10
1521-0111/90/4/427436$25.00 http://dx.doi.org/10.1124/mol.116.104182 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:427436, October 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Molecular Mechanisms of Action of M 5 Muscarinic Acetylcholine Receptor Allosteric Modulators s Alice E. Berizzi, Patrick R. Gentry, Patricia Rueda, Sandra Den Hoedt, Patrick M. Sexton, Christopher J. Langmead, and Arthur Christopoulos Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia Received March 3, 2016; accepted July 18, 2016 ABSTRACT Recently, the first subtype-selective allosteric modulators of the M 5 muscarinic acetylcholine receptor (mAChR) have been described, but their molecular mechanisms of action remain unknown. Using radioligand-binding and functional assays of inositol phosphate (IP) accumulation and Ca 21 mobilization in a recombinant cell line stably expressing the human M 5 mAChR, we investigated the effects of the positive allosteric modulator (PAM), ML380, and negative allosteric modulator, ML375. In functional assays, ML380 caused robust enhance- ments in the potency of the full agonists, acetylcholine (ACh), carbachol, and oxotremorine-M, while significantly increasing the maximal response to the partial agonist, pilocarpine. ML380 also demonstrated direct allosteric agonist activity. In contrast, ML375 displayed negative cooperativity with each of the agonists in a manner that varied with the path- way investigated and progressively reduced the maximal pilocarpine response. Radioligand-binding affinity coopera- tivity estimates were consistent with values derived from functional assays in some instances but not others, suggest- ing additional allosteric effects on orthosteric ligand efficacy. For ML375 this was confirmed in IP assays performed after reduction of receptor reserve by the alkylating agent, phenox- ybenzamine, as it reduced the maximal ACh response. In contrast, ML380 enhanced only ACh potency after receptor alkylation, with no effect on maximal response, consistent with studies of the M 1 mAChR with the prototypical PAM, BQZ12. Interaction studies between ML380 and ML375 also indicated that they most likely used an overlapping allosteric site. Our findings indicate that novel small-molecule modula- tors of the M 5 mAChR display mixed mechanisms of action compared with previously characterized modulators of other mAChRs. Introduction The muscarinic acetylcholine receptors (mAChRs) are pro- totypical family A guanine nucleotide-binding protein-coupled receptors (GPCRs); the M 1 ,M 3 , and M 5 mAChR subtypes primarily couple to G q/11 proteins to activate the inositol phosphate (IP) pathway, whereas M 2 and M 4 mAChR sub- types primarily couple to G i/o proteins to mediate the in- hibition of adenylyl cyclase (Porter et al., 2002; Matsui et al., 2004; Langmead et al., 2008). The mAChRs are widely expressed in the central nervous system (CNS) and periphery, where they control a number of physiologic processes. In the CNS, they have been implicated as important therapeutic targets for a range of disorders, including schizophrenia, Alzheimers disease, addiction, and Parkinsons disease (Wess et al., 2007; Langmead et al., 2008). To date, the majority of studies focusing on mAChRs in CNS disorders have targeted the M 1 and M 4 subtypes (Bymaster et al., 1997; Shannon et al., 2000; Brady et al., 2008; Chan et al., 2008; Ma et al., 2009; Shirey et al., 2009). However, despite only representing 2% of the total mAChR population in the brain (Yamada et al., 2003), the M 5 mAChR is expressed in highly discrete regions of therapeutic interest. For example, it is the only mAChR subtype for which mRNA transcript can be identified in dopamine-containing neurons of the substantia nigra pars compacta and the ventral tegmental area (Vilaró et al., 1990; Weiner et al., 1990; Yasuda et al., 1993), leading to the hypothesis that the M 5 mAChR may regulate midbrain dopamine trans- mission and reward mechanisms and thus represent a target for treating drug addiction (Vilaró et al., 1990; Weiner et al., 1990). The phenotype of M 5 mAChR knockout (KO) mice supports the validity of the receptor as an addiction target; KO mice self-administer less cocaine and morphine, and are unable to develop conditioned place preference to the same drugs, with blunted withdrawal symptoms (Basile et al., 2002; Fink- Jensen et al., 2003; Thomsen et al., 2005). However, until recently, there has been a lack of sufficiently potent and selective tool compounds with which to probe receptor function and augment findings from constitutive M 5 mAChR KO mice. This work was supported by the National Health and Medical Research Council of Australia [Grant APP1055134]. A.C. is a Senior Principal Research Fellow, and P.M.S. is a Principal Research Fellow of the National Health and Medical Research Council of Australia. dx.doi.org/10.1124/mol.116.104182. s This article has supplemental material available at molpharm. aspetjournals.org. ABBREVIATIONS: ACh, acetylcholine; CCh, carbachol; CHO, Chinese hamster ovary; CNS, central nervous system; GPCR, guanine nucleotide- binding protein-coupled receptor; IP, inositol phosphate; KO, knockout; mAChR, muscarinic ACh receptor; NAM, negative allosteric modulator; NMS, N-methylscopolamine; oxo-M, oxotremorine-M; PAM, positive allosteric modulator; PBZ, phenoxybenzamine. 427 http://molpharm.aspetjournals.org/content/suppl/2016/07/26/mol.116.104182.DC1 Supplemental material to this article can be found at: at ASPET Journals on March 26, 2021 molpharm.aspetjournals.org Downloaded from

