cells. Anat Rec 236:231–240.

Liu J-F, Seaman R Jr, Johnson B, Zhang B, and Li J-X (2017a) Effects of theTAAR1 partial agonist RO5263397 on morphine-related behaviors. Neuro-psychopharmacology 42:S637.

Liu J-F, Seaman R Jr, Siemian JN, Bhimani R, Johnson B, Zhang Y, Zhu Q, HoenerMC, Park J, Dietz DM, et al. (2018) Role of trace amine-associated receptor 1 innicotine’s behavioral and neurochemical effects. Neuropsychopharmacology [pub-lished ahead of print].

Liu JF, Siemian JN, Seaman R Jr, Zhang Y, and Li JX (2017b) Role of TAAR1 withinthe subregions of the mesocorticolimbic dopaminergic system in cocaine-seekingbehavior. J Neurosci 37:882–892.

Liu JF, Thorn DA, Zhang Y, and Li JX (2016) Effects of trace amine-associatedreceptor 1 agonists on the expression, reconsolidation, and extinction of cocainereward memory. Int J Neuropsychopharmacol 19:pyw009.

Liu X, Grandy DK, and Janowsky A (2014) Ractopamine, a livestock feed additive, isa full agonist at trace amine-associated receptor 1. J Pharmacol Exp Ther 350:124–129.

Livingstone MS and Tempel BL (1983) Genetic dissection of monoamine neuro-transmitter synthesis in Drosophila. Nature 303:67–70.

Lovenberg W, Weissbach H, and Udenfriend S (1962) Aromatic L-amino aciddecarboxylase. J Biol Chem 237:89–93.

Lucius A, Khajavi N, Reinach PS, Köhrle J, Dhandapani P, Huimann P, Ljubojevic N,Grötzinger C, and Mergler S (2016) 3-Iodothyronamine increases transient re-ceptor potential melastatin channel 8 (TRPM8) activity in immortalized humancorneal epithelial cells. Cell Signal 28:136–147.

Ludewick HP, Schwab SG, Albus M, Lerer B, Maier W, Trixler M, and WildenauerDB (2008) No support for an association with TAAR6 and schizophrenia in a linkedpopulation of European ancestry. Psychiatr Genet 18:208–210.

Luethi D, Trachsel D, Hoener MC, and Liechti ME (2017) Monoamine receptor in-teraction profiles of 4-thio-substituted phenethylamines (2C-T drugs). Neuro-pharmacology [published ahead of print].

Luo A, Leach ST, Barres R, Hesson LB, Grimm MC, and Simar D (2017) Themicrobiota and epigenetic regulation of T helper 17/regulatory T cells: in search ofa balanced immune system. Front Immunol 8:417.

Luo Y, Zhu J, and Gao Y (2012) Metabolomic analysis of the plasma of patients withhigh-altitude pulmonary edema (HAPE) using 1H NMR. Mol Biosyst 8:1783–1788.

Luqman A, Nega M, Nguyen M-T, Ebner P, and Götz F (2018) SadA-expressingstaphylococci in the human gut show increased cell adherence and internalization.Cell Reports 22:535–545.

Lynch LJ, Sullivan KA, Vallender EJ, Rowlett JK, Platt DM, and Miller GM (2013)Trace amine associated receptor 1 modulates behavioral effects of ethanol. SubstAbuse 7:117–126.

Ma JZ, Beuten J, Payne TJ, Dupont RT, Elston RC, and Li MD (2005) Haplotypeanalysis indicates an association between the DOPA decarboxylase (DDC) geneand nicotine dependence. Hum Mol Genet 14:1691–1698.

Ma Z, Guo X, Lei H, Li T, Hao S, and Kang L (2015) Octopamine and tyraminerespectively regulate attractive and repulsive behavior in locust phase changes. SciRep 5:8036.

Maguire JJ, Parker WA, Foord SM, Bonner TI, Neubig RR, and Davenport AP (2009)International Union of Pharmacology. LXXII. Recommendations for trace aminereceptor nomenclature. Pharmacol Rev 61:1–8.

Maitre L, Fthenou E, Athersuch T, Coen M, Toledano MB, Holmes E, Kogevinas M,Chatzi L, and Keun HC (2014) Urinary metabolic profiles in early pregnancy areassociated with preterm birth and fetal growth restriction in the Rhea mother-childcohort study. BMC Med 12:110.

Maitre L, Lau CE, Vizcaino E, Robinson O, Casas M, Siskos AP, Want EJ, AthersuchT, Slama R, Vrijheid M, et al. (2017) Assessment of metabolic phenotypic vari-ability in children’s urine using 1H NMR spectroscopy. Sci Rep 7:46082.

Manni ME, De Siena G, Saba A, Marchini M, Dicembrini I, Bigagli E, Cinci L,Lodovici M, Chiellini G, Zucchi R, et al. (2012) 3-Iodothyronamine: a modulator ofthe hypothalamus-pancreas-thyroid axes in mice. Br J Pharmacol 166:650–658.

Manni ME, De Siena G, Saba A, Marchini M, Landucci E, Gerace E, Zazzeri M,Musilli C, Pellegrini-Giampietro D, Matucci R, et al. (2013) Pharmacologicaleffects of 3-iodothyronamine (T1AM) in mice include facilitation of memory ac-quisition and retention and reduction of pain threshold. Br J Pharmacol 168:354–362.

Maqueira B, Chatwin H, and Evans PD (2005) Identification and characterization ofa novel family of Drosophila beta-adrenergic-like octopamine G-protein coupledreceptors. J Neurochem 94:547–560.

Marcobal A, de las Rivas B, and Muñoz R (2006) First genetic characterization of abacterial beta-phenylethylamine biosynthetic enzyme in Enterococcus faeciumRM58. FEMS Microbiol Lett 258:144–149.

Mariotti V, Melissari E, Iofrida C, Righi M, Di Russo M, Donzelli R, Saba A, Fras-carelli S, Chiellini G, Zucchi R, et al. (2014) Modulation of gene expression by3-iodothyronamine: genetic evidence for a lipolytic pattern. PLoS One 9:e106923.

Maxwell GD, Moore MM, and Hildebrand JG (1980) Metabolism of tyramine inthe central nervous system of the moth Manduca sexta. Insect Biochem 10:657–665.

McClung C and Hirsh J (1998) Stereotypic behavioral responses to free-base cocaineand the development of behavioral sensitization in Drosophila. Curr Biol 8:109–112.

McConnell HW, Mitchell SC, Smith RL, and Brewster M (1997) Trimethylaminuriaassociated with seizures and behavioural disturbance: a case report. Seizure 6:317–321.

McCoole MD, Atkinson NJ, Graham DI, Grasser EB, Joselow AL, McCall NM,Welker AM, Wilsterman EJ Jr, Baer KN, Tilden AR, et al. (2012) Genomic analysesof aminergic signaling systems (dopamine, octopamine and serotonin) in Daphniapulex. Comp Biochem Physiol Part D Genomics Proteomics 7:35–58.

McCormack JK, Beitz AJ, and Larson AA (1986) Autoradiographic localization oftryptamine binding sites in the rat and dog central nervous system. J Neurosci 6:94–101.

McGuire PS and Seiden LS (1980) Differential effects of imipramine in rats as afunction of DRL schedule value. Pharmacol Biochem Behav 13:691–694.

Meana C, Rubin JM, Bordallo C, Suarez L, Bordallo J, and Sanchez M (2016) Cor-relation between endogenous polyamines in human cardiac tissues and clinicalparameters in patients with heart failure. J Cell Mol Med 20:302–312.

Meyer JM and Stahl SM (2009) The metabolic syndrome and schizophrenia. ActaPsychiatr Scand 119:4–14.

Meyer T and Hesch RD (1983) Triiodothyronamine–a beta-adrenergic metabolite oftriiodothyronine? Horm Metab Res 15:602–606.

Miao J, Ling AV, Manthena PV, Gearing ME, Graham MJ, Crooke RM, Croce KJ,Esquejo RM, Clish CB, Vicent D, et al.; Morbid Obesity Study Group (2015) Flavin-containing monooxygenase 3 as a potential player in diabetes-associated athero-sclerosis. Nat Commun 6:6498.

Michaelides M, Thanos PK, Volkow ND, and Wang GJ (2012) Dopamine-relatedfrontostriatal abnormalities in obesity and binge-eating disorder: emerging evi-dence for developmental psychopathology. Int Rev Psychiatry 24:211–218.