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1521-0111/90/4/427–436$25.00 http://dx.doi.org/10.1124/mol.116.104182MOLECULAR PHARMACOLOGY Mol Pharmacol 90:427–436, October 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Molecular Mechanisms of Action of M5 Muscarinic AcetylcholineReceptor Allosteric Modulators s

Alice E. Berizzi, Patrick R. Gentry, Patricia Rueda, Sandra Den Hoedt, Patrick M. Sexton,Christopher J. Langmead, and Arthur ChristopoulosDrug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

Received March 3, 2016; accepted July 18, 2016

ABSTRACTRecently, the first subtype-selective allosteric modulators ofthe M5 muscarinic acetylcholine receptor (mAChR) have beendescribed, but their molecular mechanisms of action remainunknown. Using radioligand-binding and functional assays ofinositol phosphate (IP) accumulation and Ca21 mobilization ina recombinant cell line stably expressing the human M5mAChR, we investigated the effects of the positive allostericmodulator (PAM), ML380, and negative allosteric modulator,ML375. In functional assays, ML380 caused robust enhance-ments in the potency of the full agonists, acetylcholine (ACh),carbachol, and oxotremorine-M, while significantly increasingthe maximal response to the partial agonist, pilocarpine.ML380 also demonstrated direct allosteric agonist activity.In contrast, ML375 displayed negative cooperativity witheach of the agonists in a manner that varied with the path-way investigated and progressively reduced the maximal

pilocarpine response. Radioligand-binding affinity coopera-tivity estimates were consistent with values derived fromfunctional assays in some instances but not others, suggest-ing additional allosteric effects on orthosteric ligand efficacy.For ML375 this was confirmed in IP assays performed afterreduction of receptor reserve by the alkylating agent, phenox-ybenzamine, as it reduced the maximal ACh response. Incontrast, ML380 enhanced only ACh potency after receptoralkylation, with no effect on maximal response, consistentwith studies of the M1 mAChR with the prototypical PAM,BQZ12. Interaction studies between ML380 and ML375 alsoindicated that they most likely used an overlapping allostericsite. Our findings indicate that novel small-molecule modula-tors of the M5 mAChR display mixed mechanisms of actioncompared with previously characterized modulators of othermAChRs.

IntroductionThe muscarinic acetylcholine receptors (mAChRs) are pro-

totypical family A guanine nucleotide-binding protein-coupledreceptors (GPCRs); the M1, M3, and M5 mAChR subtypesprimarily couple to Gq/11 proteins to activate the inositolphosphate (IP) pathway, whereas M2 and M4 mAChR sub-types primarily couple to Gi/o proteins to mediate the in-hibition of adenylyl cyclase (Porter et al., 2002; Matsui et al.,2004; Langmead et al., 2008). The mAChRs are widelyexpressed in the central nervous system (CNS) and periphery,where they control a number of physiologic processes. In theCNS, they have been implicated as important therapeutictargets for a range of disorders, including schizophrenia,Alzheimer’s disease, addiction, and Parkinson’s disease(Wess et al., 2007; Langmead et al., 2008).

To date, the majority of studies focusing on mAChRs in CNSdisorders have targeted theM1andM4 subtypes (Bymaster et al.,1997; Shannon et al., 2000; Brady et al., 2008; Chan et al., 2008;Ma et al., 2009; Shirey et al., 2009). However, despite onlyrepresenting 2% of the total mAChR population in the brain(Yamada et al., 2003), the M5 mAChR is expressed in highlydiscrete regions of therapeutic interest. For example, it is the onlymAChR subtype for which mRNA transcript can be identified indopamine-containing neurons of the substantia nigra parscompacta and the ventral tegmental area (Vilaró et al., 1990;Weiner et al., 1990;Yasudaet al., 1993), leading to thehypothesisthat the M5 mAChR may regulate midbrain dopamine trans-mission and reward mechanisms and thus represent a target fortreating drug addiction (Vilaró et al., 1990; Weiner et al., 1990).The phenotype of M5 mAChR knockout (KO) mice supports

the validity of the receptor as an addiction target; KO miceself-administer less cocaine and morphine, and are unable todevelop conditioned place preference to the same drugs, withblunted withdrawal symptoms (Basile et al., 2002; Fink-Jensen et al., 2003; Thomsen et al., 2005). However, untilrecently, there has been a lack of sufficiently potent andselective tool compoundswithwhich to probe receptor functionand augment findings from constitutive M5 mAChR KO mice.

This work was supported by the National Health and Medical ResearchCouncil of Australia [Grant APP1055134]. A.C. is a Senior Principal ResearchFellow, and P.M.S. is a Principal Research Fellow of the National Health andMedical Research Council of Australia.

dx.doi.org/10.1124/mol.116.104182.s This article has supplemental material available at molpharm.

aspetjournals.org.

ABBREVIATIONS: ACh, acetylcholine; CCh, carbachol; CHO, Chinese hamster ovary; CNS, central nervous system; GPCR, guanine nucleotide-binding protein-coupled receptor; IP, inositol phosphate; KO, knockout; mAChR, muscarinic ACh receptor; NAM, negative allosteric modulator;NMS, N-methylscopolamine; oxo-M, oxotremorine-M; PAM, positive allosteric modulator; PBZ, phenoxybenzamine.

427

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Amajor breakthrough in this regard has been the discovery ofsmall-molecule allosteric modulators for these mAChRs. Bytargeting topographically distinct binding sites from those usedby orthosteric ligands, allosteric modulators can selectivelytarget receptor subtypes to enhance or inhibit the function ofacetylcholine (ACh)whilemaintaining the spatial and temporalnature of endogenous neurotransmission. Allosteric modula-tors also offer the potential of reduced propensity for on-targetmediated toxicity and engendering signal-pathway bias(Gregory et al., 2007, 2010; Leach et al., 2007; Conn et al.,2009; Valant et al., 2012; Kruse et al., 2014). Highly selectivepositive allosteric modulators (PAMs) have been identified forboth the M1 and M4 mAChR subtypes (Chan et al., 2008;Shirey et al., 2008, 2009;Ma et al., 2009), enabling their role inanimalmodels of cognition and schizophrenia to be elucidated.Most recently, screening and medicinal chemistry studies

identified the first M5 mAChR-selective allosteric ligands,including the exemplar PAM, ML380 [1-((1H-indazol-5-yl)sulfonyl)-N-ethyl-N-(2-(trifluoromethyl)benzyl)piperidine-4-carboxamide], and the negative allosteric modulator (NAM),ML375 [(S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-5(9bH)-one] (Gentry et al.,2013, 2014) (Fig. 1). However, the molecular mechanismsunderlying their activity remain largely unexplored. Giventhat GPCR allosteric modulators can display a range ofactivities, and that the type of activity can determine thedegree of in vivo efficacy in a context-sensitive manner(Christopoulos, 2014), the aim of the current study was tocharacterize the molecular mechanisms of action of ML380andML375 in combinationwith different orthosteric probes atthe human M5 mAChR. Our studies confirm that ML380 andML375 exhibit PAM agonist and NAM activity, respectively,with the potential to modulate orthosteric ligand affinity and/or intrinsic efficacy, a mechanism not observed with allostericmodulators of the related M1 mAChR subtype. Moreover,functional interaction studies between the PAM and NAMsuggest that they exert their opposing effects on receptorfunction via a common, albeit currently uncharacterized,allosteric binding site.

Materials and MethodsMaterials. Flp-In-Chinese hamster ovary (CHO) cells were

obtained from Life Technologies (Mulgrave, Australia). Dulbecco’smodified Eagle medium was purchased from Invitrogen (Carlsbad,CA). Fetal bovine serum was purchased from ThermoTrace (Melbourne,Australia). [3H]-N-methylscopolamine ([3H]-NMS; specific activity,84 Ci/mmol) and MicroScint scintillation liquid were purchased fromPerkin-Elmer Life Sciences. The IP-One assay kit was purchased fromCisbio (Codolet, France). ACh, carbachol (CCh), pilocarpine, oxotre-morine-M (oxo-M), atropine, and phenoxybenzamine (PBZ) werepurchased from Sigma-Aldrich (St. Louis, MO). ML380 and ML375

were generous gifts of C. Lindsley (Vanderbilt University, Nashville,TN). BQZ12 was synthesized in-house, as described previously(Abdul-Ridha et al., 2014a,b). All other chemicals were purchasedfrom Sigma-Aldrich. For all procedures, purified water (18.2 MV cm)from a Milli-Q PF Plus system was used.