Miller GM, Verrico CD, Jassen A, Konar M, Yang H, Panas H, Bahn M, Johnson R,and Madras BK (2005) Primate trace amine receptor 1 modulation by the dopa-mine transporter. J Pharmacol Exp Ther 313:983–994.

Miller-Fleming L, Olin-Sandoval V, Campbell K, and Ralser M (2015) Remainingmysteries of molecular biology: the role of polyamines in the cell. J Mol Biol 427:3389–3406.

Miner NB, Elmore JS, Baumann MH, Phillips TJ, and Janowsky A (2017) Traceamine-associated receptor 1 regulation of methamphetamine-induced neurotoxic-ity. Neurotoxicology 63:57–69.

Mitchell SC and Smith RL (2001) Trimethylaminuria: the fish malodor syndrome.Drug Metab Dispos 29:517–521.

Mitchell SC and Smith RL (2010) A physiological role for flavin-containing mono-oxygenase (FMO3) in humans? Xenobiotica 40:301–305.

Mitchell SC and Smith RL (2016) Trimethylamine-the extracorporeal envoy. ChemSenses 41:275–279.

Miyamoto S, Miyake N, Jarskog LF, Fleischhacker WW, and Lieberman JA (2012)Pharmacological treatment of schizophrenia: a critical review of the pharmacologyand clinical effects of current and future therapeutic agents. Mol Psychiatry 17:1206–1227.

Moghaddam B and Javitt D (2012) From revolution to evolution: the glutamate hy-pothesis of schizophrenia and its implication for treatment. Neuro-psychopharmacology 37:4–15.

Molderings GJ and Haenisch B (2012) Agmatine (decarboxylated L-arginine): phys-iological role and therapeutic potential. Pharmacol Ther 133:351–365.

Molinoff PB and Axelrod J (1972) Distribution and turnover of octopamine in tissues.J Neurochem 19:157–163.

Monastirioti M, Linn CE Jr, and White K (1996) Characterization of Drosophilatyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine.J Neurosci 16:3900–3911.

Montioli R, Paiardini A, Kurian MA, Dindo M, Rossignoli G, Heales SJR, Pope S,Voltattorni CB, and Bertoldi M (2016) The novel R347g pathogenic mutation ofaromatic amino acid decarboxylase provides additional molecular insights intoenzyme catalysis and deficiency. Biochim Biophys Acta 1864:676–682.

Moore CF, Sabino V, and Cottone P (2018) Trace amine associated receptor1 (TAAR1) modulation of food reward. Front Pharmacol 9:129.

Morton DB and Evans PD (1984) Octopamine release from an identified neurone inthe locust. J Exp Biol 113:269–287.

Mosnaim AD, Callaghan OH, Hudzik T, and Wolf ME (2013) Rat brain-uptake indexfor phenylethylamine and various monomethylated derivatives.Neurochem Res 38:842–846.

Mueller JC, Steiger S, Fidler AE, and Kempenaers B (2008) Biogenic trace amine-associated receptors (TAARS) are encoded in avian genomes: evidence and possibleimplications. J Hered 99:174–176.

Mühlhaus J, Dinter J, Jyrch S, Teumer A, Jacobi SF, Homuth G, Kühnen P, WiegandS, Grüters A, Völzke H, et al. (2017) Investigation of naturally occurring single-nucleotide variants in human TAAR1. Front Pharmacol 8:807.

Mühlhaus J, Dinter J, Nürnberg D, Rehders M, Depke M, Golchert J, Homuth G, YiCX, Morin S, Köhrle J, et al. (2014) Analysis of human TAAR8 and murine Taar8bmediated signaling pathways and expression profile. Int J Mol Sci 15:20638–20655.

Müller DJ, Chiesa A, Mandelli L, De Luca V, De Ronchi D, Jain U, Serretti A,and Kennedy JL (2010) Correlation of a set of gene variants, life events and per-sonality features on adult ADHD severity. J Psychiatr Res 44:598–604.

Murdoch TB, Fu H, MacFarlane S, Sydora BC, Fedorak RN, and Slupsky CM (2008)Urinary metabolic profiles of inflammatory bowel disease in interleukin-10 gene-deficient mice. Anal Chem 80:5524–5531.

Nagaya Y, Kutsukake M, Chigusa SI, and Komatsu A (2002) A trace amine, tyra-mine, functions as a neuromodulator in Drosophila melanogaster. Neurosci Lett329:324–328.

Naila A, Flint S, Fletcher G, Bremer P, and Meerdink G (2010) Control of biogenicamines in food–existing and emerging approaches. J Food Sci 75:R139–R150.


Nassiri-Asl M, Zamansoltani F, and Zangivand AA (2008) The inhibitory effect oftrimethylamine on the anticonvulsant activities of quinine in the pentylenete-trazole model in rats. Prog Neuropsychopharmacol Biol Psychiatry 32:1496–1500.

Nazimek J, Perini F, Capitao L, McKie S, Browning M, Harmer C, and Deakin B(2016) A phase I functional neuroimaging study of SEP-363856 in healthy volun-teers with high or low stereotypy. Neuropsychopharmacology 41:S393–S394.

Neff NH, Wemlinger TA, Duchemin AM, and Hadjiconstantinou M (2006) Clozapinemodulates aromatic L-amino acid decarboxylase activity in mouse striatum.J Pharmacol Exp Ther 317:480–487.

Nelson DA, Tolbert MD, Singh SJ, and Bost KL (2007) Expression of neuronal traceamine-associated receptor (Taar) mRNAs in leukocytes. J Neuroimmunol 192:21–30.

Nelson TM, Borgogna JL, Brotman RM, Ravel J, Walk ST, and Yeoman CJ (2015)Vaginal biogenic amines: biomarkers of bacterial vaginosis or precursors to vaginaldysbiosis? Front Physiol 6:253.

Nishimura K, Kitamura Y, Inoue T, Umesono Y, Yoshimoto K, Taniguchi T,and Agata K (2008) Characterization of tyramine beta-hydroxylase in planarianDugesia japonica: cloning and expression. Neurochem Int 53:184–192.

Nishimura K, Utsumi K, Yuhara M, Fujitani Y, and Iritani A (1989) Identification ofpuberty-accelerating pheromones in male mouse urine. J Exp Zool 251:300–305.

Niwa T, Murayama N, Umeyama H, Shimizu M, and Yamazaki H (2011) Humanliver enzymes responsible for metabolic elimination of tyramine; a vasopressoragent from daily food. Drug Metab Lett 5:216–219.

Noble EP (2003) D2 dopamine receptor gene in psychiatric and neurologic disordersand its phenotypes. Am J Med Genet B Neuropsychiatr Genet 116B:103–125.

O’Donnell JM and Seiden LS (1983) Differential-reinforcement-of-low-rate 72-secondschedule: selective effects of antidepressant drugs. J Pharmacol Exp Ther 224:80–88.

O’Hara BF, Smith SS, Bird G, Persico AM, Suarez BK, Cutting GR, and Uhl GR(1993) Dopamine D2 receptor RFLPs, haplotypes and their association with sub-stance use in black and Caucasian research volunteers. Hum Hered 43:209–218.

O’Malley KL, Harmon S, Moffat M, Uhland-Smith A, and Wong S (1995) The humanaromatic L-amino acid decarboxylase gene can be alternatively spliced to generateunique protein isoforms. J Neurochem 65:2409–2416.

Ohta H, Takebe Y, Murakami Y, Takahama Y, and Morimura S (2017) Tyramine andb-phenylethylamine, from fermented food products, as agonists for the humantrace amine-associated receptor 1 (hTAAR1) in the stomach. Biosci BiotechnolBiochem 81:1002–1006.

Orchard I and Lange AB (1987) The release of octopamine and proctolin from aninsect visceral muscle: effects of high-potassium saline and neural stimulation.Brain Res 413:251–258.

Pacifico R, Dewan A, Cawley D, Guo C, and Bozza T (2012) An olfactory subsystemthat mediates high-sensitivity detection of volatile amines. Cell Reports 2:76–88.

Pae CU, Drago A, Kim JJ, Patkar AA, Jun TY, De Ronchi D, and Serretti A (2010)TAAR6 variations possibly associated with antidepressant response and suicidalbehavior. Psychiatry Res 180:20–24.

Pae CU, Drago A, Kim JJ, Patkar AA, Jun TY, Lee C, Mandelli L, De Ronchi D, PaikIH, and Serretti A (2008a) TAAR6 variation effect on clinic presentation andoutcome in a sample of schizophrenic in-patients: an open label study. Eur Psy-chiatry 23:390–395.

Pae CU, Drago A, Patkar AA, Jun TY, and Serretti A (2009) Epistasis between a setof variations located in the TAAR6 and HSP-70 genes toward schizophrenia andresponse to antipsychotic treatment. Eur Neuropsychopharmacol 19:806–811.

Pae CU, Yu HS, Amann D, Kim JJ, Lee CU, Lee SJ, Jun TY, Lee C, Paik IH, PatkarAA, et al. (2008b) Association of the trace amine associated receptor 6 (TAAR6)gene with schizophrenia and bipolar disorder in a Korean case control sample.J Psychiatr Res 42:35–40.

Paetsch PR, Baker GB, and Greenshaw AJ (1993) Induction of functional down-regulationof beta-adrenoceptors in rats by 2-phenylethylamine. J Pharm Sci 82:22–24.

Paetsch PR and Greenshaw AJ (1993) Down-regulation of beta-adrenergic and do-paminergic receptors induced by 2-phenylethylamine. Cell Mol Neurobiol 13:203–215.

Panas HN, Lynch LJ, Vallender EJ, Xie Z, Chen GL, Lynn SK, Scanlan TS,and Miller GM (2010) Normal thermoregulatory responses to 3-iodothyronamine,trace amines and amphetamine-like psychostimulants in trace amine associatedreceptor 1 knockout mice. J Neurosci Res 88:1962–1969.

Panas MW, Xie Z, Panas HN, Hoener MC, Vallender EJ, and Miller GM (2012) Traceamine associated receptor 1 signaling in activated lymphocytes. J NeuroimmunePharmacol 7:866–876.

Pankiw T and Page RE Jr (2003) Effect of pheromones, hormones, and handling onsucrose response thresholds of honey bees (Apis mellifera L.). J Comp Physiol ANeuroethol Sens Neural Behav Physiol 189:675–684.

Parker EM and Cubeddu LX (1988) Comparative effects of amphetamine, phenyl-ethylamine and related drugs on dopamine efflux, dopamine uptake and mazindolbinding. J Pharmacol Exp Ther 245:199–210.

Paterson IA (1988) The potentiation of cortical neurone responses to noradrenalineby beta-phenylethylamine: effects of lesions of the locus coeruleus. Neurosci Lett87:139–144.

Paterson IA (1993) The potentiation of cortical neuron responses to noradrenaline by2-phenylethylamine is independent of endogenous noradrenaline. Neurochem Res18:1329–1336.

Paterson IA and Boulton AA (1988) beta-Phenylethylamine enhances single corticalneurone responses to noradrenaline in the rat. Brain Res Bull 20:173–177.

Paterson IA, Juorio AV, Berry MD, and Zhu MY (1991) Inhibition of monoamineoxidase-B by (-)-deprenyl potentiates neuronal responses to dopamine agonists butdoes not inhibit dopamine catabolism in the rat striatum. J Pharmacol Exp Ther258:1019–1026.

Paulos MA and Tessel RE (1982) Excretion of beta-phenethylamine is elevated inhumans after profound stress. Science 215:1127–1129.

Peers EM, Lyles GA, and Callingham BA (1980) The deamination of isoamylamine bymonamine oxidase in mitochondrial preparations from rat liver and heart: acomparison with phenylethylamine. Biochem Pharmacol 29:1097–1102.

Pegg AE (2016) Functions of polyamines in mammals. J Biol Chem 291:14904–14912.

Pei Y, Asif-Malik A, and Canales JJ (2016) Trace amines and the trace amine-associated receptor 1: pharmacology, neurochemistry, and clinical implications.Front Neurosci 10:148.

Pei Y, Asif-Malik A, Hoener M, and Canales JJ (2017) A partial trace amine-associated receptor 1 agonist exhibits properties consistent with a methamphet-amine substitution treatment. Addict Biol 22:1246–1256.

Pei Y, Lee J, Leo D, Gainetdinov RR, Hoener MC, and Canales JJ (2014) Activation ofthe trace amine-associated receptor 1 prevents relapse to cocaine seeking. Neuro-psychopharmacology 39:2299–2308.

Pei Y, Mortas P, Hoener MC, and Canales JJ (2015) Selective activation of the traceamine-associated receptor 1 decreases cocaine’s reinforcing efficacy and preventscocaine-induced changes in brain reward thresholds. Prog NeuropsychopharmacolBiol Psychiatry 63:70–75.

Pellicciari A, Posar A, Cremonini MA, and Parmeggiani A (2011) Epilepsy and tri-methylaminuria: a new case report and literature review. Brain Dev 33:593–596.

Perälä J, Suvisaari J, Saarni SI, Kuoppasalmi K, Isometsä E, Pirkola S, Partonen T,Tuulio-Henriksson A, Hintikka J, Kieseppä T, et al. (2007) Lifetime prevalence ofpsychotic and bipolar I disorders in a general population. Arch Gen Psychiatry 64:19–28.

Pérez-Gómez A, Bleymehl K, Stein B, Pyrski M, Birnbaumer L, Munger SD,Leinders-Zufall T, Zufall F, and Chamero P (2015) Innate predator odor aversiondriven by parallel olfactory subsystems that converge in the ventromedial hypo-thalamus. Curr Biol 25:1340–1346.

Perry DC (1986) [3H]tryptamine autoradiography in rat brain and choroid plexusreveals two distinct sites. J Pharmacol Exp Ther 236:548–559.

Persico AM, Bird G, Gabbay FH, and Uhl GR (1996) D2 dopamine receptor gene TaqIA1 and B1 restriction fragment length polymorphisms: enhanced frequencies inpsychostimulant-preferring polysubstance abusers. Biol Psychiatry 40:776–784.

Pessione E and Cirrincione S (2016) Bioactive molecules released in food by lacticacid bacteria: encrypted peptides and biogenic amines. Front Microbiol 7:876.

Philips SR and Boulton AA (1979) The effect of monoamine oxidase inhibitors onsome arylalkylamines in rate striatum. J Neurochem 33:159–167.

Piehl S, Heberer T, Balizs G, Scanlan TS, Smits R, Koksch B, and Köhrle J (2008)Thyronamines are isozyme-specific substrates of deiodinases. Endocrinology 149:3037–3045.

Piehl S, Hoefig CS, Scanlan TS, and Köhrle J (2011) Thyronamines–past, present,and future. Endocr Rev 32:64–80.

Pietsch CA, Scanlan TS, and Anderson RJ (2007) Thyronamines are substrates forhuman liver sulfotransferases. Endocrinology 148:1921–1927.

Piletz JE, Aricioglu F, Cheng JT, Fairbanks CA, Gilad VH, Haenisch B, Halaris A,Hong S, Lee JE, Li J, et al. (2013) Agmatine: clinical applications after 100 years intranslation. Drug Discov Today 18:880–893.

Pirri JK, McPherson AD, Donnelly JL, Francis MM, and Alkema MJ (2009) Atyramine-gated chloride channel coordinates distinct motor programs of a Caeno-rhabditis elegans escape response. Neuron 62:526–538.

Pribbenow B and Erber J (1996) Modulation of antennal scanning in the honeybee bysucrose stimuli, serotonin, and octopamine: behavior and electrophysiology. Neu-robiol Learn Mem 66:109–120.

Price MA and Vandenbergh JG (1992) Analysis of puberty-accelerating pheromones.J Exp Zool 264:42–45.

Pugh CEM and Quastel JH (1937) Oxidation of aliphatic amines by brain and othertissues. Biochem J 31:286–291.

Qi YX, Xu G, Gu GX, Mao F, Ye GY, Liu W, and Huang J (2017) A new Drosophilaoctopamine receptor responds to serotonin. Insect Biochem Mol Biol 90:61–70.

Raab S, Wang H, Uhles S, Cole N, Alvarez-Sanchez R, Künnecke B, Ullmer C, MatileH, Bedoucha M, Norcross RD, et al. (2015) Incretin-like effects of small moleculetrace amine-associated receptor 1 agonists. Mol Metab 5:47–56.

Raasch W, Schäfer U, Chun J, and Dominiak P (2001) Biological significance ofagmatine, an endogenous ligand at imidazoline binding sites. Br J Pharmacol 133:755–780.

Rahman MK, Nagatsu T, and Kato T (1981) Aromatic L-amino acid decarboxylaseactivity in central and peripheral tissues and serum of rats with L-DOPA and L-5-hydroxytryptophan as substrates. Biochem Pharmacol 30:645–649.