Cell Culture. The wild-type human (hM5) mAChR construct wasisogenically integrated into Flp-In CHO cells (Invitrogen), and cellswere selected in the presence of 600 mg/mL hygromycin B at 37°C, 5%CO2, as previously described for the hM1 mAChR (Abdul-Ridha et al.,2014a). CHO-hM1 and CHO-hM5 cells were subcultured in a 1:3 ratioevery 2 days after reaching 80% confluence with 6–10 mL Versene todetach cells and incubated in a humidified atmosphere at 37°C, 5%CO2, for 2–5 minutes. Cells were counted by a hemocytometer to beseeded at 25,000 cells/well in 96-well cell culture plates 24 hours priorto assay, unless otherwise stated.

[3H]-NMS Equilibrium Binding. CHO-hM5 cells were seededinto 96-well Isoplates (PerkinElmer Life Sciences) in 100 mL culturemedia and incubated in a humidified atmosphere at 37°C, 5% CO2, forat least 6 hours. After 6 hours, media was removed and cells werewashed with 100 mL phosphate-buffered saline, before physiologicHEPES buffer (100 mL; 110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2,1 mMMgSO4, 25mMglucose, 50mMHEPES, and 58mM sucrose, pH7.4) was added. Binding was determined in the presence of 0.3 nM[3H]-NMS. Nonspecific binding of the radioligand was assessed in thepresence of 10 mM atropine and total binding by radioligand in thepresence of HEPES buffer alone. Binding assays were terminatedafter a 2-hour incubation with radioligand at 37°C by removal of anyunbound radioligand and the repeated washing of cells (3 times) with100 mL 0.9% NaCl at 4°C, followed by addition of 100 mL Microscintscintillation liquid. Bound radioactivity was assessed by liquidscintillation counting by a MicroBeta2 Plate Counter (PerkinElmerLife Sciences, Glen Waverley, Australia). For inhibition-bindingexperiments, varying concentrations of individual (or combinationsof) agonists, allosteric modulators, and antagonist in HEPES bufferwere added to the cells in the presence of [3H]-NMS in a final assay ofvolume of 100 mL.

IP Accumulation. The IP-One assay kit (Cisbio) was used for thedirect quantitativemeasurement ofmyo-inositol 1-phosphate in CHO-hM5 or CHO-hM1 cells, as described previously (Abdul-Ridha et al.,2014a), with the exception that cells were seeded at 25,000 per wellonto a transparent 96-well cell culture plate, and the following daycells were stimulatedwith various concentrations of orthosteric and/orallosteric ligands, and were then incubated for 40 minutes at 37°C, 5%CO2. Responses were normalized to that to a single, maximalconcentration of ACh (10 mM).

Calcium Mobilization. CHO-hM5 cells were cultured overnightin 96-well cell culture plates at 50,000 cells/well. The following day,media was removed and replaced with 50 mL 1 mM Fluo-4-AM Ca21

sensitive dye in assay buffer (Hank’s balanced salt solution supple-mented with 1.26 mM CaCl2, 20 mM HEPES, and 2.5 mM pro-benecid, pH 7.4), and the cells were incubated 45 minutes at 37°C,5% CO2. Dye was then removed and replaced with 50 mL fresh assaybuffer. Test compounds were added to the cells in a FLEXStation II(Molecular Devices, Sunnyvale, CA). For interaction studies, ligandswere added simultaneously to cells. Ca21/Fluo-4 fluorescence wasmeasured at an emission wavelength of 525 nm for 90 seconds(19 seconds prior to and 71 seconds following the addition of ligand)at room temperature.

Receptor Alkylation Studies. To determine functional affinityand efficacy estimates for the agonists, additional IP accumulationassays were performed, as described above, but on cells pretreated for30 minutes at 37°C with the irreversible orthosteric-site alkylatingagent, PBZ (or vehicle buffer control), followed by three washes with10% phosphate-buffered saline. PBZ reduces functional receptoravailability by irreversible steric hindrance for orthosteric agonists(ACh, CCh, oxo-M) or due to the irreversible formation of an inactivestate for which the positive allosteric modulator, ML380, has minimalaffinity due to high negative cooperativity.Fig. 1. Structures of (A) ML380 and (B) ML375.