Raiteri M, Del Carmine R, Bertollini A, and Levi G (1977) Effect of sympathomimeticamines on the synaptosomal transport of noradrenaline, dopamine and5-hydroxytryptamine. Eur J Pharmacol 41:133–143.

Rajput AH, Fenton ME, Di Paolo T, Sitte H, Pifl C, and Hornykiewicz O (2004)Human brain dopamine metabolism in levodopa-induced dyskinesia and wearing-off. Parkinsonism Relat Disord 10:221–226.

Reale V, Hannan F, Midgley JM, and Evans PD (1997) The expression of a clonedDrosophila octopamine/tyramine receptor in Xenopus oocytes. Brain Res 769:309–320.

Reed C, Baba H, Zhu Z, Erk J, Mootz JR, Varra NM, Williams RW, and Phillips TJ(2018) A spontaneous mutation in Taar1 impacts methamphetamine-related traitsexclusively in DBA/2 mice from a single vendor. Front Pharmacol 8:993.

Reese EA, Bunzow JR, Arttamangkul S, Sonders MS, and Grandy DK (2007) Traceamine-associated receptor 1 displays species-dependent stereoselectivity for iso-mers of methamphetamine, amphetamine, and para-hydroxyamphetamine. JPharmacol Exp Ther 321:178–186.

Regard JB, Kataoka H, Cano DA, Camerer E, Yin L, Zheng YW, Scanlan TS, HebrokM, and Coughlin SR (2007) Probing cell type-specific functions of Gi in vivo iden-tifies GPCR regulators of insulin secretion. J Clin Invest 117:4034–4043.

Regard JB, Sato IT, and Coughlin SR (2008) Anatomical profiling of G protein-coupled receptor expression. Cell 135:561–571.


Reith J, Benkelfat C, Sherwin A, Yasuhara Y, Kuwabara H, Andermann F, BachneffS, Cumming P, Diksic M, Dyve SE, et al. (1994) Elevated dopa decarboxylaseactivity in living brain of patients with psychosis. Proc Natl Acad Sci USA 91:11651–11654.

Revel FG, Meyer CA, Bradaia A, Jeanneau K, Calcagno E, Andre CB, Haenggi M,Miss MT, Galley G, Norcross RD, et al. (2012a) Brain-specific overexpression oftrace amine-associated receptor 1 alters monoaminergic neurotransmission anddecreases sensitivity to amphetamine. Neuropsychopharmacology 37:2580–2592.

Revel FG, Moreau JL, Gainetdinov RR, Bradaia A, Sotnikova TD, Mory R, Durkin S,Zbinden KG, Norcross R, Meyer CA, et al. (2011) TAAR1 activation modulatesmonoaminergic neurotransmission, preventing hyperdopaminergic and hypo-glutamatergic activity. Proc Natl Acad Sci USA 108:8485–8490.

Revel FG, Moreau JL, Gainetdinov RR, Ferragud A, Velázquez-Sánchez C, SotnikovaTD, Morairty SR, Harmeier A, Groebke Zbinden K, Norcross RD, et al. (2012b)Trace amine-associated receptor 1 partial agonism reveals novel paradigm forneuropsychiatric therapeutics. Biol Psychiatry 72:934–942.

Revel FG, Moreau JL, Pouzet B, Mory R, Bradaia A, Buchy D, Metzler V, Chaboz S,Groebke Zbinden K, Galley G, et al. (2013) A new perspective for schizophrenia:TAAR1 agonists reveal antipsychotic- and antidepressant-like activity, improvecognition and control body weight. Mol Psychiatry 18:543–556.

Reynolds GP and Gray DO (1978) Gas chromatographic detection of N-methyl-2-phenylethylamine: a new component of human urine. J Chromatogr A 145:137–140.

Reynolds GP and Kirk SL (2010) Metabolic side effects of antipsychotic drug treat-ment–pharmacological mechanisms. Pharmacol Ther 125:169–179.

Reynolds GP, Seakins JW, and Gray DO (1978) The urinary excretion of2-phenylethylamine in phenylketonuria. Clin Chim Acta 83:33–39.

Riba J, Valle M, Urbano G, Yritia M, Morte A, and Barbanoj MJ (2003) Humanpharmacology of ayahuasca: subjective and cardiovascular effects, monoaminemetabolite excretion, and pharmacokinetics. J Pharmacol Exp Ther 306:73–83.

Rigby LM and Merritt DJ (2011) Roles of biogenic amines in regulating bio-luminescence in the Australian glowworm Arachnocampa flava. J Exp Biol 214:3286–3293.

Rillich J, Schildberger K, and Stevenson PA (2011) Octopamine and occupancy: anaminergic mechanism for intruder-resident aggression in crickets. Proc Biol Sci278:1873–1880.

Ringstad N, Abe N, and Horvitz HR (2009) Ligand-gated chloride channels are re-ceptors for biogenic amines in C. elegans. Science 325:96–100.

Robacker DC, Garcia JA, and Bartelt RJ (2000) Volatiles from duck feces attractive toMexican fruit fly. J Chem Ecol 26:1849–1867.

Robb S, Cheek TR, Hannan FL, Hall LM, Midgley JM, and Evans PD (1994) Agonist-specific coupling of a cloned Drosophila octopamine/tyramine receptor to multiplesecond messenger systems. EMBO J 13:1325–1330.

Roeder T (2005) Tyramine and octopamine: ruling behavior and metabolism. AnnuRev Entomol 50:447–477.

Roeder T (2016) Trace amines: an overview, in Trace Amines and Neurological Dis-orders: Potential Mechanisms and Risk Factors (Farooqui T and Farooqui AA eds)pp 3–9, Academic Press, Boston.

Roeder T, Degen J, Dyczkowski C, and Gewecke M (1995) Pharmacology and mo-lecular biology of octopamine receptors from different insect species. Prog BrainRes 106:249–258.

Roeder T and Gewecke M (1989) Octopamine uptake systems in thoracic ganglia andleg muscles of Locusta migratoria. Comp Biochem Physiol C Toxicol Pharmacol 94:143–147.

Roeder T, Seifert M, Kähler C, and Gewecke M (2003) Tyramine and octopamine:antagonistic modulators of behavior and metabolism. Arch Insect Biochem Physiol54:1–13.

Rorsman F, Husebye ES, Winqvist O, Björk E, Karlsson FA, and Kämpe O (1995)Aromatic-L-amino-acid decarboxylase, a pyridoxal phosphate-dependent enzyme,is a beta-cell autoantigen. Proc Natl Acad Sci USA 92:8626–8629.

Rossetti Z, Krajnc D, Neff NH, and Hadjiconstantinou M (1989) Modulation of retinalaromatic L-amino acid decarboxylase via alpha 2 adrenoceptors. J Neurochem 52:647–652.

Rossetti ZL, Silvia CP, Krajnc D, Neff NH, and Hadjiconstantinou M (1990) AromaticL-amino acid decarboxylase is modulated by D1 dopamine receptors in rat retina.J Neurochem 54:787–791.

Roy G, Placzek E, and Scanlan TS (2012) ApoB-100-containing lipoproteins are majorcarriers of 3-iodothyronamine in circulation. J Biol Chem 287:1790–1800.

Roy S and Trinchieri G (2017) Microbiota: a key orchestrator of cancer therapy. NatRev Cancer 17:271–285.

Saavedra JM (1974) Enzymatic isotopic assay for and presence of beta-phenylethylamine in brain. J Neurochem 22:211–216.

Saavedra JM and Axelrod J (1973) Effect of drugs on the tryptamine content of rattissues. J Pharmacol Exp Ther 185:523–529.

Saba A, Chiellini G, Frascarelli S, Marchini M, Ghelardoni S, Raffaelli A, TonaccheraM, Vitti P, Scanlan TS, and Zucchi R (2010) Tissue distribution and cardiac me-tabolism of 3-iodothyronamine. Endocrinology 151:5063–5073.

Sabelli H, Fink P, Fawcett J, and Tom C (1996) Sustained antidepressant effect ofPEA replacement. J Neuropsychiatry Clin Neurosci 8:168–171.

Sabelli HC and Javaid JI (1995) Phenylethylamine modulation of affect: therapeuticand diagnostic implications. J Neuropsychiatry Clin Neurosci 7:6–14.

Sabelli HC and Mosnaim AD (1974) Phenylethylamine hypothesis of affective be-havior. Am J Psychiatry 131:695–699.