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Data Analysis. GraphPad Prism version 6.04 (SanDiego, CA) wasused for all statistical analysis and curve fitting. For the empiricalanalysis of functional concentration–response data, agonistconcentration–response curves were analyzed using a standardlogistic function to determine measures of potency (as negativelogarithms; pEC50) and maximal response (Emax). For direct de-termination of functional agonist dissociation constants (KA) andoperational efficacies (t) from the alkylation experiments, agonist IPaccumulation concentration–response curves (in the presence orabsence of PBZ pretreatment) were globally fitted to the operationalmodel of agonism (Black and Leff, 1983):

Y5Basal1

(ðEM 2BasalÞ

��11

��KA 110Log½A�

�.�t110Log½A�

�n�)

(1)

where [A] represents the concentration of the agonist, KA repre-sents the agonist equilibrium dissociation constant, Basal is theresponse in the absence of ligand, EM is themaximal system response,t represents the agonist operational efficacy, which subsumes bothreceptor density and stimulus–response coupling efficiency, and n isthe slope of the transducer function linking occupancy to response. Amodified form of eq. 1 was also applied to the data for the partialagonist, pilocarpine, using a four-parameter logistic fit of the AChcontrol (full agonist) concentration–response curve to provide esti-mates of EM, transducer slope (n), and Basal for fitting the pilocarpinedata.

For functional interaction studies between full orthosteric agonistsand allosteric ligands in the IP accumulation or calcium assays, thefollowing simplified operational model of allosterism was applied(Leach et al., 2007):

Y5Basal1ðEM 2BasalÞ×��½A�ðKB 1ab½B�Þ1 tB½B�×EC50

n��½A�ðKB 1ab½B�Þ1 tB½B�×EC50n

1�EC50

n×ðKB½B�Þn(2)

where Basal is the response in the absence of ligand, EC50 is themidpoint of the full agonist concentration–response curve, KB is theequilibrium dissociation constant of the allosteric ligand (fixed tothe KA value from alkylation studies for ML380 for the IP assay), tBdenotes the capacity of the allosteric ligand to exhibit agonism(constrained to 0 for ML375), and ab is the net affinity/efficacycooperativity parameter describing the combined effect of the alloste-ric modulator on agonist function (both affinity and efficacy). EM and nare as described above. As this model is only suitable for full agoniststhat remain full agonists in the presence of allosteric modulator(Aurelio et al., 2009; Leach et al., 2010), the pilocarpine datasets wereanalyzed according to the complete model of allostery and agonism(Leach et al., 2007):

Y5Basal1ðEM 2BasalÞ×��tA½A�ðKB 1ab½B�Þ1 tBKB

n�½A�KB 1KAKB 1KA½B�1a½A�½B�n 1 �tA ½A�

�KB 1ab½B�1 tB½B�×KB

n(3)

where [A] and [B] represent the concentrations of the orthostericagonist and allosteric ligand, respectively, and KA and KB representtheir respective equilibrium dissociation constants (constrained asabove for ML380). tA and tB represent the relative efficacies of theorthosteric and allosteric ligands, receptively, and n represents theslope of the transducer function that links occupancy to response. arepresents the affinity cooperativity between the orthosteric agonistand allosteric modulator, and b represents a scaling factor thatdescribes the magnitude and direction of the effect of the allostericmodulator on orthosteric agonist efficacy. Where required to facilitateconvergence of global model fitting, the value of n was constrained tounity and indicated as such.

All interaction radioligand-binding studies were analyzed accord-ing to the following adapted form of an allosteric ternary complexmodel that accounts for the interaction of two orthosteric ligands andone allosteric ligand on a receptor (Christopoulos and Mitchelson,1997):

Y5Bmax×½A�

½A�1 ðKA×KBÞ=ðaA ½B�1KBÞ�× 11 ð½I�=KIÞ1 ð½B�=KBÞ1 ½ðaI ½I�½B�Þ=ðKI ×KBÞ�f g

(4)

where [A], [B], and [I] represent the concentrations of the radio-ligand ([3H]-NMS), allosteric ligand, and orthosteric inhibitor, re-spectively, and KA, KB, and KI represent their respective equilibriumdissociation constants, and Bmax is as defined above. The valueKAwasfixed to 0.3 nM. aA and aI represent the affinity cooperativity valuesbetween the allosteric ligand and the radioligand or orthostericinhibitor, respectively; values greater than 1 indicate positive cooper-ativity; values ,1 (but .0) indicate negative cooperativity; andvalues of unity indicate neutral cooperativity. All potency, affinity,and cooperativity parameters were estimated as logarithms(Christopoulos, 1998). Where appropriate, fitted parameters werecompared by extra sum-of-squares F-test (Motulsky and Christopoulos,2004).