Sabelli HC, Vazquez AJ, Mosnaim AD, and Madrid-Pedemonte L (1974) 2-Phenyl-ethylamine as a possible mediator for delta9-tetrahydrocannabinol-induced stim-ulation. Nature 248:144–145.

Safratowich BD, Hossain M, Bianchi L, and Carvelli L (2014) Amphetamine poten-tiates the effects of b-phenylethylamine through activation of an amine-gatedchloride channel. J Neurosci 34:4686–4691.

Sánchez M, Suárez L, Andrés MT, Flórez BH, Bordallo J, Riestra S, and CantabranaB (2017) Modulatory effect of intestinal polyamines and trace amines on thespontaneous phasic contractions of the isolated ileum and colon rings of mice. FoodNutr Res 61:1321948.

Sanders AR, Duan J, Levinson DF, Shi J, He D, Hou C, Burrell GJ, Rice JP, NertneyDA, Olincy A, et al. (2008) No significant association of 14 candidate genes withschizophrenia in a large European ancestry sample: implications for psychiatricgenetics. Am J Psychiatry 165:497–506.

Santoru ML, Piras C, Murgia A, Palmas V, Camboni T, Liggi S, Ibba I, Lai MA, OrrùS, Blois S, et al. (2017) Cross sectional evaluation of the gut-microbiome metab-olome axis in an Italian cohort of IBD patients. Sci Rep 7:9523.

Santos PS, Courtiol A, Heidel AJ, Höner OP, Heckmann I, Nagy M, Mayer F, PlatzerM, Voigt CC, and Sommer S (2016) MHC-dependent mate choice is linked to atrace-amine-associated receptor gene in a mammal. Sci Rep 6:38490.

Saraiva LR, Kondoh K, Ye X, Yoon KH, Hernandez M, and Buck LB (2016) Combinatorialeffects of odorants on mouse behavior. Proc Natl Acad Sci USA 113:E3300–E3306.

Saraswati S, Fox LE, Soll DR, and Wu CF (2004) Tyramine and octopamine haveopposite effects on the locomotion of Drosophila larvae. J Neurobiol 58:425–441.

Saudou F, Amlaiky N, Plassat JL, Borrelli E, and Hen R (1990) Cloning and char-acterization of a Drosophila tyramine receptor. EMBO J 9:3611–3617.

Sayyah M, Rezaie M, Haghighi S, and Amanzadeh A (2007) Intra-amygdala all-transretinoic acid inhibits amygdala-kindled seizures in rats. Epilepsy Res 75:97–103.

Scanlan TS, Suchland KL, Hart ME, Chiellini G, Huang Y, Kruzich PJ, Frascarelli S,Crossley DA, Bunzow JR, Ronca-Testoni S, et al. (2004) 3-Iodothyronamine is anendogenous and rapid-acting derivative of thyroid hormone. Nat Med 10:638–642.

Scheiner R, Plückhahn S, Oney B, Blenau W, and Erber J (2002) Behaviouralpharmacology of octopamine, tyramine and dopamine in honey bees. Behav BrainRes 136:545–553.

Scheiner R, Toteva A, Reim T, Søvik E, and Barron AB (2014) Differences in thephototaxis of pollen and nectar foraging honey bees are related to their octopaminebrain titers. Front Physiol 5:116.

Schulz DJ, Elekonich MM, and Robinson GE (2003) Biogenic amines in the antennallobes and the initiation and maintenance of foraging behavior in honey bees.J Neurobiol 54:406–416.

Schulz DJ and Robinson GE (2001) Octopamine influences division of labor in honeybee colonies. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 187:53–61.

Schwartz MD, Black SW, Fisher SP, Palmerston JB, Morairty SR, Hoener MC,and Kilduff TS (2017) Trace amine-associated receptor 1 regulates wakefulnessand EEG spectral composition. Neuropsychopharmacology 42:1305–1314.

Schwartz MD, Palmerston JB, Lee DL, Hoener MC, and Kilduff TS (2018) Deletion oftrace amine-associated receptor 1 attenuates behavioral responses to caffeine.Front Pharmacol 9:35.

Serretti A, Pae CU, Chiesa A, Mandelli L, and De Ronchi D (2009) Influence ofTAAR6 polymorphisms on response to aripiprazole. Prog NeuropsychopharmacolBiol Psychiatry 33:822–826.

Sessa B (2017) MDMA and PTSD treatment: “PTSD: from novel pathophysiology toinnovative therapeutics”. Neurosci Lett 649:176–180.

Shannon HE and Thompson WA (1984) Behavior maintained under fixed-intervaland second-order schedules by intravenous injections of endogenous noncatecholicphenylethylamines in dogs. J Pharmacol Exp Ther 228:691–695.

Shao L, Campbell JE, Hewitt MC, Campbell U, and Hanania TG (2011) inventors,Sunovion Pharmaceuticals Inc. and Psychogenics Inc., assignees. Multicycliccompounds and methods of use thereof. Patent WO 2011/069063 A2. 2011 Jun 9.

Shao X, Lakhina V, Dang P, Cheng RP, Marcaccio CL, and Raper JA (2017) Olfactorysensory axons target specific protoglomeruli in the olfactory bulb of zebrafish.Neural Dev 12:18.

Shaw JE, Sicree RA, and Zimmet PZ (2010) Global estimates of the prevalence ofdiabetes for 2010 and 2030. Diabetes Res Clin Pract 87:4–14.

Shepherd RM, Hashmi MN, Kane C, Squires PE, and Dunne MJ (1996) Elevation ofcytosolic calcium by imidazolines in mouse islets of Langerhans: implications forstimulus-response coupling of insulin release. Br J Pharmacol 119:911–916.

Shi W, Mersfelder J, and Hille R (2005) The interaction of trimethylamine de-hydrogenase and electron-transferring flavoprotein. J Biol Chem 280:20239–20246.

Shifman S, Levit A, Chen ML, Chen CH, Bronstein M, Weizman A, Yakir B, Navon R,and Darvasi A (2006) A complete genetic association scan of the 22q11 deletionregion and functional evidence reveal an association between DGCR2 and schizo-phrenia. Hum Genet 120:160–170.

Shimizu M, Cashman JR, and Yamazaki H (2007) Transient trimethylaminuria re-lated to menstruation. BMC Med Genet 8:2.

Siemian JN, Zhang Y, and Li JX (2017) Trace amine-associated receptor 1 agonistsRO5263397 and RO5166017 attenuate quinpirole-induced yawning but not hypo-thermia in rats. Behav Pharmacol 28:590–593.

Sikkema JM, Franx A, Fijnheer R, Nikkels PGJ, Bruinse HW, and Boomsma F (2002)Semicarbazide-sensitive amine oxidase in pre-eclampsia: no relation with markersof endothelial cell activation. Clin Chim Acta 324:31–38.

Silkaitis RP and Mosnaim AD (1976) Pathways linking L-phenylalanine and2-phenylethylamine with p-tyramine in rabbit brain. Brain Res 114:105–115.

Simmler LD, Buchy D, Chaboz S, Hoener MC, and Liechti ME (2016) In vitrocharacterization of psychoactive substances at rat, mouse, and human trace amine-associated receptor 1. J Pharmacol Exp Ther 357:134–144.

Sims KL and Bloom FE (1973) Rat brain L-3,4-dihydroxyphenylalanine andL-5-hydroxytryptophan decarboxylase activities: differential effect of6-hydroxydopamine. Brain Res 49:165–175.

Sims KL, Davis GA, and Bloom FE (1973) Activities of 3,4-dihydroxy-L-phenylalanine and 5-hydroxy-L-tryptophan decarboxylases in rat brain: assaycharacteristics and distribution. J Neurochem 20:449–464.

Siow YL and Dakshinamurti K (1985) Effect of pyridoxine deficiency on aromaticL-amino acid decarboxylase in adult rat brain. Exp Brain Res 59:575–581.


Skorupski P (1996) Octopamine induces steady-state reflex reversal in crayfishthoracic ganglia. J Neurophysiol 76:93–108.

Sloley BD (2004) Metabolism of monoamines in invertebrates: the relative impor-tance of monoamine oxidase in different phyla. Neurotoxicology 25:175–183.

Smith I, Kellow AH, and Hanington E (1970) A clinical and biochemical correlationbetween tyramine and migraine headache. Headache 10:43–52.

Smith SB, Maixner DW, Fillingim RB, Slade G, Gracely RH, Ambrose K, Zaykin DV, HydeC, John S, Tan K, et al. (2012) Large candidate gene association study reveals genetic riskfactors and therapeutic targets for fibromyalgia. Arthritis Rheum 64:584–593.

Smith TA (1977) Phenethylamine and related compounds in plants. Phytochemistry 16:9–18.Snead AN, Santos MS, Seal RP, Miyakawa M, Edwards RH, and Scanlan TS (2007)Thyronamines inhibit plasma membrane and vesicular monoamine transport. ACSChem Biol 2:390–398.

Snoddy AM, Heckathorn D, and Tessel RE (1985) Cold-restraint stress and urinaryendogenous beta-phenylethylamine excretion in rats. Pharmacol Biochem Behav22:497–500.

Snodgrass SR and Iversen LL (1974) Formation and release of 3H-tryptamine from3H-tryptophan in rat spinal cord slices. Adv Biochem Psychopharmacol 10:141–150.

Sombati S and Hoyle G (1984) Central nervous sensitization and dishabituation of reflexaction in an insect by the neuromodulator octopamine. J Neurobiol 15:455–480.

Sonnenburg JL and Bäckhed F (2016) Diet-microbiota interactions as moderators ofhuman metabolism. Nature 535:56–64.

Sotnikova TD, Beaulieu JM, Espinoza S, Masri B, Zhang X, Salahpour A, Barak LS,Caron MG, and Gainetdinov RR (2010) The dopamine metabolite3-methoxytyramine is a neuromodulator. PLoS One 5:e13452.

Sotnikova TD, Budygin EA, Jones SR, Dykstra LA, Caron MG, and Gainetdinov RR(2004) Dopamine transporter-dependent and -independent actions of trace aminebeta-phenylethylamine. J Neurochem 91:362–373.

Sotnikova TD, Caron MG, and Gainetdinov RR (2009) Trace amine-associated re-ceptors as emerging therapeutic targets. Mol Pharmacol 76:229–235.

Sotnikova TD and Gainetdinov RR (2009) Octopamine and other monoamines ininvertebrates, in Encyclopedia of Neuroscience (Squire LR ed) pp 9–17, AcademicPress, Cambridge, MA.

Sotnikova TD, Zorina OI, Ghisi V, Caron MG, and Gainetdinov RR (2008) Traceamine associated receptor 1 and movement control. Parkinsonism Relat Disord 14(Suppl 2):S99–S102.

Spitz MR, Shi H, Yang F, Hudmon KS, Jiang H, Chamberlain RM, Amos CI, WanY, Cinciripini P, Hong WK, et al. (1998) Case-control study of the D2 dopaminereceptor gene and smoking status in lung cancer patients. J Natl Cancer Inst 90:358–363.

Spivak M, Masterman R, Ross R, and Mesce KA (2003) Hygienic behavior in the honeybee (Apis mellifera L.) and the modulatory role of octopamine. J Neurobiol 55:341–354.

Srinivasa S, Fitch KV, Lo J, Kadar H, Knight R, Wong K, Abbara S, Gauguier D, CapeauJ, Boccara F, et al. (2015) Plaque burden in HIV-infected patients is associated withserum intestinal microbiota-generated trimethylamine. AIDS 29:443–452.

Sriram U, Cenna JM, Haldar B, Fernandes NC, Razmpour R, Fan S, Ramirez SH,and Potula R (2016) Methamphetamine induces trace amine-associated receptor1 (TAAR1) expression in human T lymphocytes: role in immunomodulation.J Leukoc Biol 99:213–223.

Sriram U, Haldar B, Cenna JM, Gofman L, and Potula R (2015) Methamphetaminemediates immune dysregulation in a murine model of chronic viral infection. FrontMicrobiol 6:793.

Stalder H, Hoener MC, and Norcross RD (2011) Selective antagonists of mouse traceamine-associated receptor 1 (mTAAR1): discovery of EPPTB (RO5212773). BioorgMed Chem Lett 21:1227–1231.

Stäubert C, Böselt I, Bohnekamp J, Römpler H, Enard W, and Schöneberg T (2010)Structural and functional evolution of the trace amine-associated receptorsTAAR3, TAAR4 and TAAR5 in primates. PLoS One 5:e11133.

Stavrou S, Gratz M, Tremmel E, Kuhn C, Hofmann S, Heidegger H, Peryanova M,Hermelink K, Hutter S, Toth B, et al. (2018) TAAR1 induces a disturbed GSK3bphosphorylation in recurrent miscarriages through the ODC. Endocr Connect 7:372–384.

Stelinski LL, Miller JR, Ressa NE, and Gut LJ (2003) Increased EAG responses oftortricid moths after prolonged exposure to plant volatiles: evidence foroctopamine-mediated sensitization. J Insect Physiol 49:845–856.

Stevenson PA, Dyakonova V, Rillich J, and Schildberger K (2005) Octopamine andexperience-dependent modulation of aggression in crickets. J Neurosci 25:1431–1441.

Stoff DM, Jeste DV, Gillin JC, Moja EA, Cohen L, Stauderman KA, and Wyatt RJ(1984) Behavioral supersensitivity to beta-phenylethylamine after chronic ad-ministration of haloperidol. Biol Psychiatry 19:101–106.

Stohs SJ and Hartman MJ (2015) A review of the receptor binding and pharmaco-logical effects of N-methyltyramine. Phytother Res 29:14–16.

Sugimoto M, Saruta J, Matsuki C, To M, Onuma H, Kaneko M, Soga T, Tomita M,and Tsukinoki K (2013) Physiological and environmental parameters associated withmass spectrometry-based salivary metabolomic profiles. Metabolomics 9:454–463.

Sukhanov I, Caffino L, Efimova EV, Espinoza S, Sotnikova TD, Cervo L, Fumagalli F,and Gainetdinov RR (2016) Increased context-dependent conditioning to amphet-amine in mice lacking TAAR1. Pharmacol Res 103:206–214.

Sukhanov I, Dorofeikova M, Dolgorukova A, Dorotenko A, and Gainetdinov RR (2018)Trace amine-associated receptor 1 modulates the locomotor and sensitization ef-fects of nicotine. Front Pharmacol 9:329.

Sukhanov I, Espinoza S, Yakovlev DS, Hoener MC, Sotnikova TD, and GainetdinovRR (2014) TAAR1-dependent effects of apomorphine in mice. Int J Neuro-psychopharmacol 17:1683–1693.

Sumi-Ichinose C, Hasegawa S, Ichinose H, Sawada H, Kobayashi K, Sakai M, Fujii T,Nomura H, Nomura T, Nagatsu I, et al. (1995) Analysis of the alternative pro-moters that regulate tissue-specific expression of human aromatic L-amino aciddecarboxylase. J Neurochem 64:514–524.

Syed AS, Sansone A, Röner S, Bozorg Nia S, Manzini I, and Korsching SI (2015) Differentexpression domains for two closely related amphibian TAARs generate a bimodal dis-tribution similar to neuronal responses to amine odors. Sci Rep 5:13935.

Szabo A (2015) Psychedelics and immunomodulation: novel approaches and thera-peutic opportunities. Front Immunol 6:358.

Szabo A, Billett E, and Turner J (2001) Phenylethylamine, a possible link to theantidepressant effects of exercise? Br J Sports Med 35:342–343.

Szumska J, Qatato M, Rehders M, Führer D, Biebermann H, Grandy DK, Köhrle J,and Brix K (2015) Trace amine-associated receptor 1 localization at the apicalplasma membrane domain of fisher rat thyroid epithelial cells is confined to cilia.Eur Thyroid J 4 (Suppl 1):30–41.

Taksande BG, Kotagale NR, Tripathi SJ, Ugale RR, and Chopde CT (2009) Antide-pressant like effect of selective serotonin reuptake inhibitors involve modulation ofimidazoline receptors by agmatine. Neuropharmacology 57:415–424.

Taquet N, Philippe C, Reimund J-M, and Muller CD (2012) Inflammatory boweldisease G-protein coupled receptors (GPCRs) expression profiling with microfluidiccards, in Crohn’s Disease (Karoui S ed) pp 59–86, IntechOpen, London.

Tashiro S, Taniguchi E, Eto M, and Maekawa K (1974) Isoamylamine as the possibleneuroactive metabolite of l-leucine. Agric Biol Chem 39:569–570.

Tchercansky DM, Acevedo C, and Rubio MC (1994) Studies of tyramine transfer andmetabolism using an in vitro intestinal preparation. J Pharm Sci 83:549–552.