ResultsThe effects of ML380 and ML375 were studied in canonical

Gq/11-linked IP accumulation and Ca21 mobilization func-tional assays. ACh stimulates both IP accumulation (pEC50 56.68 6 0.05; nH 5 1.0 6 0.1; n 5 3) and Ca21 mobilization(pEC50 5 7.636 0.06; nH 5 1.36 0.2; n5 4) in CHO-hM5 cells(Fig. 2). The PAM, ML380, also robustly stimulated IPaccumulation and Ca21 mobilization (pEC50 values 5.33 60.06; nH 5 1.4 6 0.2; n 5 5 and 5.71 6 0.07; nH 5 1.4 6 0.3;n5 3, respectively; Fig. 2), appearing as a high efficacy agonistin its own right. The NAM, ML375, was inactive on its own ineither signaling assay.To determine the degree of relative intrinsic efficacy of the

allosteric agonist, ML380, relative to that of the orthostericagonists, concentration–response curves to ACh, CCh, oxo-M,or ML380 in IP accumulation assays were re-established afterpretreatment (followed bywashout) of the CHO-hM5 cells withPBZ to irreversibly reduce receptor accessibility throughdirect steric hindrance (orthosteric agonists) or high negativecooperativity (ML380). As shown in Fig. 3, this resulted in areduction in both potencies and maximal responses for allagonists. Application of the operational model of agonism tothe resulting families of curves for each agonist (Table 1)

Fig. 2. Effect of ACh, ML380, and ML375 on (A) IP accumulation and (B)Ca2+mobilization in CHO-hM5 cells. Data are expressed as a percentage ofmaximal ACh response and represent the mean 6 S.E.M. of at least fourindependent experiments performed in triplicate; fitted curves are from afour-parameter logistic equation.

Mechanisms of M5 Muscarinic Receptor Allosteric Modulation 429

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revealed that PBZ reduces the freely accessible receptorpopulation by 48 6 13% at 1 mM, 88 6 4.4% at 10 mM, and99 6 0.4% at 100 mM. All of the orthosteric and allostericligands had similar functional affinities for their respectivesites on the receptor (pKA5 4.8–5.3), butML380 displayed thelowest operational efficacy (t 5 4) compared with the orthos-teric agonists (t 5 20–33; Table 1).As well as exhibiting intrinsic agonist activity, ML380

potentiated the activity of ACh in both IP and Ca21

mobilization assays (Fig. 4, A and B), thus acting as aPAM agonist (Christopoulos, 2014) with net cooperativity(Log ab) values of 0.966 0.26 (ab 5 9.1) and 1.696 0.26 (ab 549), respectively (Table 2). The stronger positive cooperativityfor ML380 in the calcium assay is consistent with its higherdegree of stimulus–response coupling, also reflected by thegreater intrinsic agonism for ML380 at this endpoint versusthe IP assay (Log tB 5 1.18 6 0.18; tB 5 15, versus Log tB 50.69 6 0.06; tB 5 4.9). To evaluate potential probe de-pendence of the PAM activity, interaction studies were alsoconducted with CCh, oxo-M, and the partial agonist, pilo-carpine, in the IP assay. The positive modulation of CCh-and oxo-M–mediated responses by ML380 was similar tothat for ACh (Fig. 5; Table 2), indicating little probedependence with full agonists. Interestingly, ML380 mark-edly potentiated the potency and maximal response tothe partial agonist, pilocarpine, with a Log ab value of

1.78 6 0.15 (ab 5 60), which was significantly greater thanthat observed against the full agonists and not consistentwith the predictions of a simple two-state model of allosteryas exemplified by prior studies of the related M1 mAChRwith PAMs such as BQCA and BQZ12 (Canals et al., 2012;Abdul-Ridha et al., 2014a,b).The NAM, ML375, produced parallel, rightward shifts in

concentration–response curves to ACh in both IP and Ca21

mobilization assays (Fig. 4, C and D). Analysis of the latteryielded estimates of allosteric site affinity (pKB 5 6.626 0.06;n5 4) and saturable negative cooperativity [Log ab521.3460.04 (ab 5 0.05); n 5 4]. In the IP assay, however, thereduction in agonist potency did not appear to reach a ceilinglevel over the entire concentration range tested, indicative ofeither a competitive interaction or, more likely, a very highdegree of negative cooperativity. As such, the data couldbe fitted to an allosteric model in which the negativecooperativity could be constrained to a very small value (Logab 5 23.0) under which the allosteric ternary complex modeland a competitive interaction model become indistinguish-able (Keov et al., 2014). This yielded an estimate of theML375pKB 5 6.22 6 0.13 (n 5 6), which was in excellent agreementwith the allosteric ternary complex analysis of the Ca21