Tessarolo JA, Tabesh MJ, Nesbitt M, and Davidson WS (2014) Genomic organizationand evolution of the trace amine-associated receptor (TAAR) repertoire in Atlanticsalmon (Salmo salar). G3 (Bethesda) 4:1135–1141.

Thaiss CA, Zmora N, Levy M, and Elinav E (2016) The microbiome and innateimmunity. Nature 535:65–74.

Thomas P, Alptekin K, Gheorghe M, Mauri M, Olivares JM, and Riedel M (2009)Management of patients presenting with acute psychotic episodes of schizophrenia.CNS Drugs 23:193–212.

Thorn DA, Jing L, Qiu Y, Gancarz-Kausch AM, Galuska CM, Dietz DM, Zhang Y,and Li JX (2014a) Effects of the trace amine-associated receptor 1 agonistRO5263397 on abuse-related effects of cocaine in rats. Neuropsychopharmacology39:2309–2316.

Thorn DA, Zhang C, Zhang Y, and Li JX (2014b) The trace amine associated receptor1 agonist RO5263397 attenuates the induction of cocaine behavioral sensitizationin rats. Neurosci Lett 566:67–71.

Tiihonen J, Vilkman H, Räsänen P, Ryynänen OP, Hakko H, Bergman J, Hämäläi-nen T, Laakso A, Haaparanta-Solin M, Solin O, et al. (1998) Striatal presynapticdopamine function in type 1 alcoholics measured with positron emission tomog-raphy. Mol Psychiatry 3:156–161.

Todd WA (1979) Psychosocial problems as the major complication of an adolescentwith trimethylaminuria. J Pediatr 94:936–937.

Toro-Funes N, Bosch-Fuste J, Latorre-Moratalla ML, Veciana-Nogués MT,and Vidal-Carou MC (2015) Biologically active amines in fermented and non-fermented commercial soybean products from the Spanish market. Food Chem173:1119–1124.

Toye AA, Dumas ME, Blancher C, Rothwell AR, Fearnside JF, Wilder SP, BihoreauMT, Cloarec O, Azzouzi I, Young S, et al. (2007) Subtle metabolic and liver genetranscriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice. Diabetologia 50:1867–1879.

Tsutsumi E, Kanai S, Ohta M, Suwa Y, and Miyasaka K (2010) Stimulatory effect ofN-methyltyramine, a congener of beer, on pancreatic secretion in conscious rats.Alcohol Clin Exp Res 34 (Suppl 1):S14–S17.

Ungar F, Mosnaim AD, Ungar B, and Wolf ME (1977) Tyramine-binding by synap-tosomes from rat brain: effect of centrally active drugs. Biol Psychiatry 12:661–668.

Unoki S, Matsumoto Y, and Mizunami M (2005) Participation of octopaminergicreward system and dopaminergic punishment system in insect olfactory learningrevealed by pharmacological study. Eur J Neurosci 22:1409–1416.

Vaccari A (1986) High affinity binding of [3H]-tyramine in the central nervous sys-tem. Br J Pharmacol 89:15–25.

Vallender EJ, Xie Z, Westmoreland SV, and Miller GM (2010) Functional evolution ofthe trace amine associated receptors in mammals and the loss of TAAR1 in dogs.BMC Evol Biol 10:51.

van Nguyen T, Paterson IA, Juorio AV, Greenshaw AJ, and Boulton AA (1989)Tryptamine receptors: neurochemical and electrophysiological evidence for post-synaptic and functional binding sites. Brain Res 476:85–93.

Vanden Broeck J, Vulsteke V, Huybrechts R, and De Loof A (1995) Characterizationof a cloned locust tyramine receptor cDNA by functional expression in permanentlytransformed Drosophila S2 cells. J Neurochem 64:2387–2395.

Vanti WB, Muglia P, Nguyen T, Cheng R, Kennedy JL, George SR, and O’Dowd BF(2003) Discovery of a null mutation in a human trace amine receptor gene. Geno-mics 82:531–536.

Vassilacopoulou D, Sideris DC, Vassiliou AG, and Fragoulis EG (2004) Identificationand characterization of a novel form of the human L-dopa decarboxylase mRNA.Neurochem Res 29:1817–1823.

Vattai A, Akyol E, Kuhn C, Hofmann S, Heidegger H, von Koch F, Hermelink K,Wuerstlein R, Harbeck N, Mayr D, et al. (2017) Increased trace amine-associatedreceptor 1 (TAAR1) expression is associated with a positive survival rate in pa-tients with breast cancer. J Cancer Res Clin Oncol 143:1637–1647.

Venditti P, Napolitano G, Di Stefano L, Chiellini G, Zucchi R, Scanlan TS, and DiMeo S (2011) Effects of the thyroid hormone derivatives 3-iodothyronamine andthyronamine on rat liver oxidative capacity. Mol Cell Endocrinol 341:55–62.

Venken T, Claes S, Sluijs S, Paterson AD, van Duijn C, Adolfsson R, Del-Favero J, and VanBroeckhoven C (2005) Genomewide scan for affective disorder susceptibility Loci in fam-ilies of a northern Swedish isolated population. Am J Hum Genet 76:237–248.

Verlinden H, Vleugels R, Marchal E, Badisco L, Pflüger HJ, Blenau W, and Broeck JV(2010a) The role of octopamine in locusts and other arthropods. J Insect Physiol 56:854–867.

Verlinden H, Vleugels R, Marchal E, Badisco L, Tobback J, Pflüger HJ, Blenau W,and Vanden Broeck J (2010b) The cloning, phylogenetic relationship and


distribution pattern of two new putative GPCR-type octopamine receptors in thedesert locust (Schistocerca gregaria). J Insect Physiol 56:868–875.

Vieira-Coelho MA and Soares-da-Silva P (1993) Dopamine formation, from its im-mediate precursor 3,4-dihydroxyphenylalanine, along the rat digestive tract.Fundam Clin Pharmacol 7:235–243.

Vierk R, Pflueger HJ, and Duch C (2009) Differential effects of octopamine andtyramine on the central pattern generator for Manduca flight. J Comp Physiol ANeuroethol Sens Neural Behav Physiol 195:265–277.

Vladimirov V, Thiselton DL, Kuo PH, McClay J, Fanous A, Wormley B, Vittum J,Ribble R, Moher B, van den Oord E, et al. (2007) A region of 35 kb containing thetrace amine associate receptor 6 (TAAR6) gene is associated with schizophrenia inthe Irish study of high-density schizophrenia families. Mol Psychiatry 12:842–853.

Vladimirov VI, Maher BS, Wormley B, O’Neill FA, Walsh D, Kendler KS, and RileyBP (2009) The trace amine associated receptor (TAAR6) gene is not associated withschizophrenia in the Irish Case-Control Study of Schizophrenia (ICCSS) sample.Schizophr Res 107:249–254.

Wainscott DB, Little SP, Yin T, Tu Y, Rocco VP, He JX, and Nelson DL (2007)Pharmacologic characterization of the cloned human trace amine-associated re-ceptor1 (TAAR1) and evidence for species differences with the rat TAAR1.J Pharmacol Exp Ther 320:475–485.

Walker RJ and Kerkut GA (1978) The first family (adrenaline, noradrenaline, do-pamine, octopamine, tyramine, phenylethanolamine and phenylethylamine).Comp Biochem Physiol C Toxicol Pharmacol 61:261–266.

Wallrabenstein I, Kuklan J, Weber L, Zborala S, Werner M, Altmüller J, Becker C,Schmidt A, Hatt H, Hummel T, et al. (2013) Human trace amine-associated re-ceptor TAAR5 can be activated by trimethylamine. PLoS One 8:e54950.

Wallrabenstein I, Singer M, Panten J, Hatt H, and Gisselmann G (2015) Timberol®inhibits TAAR5-mediated responses to trimethylamine and influences the olfactorythreshold in humans. PLoS One 10:e0144704.

Wang D, Ma JZ, and Li MD (2005) Mapping and verification of susceptibility loci forsmoking quantity using permutation linkage analysis. Pharmacogenomics J 5:166–172.

Wang X, Corin K, Rich C, and Zhang S (2011) Study of two G-protein coupled receptorvariants of human trace amine-associated receptor 5. Sci Rep 1:102.

Wasik AM, Millan MJ, Scanlan T, Barnes NM, and Gordon J (2012) Evidence forfunctional trace amine associated receptor-1 in normal and malignant B cells. LeukRes 36:245–249.

Wienecke J, Ren LQ, Hultborn H, Chen M, Møller M, Zhang Y, and Zhang M (2014)Spinal cord injury enables aromatic L-amino acid decarboxylase cells to synthesizemonoamines. J Neurosci 34:11984–12000.

Williams BB, Van Benschoten AH, Cimermancic P, Donia MS, Zimmermann M,Taketani M, Ishihara A, Kashyap PC, Fraser JS, and Fischbach MA (2014) Dis-covery and characterization of gut microbiota decarboxylases that can produce theneurotransmitter tryptamine. Cell Host Microbe 16:495–503.

Willoughby D, Thomas R, and Schwiening C (2001) The effects of intracellular pH changeson resting cytosolic calcium in voltage-clamped snail neurones. J Physiol 530:405–416.

Wolf EP (1921) Experimental studies on inflammation. I. The influence of chemicalsupon the chemotaxis of leucocytes in vitro. J Exp Med 34:375–396.

Wolf EP (1923) Experimental studies on inflammation: II. Experimental chemicalinflammation in vivo. J Exp Med 37:511–524.

Wolf ME and Mosnaim AD (1983) Phenylethylamine in neuropsychiatric disorders.Gen Pharmacol 14:385–390.

Wolinsky TD, Swanson CJ, Smith KE, Zhong H, Borowsky B, Seeman P, Branchek T,and Gerald CP (2007) The trace amine 1 receptor knockout mouse: an animalmodel with relevance to schizophrenia. Genes Brain Behav 6:628–639.

WoodWJ, Geraci T, Nilsen A, DeBarber AE, and Scanlan TS (2009) Iodothyronamines areoxidatively deaminated to iodothyroacetic acids in vivo. ChemBioChem 10:361–365.

Wright TR (1987) The genetics of biogenic amine metabolism, sclerotization, andmelanization in Drosophila melanogaster. Adv Genet 24:127–222.

Wu SF, Xu G, and Ye GY (2015) Characterization of a tyramine receptor type 2 fromhemocytes of rice stem borer, Chilo suppressalis. J Insect Physiol 75:39–46.

Xie Z and Miller GM (2008) Beta-phenylethylamine alters monoamine transporterfunction via trace amine-associated receptor 1: implication for modulatory roles oftrace amines in brain. J Pharmacol Exp Ther 325:617–628.

Xie Z, Vallender EJ, Yu N, Kirstein SL, Yang H, Bahn ME, Westmoreland SV,and Miller GM (2008a) Cloning, expression, and functional analysis of rhesusmonkey trace amine-associated receptor 6: evidence for lack of monoaminergicassociation. J Neurosci Res 86:3435–3446.

Xie Z, Westmoreland SV, and Miller GM (2008b) Modulation of monoamine trans-porters by common biogenic amines via trace amine-associated receptor 1 andmonoamine autoreceptors in human embryonic kidney 293 cells and brain syn-aptosomes. J Pharmacol Exp Ther 325:629–640.

Xue Z, Siemian JN, Johnson BN, Zhang Y, and Li JX (2018) Methamphetamine-induced impulsivity during chronic methamphetamine treatment in rats: effects ofthe TAAR 1 agonist RO5263397. Neuropharmacology 129:36–46.

Yang HY and Neff NH (1973) Beta-phenylethylamine: a specific substrate for type Bmonoamine oxidase of brain. J Pharmacol Exp Ther 187:365–371.

Yang XC and Reis DJ (1999) Agmatine selectively blocks the N-methyl-D-aspartatesubclass of glutamate receptor channels in rat hippocampal neurons. J PharmacolExp Ther 288:544–549.

Yang YX, Mu CL, Luo Z, and Zhu WY (2015a) Bromochloromethane, a methaneanalogue, affects the microbiota and metabolic profiles of the rat gastrointestinaltract. Appl Environ Microbiol 82:778–787.

Yang Z, Yu Y, Zhang V, Tian Y, Qi W, and Wang L (2015b) Octopamine mediatesstarvation-induced hyperactivity in adult Drosophila. Proc Natl Acad Sci USA 112:5219–5224.

Yen YR, Wang YH, Wang LN, Chen LC, Lu FJ, and Wang SR (2016) Novel function ofisoamylamine improves survival in endotoxemic mice by ameliorating coagulop-athy and attenuating MMP-9 expression through p-ERK/p-p38 signaling at earlystage. Shock 47:772–779.

Yokoo Y, Kohda H, Kusumoto A, Naoki H, Matsumoto N, Amachi T, Suwa Y,Fukazawa H, Ishida H, Tsuji K, et al. (1999) Isolation from beer and structuraldetermination of a potent stimulant of gastrin release. Alcohol Alcohol 34:161–168.

Youdim MB, Harshak N, Yoshioka M, Araki H, Mukai Y, and Gotto G (1991) Novelsubstrates and products of amine oxidase-catalysed reactions. Biochem Soc Trans19:224–228.

Young EA, Neff NH, and Hadjiconstantinou M (1993) Evidence for cyclic AMP-mediated increase of aromatic L-amino acid decarboxylase activity in the striatumand midbrain. J Neurochem 60:2331–2333.

Young EA, Neff NH, and Hadjiconstantinou M (1994) Phorbol ester administrationtransiently increases aromatic L-amino acid decarboxylase activity of the mousestriatum and midbrain. J Neurochem 63:694–697.

Yu AM, Granvil CP, Haining RL, Krausz KW, Corchero J, Küpfer A, Idle JR,and Gonzalez FJ (2003) The relative contribution of monoamine oxidase and cy-tochrome p450 isozymes to the metabolic deamination of the trace amine trypt-amine. J Pharmacol Exp Ther 304:539–546.

Zeng H, Loughton BG, and Jennings KR (1996) Tissue specific transduction sys-tems for octopamine in the locust (Locusta migratoria). J Insect Physiol 42:765–769.

Zhang A, Liu Q, Zhao H, Zhou X, Sun H, Nan Y, Zou S, Ma CW, and Wang X (2016)Phenotypic characterization of nanshi oral liquid alters metabolic signaturesduring disease prevention. Sci Rep 6:19333.

Zhang AQ, Mitchell SC, and Smith RL (1999) Dietary precursors of trimethylaminein man: a pilot study. Food Chem Toxicol 37:515–520.

Zhang J, Pacifico R, Cawley D, Feinstein P, and Bozza T (2013) Ultrasensitive de-tection of amines by a trace amine-associated receptor. J Neurosci 33:3228–3239.

Zhang LS and Davies SS (2016) Microbial metabolism of dietary components tobioactive metabolites: opportunities for new therapeutic interventions. GenomeMed 8:46.

Zheng X, Xie G, Zhao A, Zhao L, Yao C, Chiu NH, Zhou Z, Bao Y, JiaW, Nicholson JK, et al.(2011) The footprints of gut microbial-mammalian co-metabolism. J Proteome Res 10:5512–5522.

Zhu G, Wang L, Tang W, Wang X, and Wang C (2017) Identification of olfactory receptorgenes in the Japanese grenadier anchovy Coilia nasus. Genes Genomics 39:521–532.

Zhu MY, Juorio AV, Paterson IA, and Boulton AA (1992) Regulation of aromaticL-amino acid decarboxylase by dopamine receptors in the rat brain. J Neurochem58:636–641.

Zhu MY, Juorio AV, Paterson IA, and Boulton AA (1993) Regulation of striatal ar-omatic L-amino acid decarboxylase: effects of blockade or activation of dopaminereceptors. Eur J Pharmacol 238:157–164.

Zhu M-Y, Juorio AV, Paterson IA, and Boulton AA (1994) Regulation of aromaticL-amino acid decarboxylase in rat striatal synaptosomes: effects of dopamine re-ceptor agonists and antagonists. Br J Pharmacol 112:23–30.

Zhu Y, Jameson E, Crosatti M, Schäfer H, Rajakumar K, Bugg TD, and Chen Y(2014) Carnitine metabolism to trimethylamine by an unusual Rieske-type oxy-genase from human microbiota. Proc Natl Acad Sci USA 111:4268–4273.

Zornik E, Paisley K, and Nichols R (1999) Neural transmitters and a peptide mod-ulate Drosophila heart rate. Peptides 20:45–51.

Zucchi R, Accorroni A, and Chiellini G (2014) Update on 3-iodothyronamine and itsneurological and metabolic actions. Front Physiol 5:402.

Zucchi R, Ghelardoni S, and Chiellini G (2010) Cardiac effects of thyronamines.HeartFail Rev 15:171–176.


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