mobilization data. As was observed for the ML380 experi-ments, there was little difference between the observedmodulation of ACh-, CCh-, and oxo-M–stimulated IP accumu-lation, with ML375 causing parallel rightward shifts withhigh negative cooperativity and similar estimates of affinityirrespective of the agonist tested (Fig. 5; Table 2). Again, asobserved with the PAM with ML380, the modulation ofpilocarpine activity by the NAM yielded a different profile tothe full agonists, with ML375 virtually abolishing the maxi-mal agonist response (Fig. 5; Table 2).To further understand the mechanisms underlying the

positive and negative cooperativity exhibited by ML380 andML375, respectively, whole-cell [3H]-NMS–binding studieswere subsequently performed to directly determine the co-operative effects on agonist binding alone (a). [3H]-NMSbound to whole CHO-hM5 cells in a monophasic and saturablemanner with an estimated pKD 5 9.64 6 0.12 (KD 5 0.26 60.07 nM; n 5 4) and maximal binding capacity (Bmax) of 7.3 61.2 fmol/105 cells.

Fig. 3. Effect of PBZ (0.1–100 mM) on IP accumulationstimulated by (A) ACh, (B) CCh, (C) oxo-M, and (D)ML380 in CHO-hM5 cells. Data are expressed as apercentage of maximal control ACh response and repre-sent the mean 6 S.E.M. of at least five independentexperiments performed in triplicate. Fitted curves arefrom global analysis of the datasets according to eq. 1 (inMaterials and Methods), with parameter estimatesshown in Table 1.

TABLE 1Operational model parameters for agonist-stimulated IP accumulationwith and without PBZ pretreatment in CHO-hM5 cells

Agonist apKAbLog t (t) cn

ACh 5.28 6 0.11 1.29 6 0.11 (20) 0.93 6 0.06CCh 4.78 6 0.18 1.42 6 0.18 (26) 0.64 6 0.07Oxo-M 4.65 6 0.22 1.52 6 0.23 (33) 0.53 6 0.06ML380 4.79 6 0.16 0.61 6 0.11 (4) 1.39 6 0.18

Estimated parameters represent the mean 6 S.E.M. of at least four experimentsperformed in triplicate. Functional IP accumulation responses were analyzedaccording to eq. 1 (Materials and Methods).

aNegative logarithm of the agonist equilibrium dissociation constant.bNegative logarithm of the operational efficacy parameter (antilog shown in

parentheses).cSlope of the transducer function linking receptor occupancy to response.

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ML380 increased the affinity of ACh, CCh, and oxo-M toinhibit the binding of [3H]-NMS from CHO-hM5 cells withestimates of positive cooperativity (Log a) similar to the Logab values determined in the IP accumulation assays (Fig. 6;Table 3). In addition, ML380 exerted weak negative coopera-tivity with respect to the inverse agonist radioligand, [3H]-NMS,and neutral affinity cooperativity with respect to the weakpartial agonist, pilocarpine (Log a 5 0.00 6 0.18; Fig. 6;Table 3).Similar [3H]-NMS–binding studies revealed that ML375

caused a concentration-dependent decrease in the affinitiesof ACh, CCh, and, to a lesser extent, oxo-M and pilocarpine(Fig. 7; Table 3). Conversely, ML375 exhibited neutral-to-weak positive cooperativity with respect to [3H]-NMS itself.The affinity (pKB) estimates for ML375 were broadly in linewith those generated from the functional assays (Table 2).However, the degree of negative cooperativity with respectto agonist binding was not sufficient to account for thehigher negative cooperativity noted in the functional assays(Table 2).The discrepancies between cooperativity values across

binding and functional studies, and the direct effects observedwith either PAM or NAM on the maximal effect of the partialagonist, pilocarpine, thus led to the hypothesis that theseallosteric modulators may be exerting their overall effects on

agonist function not only by modulating orthosteric agonistaffinity, but also intrinsic efficacy. As these effects were notapparent in the functional assays with the full orthostericagonists [because their higher efficacies (Table 1) allowedthem to achieve the maximum cellular effect in both theabsence and presence of modulator under control conditions],we exploited the ability of PBZ to irreversibly alkylate themAChRs and effectively reduce the number of spare receptors,thus enforcing conditions in which even high efficacy agonistslike ACh would require maximal receptor occupancy toachieve maximal observed effect. Accordingly, pretreatmentwith PBZ (3 mM), followed by extensive washout, yielded adepression in the ACh maximal response and potency tostimulate IP accumulation in CHO-hM5 cells (control pEC50

5 7.006 0.13, Emax 5 98.36 3.2%; PBZ-treated pEC50 5 6.036 0.10, Emax5 47.26 1.9%; Fig. 8A). In the presence ofML380(10mM), the potency (pEC505 7.096 0.15;P, 0.0001,F-test),but not the maximal response to ACh (49.7 6 1.8%; P 5 0.35,F-test), was significantly increased compared with control.There was a similar lack of effect on the maximal AChresponse in the presence of a higher concentration of ML380(30mM; data not shown) and also a small elevation in the basalresponse, reflecting residual ML380 agonist activity (Fig. 8A).An almost identical profile was seen when the experimentalparadigmwas repeated using oxo-M as the orthosteric agonist

Fig. 4. Effect of ML380 (A, B) or ML375 (C, D) on ACh-stimulated IP accumulation (A, C) and Ca2+ mobiliza-tion (B, D) in CHO-hM5 cells. Data are expressed as apercentage of maximal ACh response and represent themean6 S.E.M. of at least five independent experimentsperformed in triplicate. Fitted curves are from globalanalysis of datasets according to eq. 2 (inMaterials andMethods) with parameter estimates shown in Table 2and Results.

TABLE 2Operational model estimates for the interactions between ML380 or ML375 and the indicated orthostericagonists in IP accumulation assays in CHO-hM5 cells

ML380 ML375

Agonist apKBbLog ab (ab) cLog tB

dn pKBbLog ab (ab) dn

ACh 4.79 0.96 6 0.26 (9.1) 0.69 6 0.06 =1.00 6.22 6 0.13 23.0 (0.001) 0.92 6 0.11CCh 4.79 1.1 6 0.32 (12.3) 0.68 6 0.07 =1.00 6.22 6 0.13 23.0 (0.001) 1.08 6 0.15Oxo-M 4.79 0.81 6 0.19 (6.5) 0.51 6 0.04 =1.00 6.35 6 0.11 23.0 (0.001) 1.29 6 0.21Pilocarpine 4.79 1.8 6 0.15 (60.2) 0.45 6 0.05 =1.00 6.62 6 0.42 23.0 (0.001) 0.56 6 0.18

Estimated parameters represent the mean 6 S.E. of at least five experiments performed in triplicate. Functional IPaccumulation responses were analyzed according to eq. 2 (or eq. 3 for pilocarpine; Materials and Methods).

aNegative logarithm of the allosteric modulator equilibrium dissociation constant (constrained to the value obtainedfrom the receptor alkylation studies; Table 1).

bLogarithm of functional cooperativity between agonist and allosteric modulator (antilog values shown in parentheses).cLogarithm of operational efficacy parameter.dSlope of the transducer function linking receptor occupancy to response (constrained to unity for ML380 datasets).

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(Fig. 8B), indicating that the ML380-mediated modulation ofpilocarpine efficacy is not seen in combination with higherefficacy agonists.To compare this mechanism with that of other well-studied

allosteric modulators of mAChRs, we performed similaralkylation and IP accumulation studies in CHO cells stablyexpressing the Gq/11-coupled M1 mAChR subtype using

BQZ12, a high-affinity congener of the M1 mAChR PAM,BQCA, that has been proposed to act according to a simpletwo-state model of allostery (Canals et al., 2012; Abdul-Ridhaet al., 2014a). As with the M5 mAChR experiments, PBZpretreatment reduced ACh potency and maximal response tostimulate IP accumulation in CHO-hM1 cells (control pEC5056.666 0.06, Emax 5 88.56 1.5%; PBZ-treated pEC50 5 5.1860.11, Emax 5 49.8 6 2.2%; Fig. 8C). In the presence of BQZ12(10–30 nM), the ACh potency was significantly enhanced byapproximately 50-fold (P , 0.0001, F-test), but there was noeffect on the maximal response (P 5 0.65, F-test; Fig. 8C),suggesting that ML380may exert its effects at theM5mAChRin a similar manner to BQZ12 at the M1 mAChR, that is,primarily by modulation of full agonist affinity.Interestingly, when profiled under identical PBZ-treated

conditions, ML375 (10–30 mM) significantly decreased bothpotency (control pEC50 5 6.85 6 0.05, Emax 5 98.8 6 1.1%;PBZ-treated5 5.456 0.09, Emax 5 48.26 1.4%; pEC50 5 3.986 0.12 at 10 mM ML375; 3.75 6 0.17 at 30 mM ML375; P ,0.01, F-test) and maximal response (27.1 6 1.6% at 10 mM;22.16 2.0% at 30 mM; P, 0.05, F-test) of ACh to stimulate IPaccumulation compared with control (Fig. 8D), indicating thatit is a NAM of both ACh affinity and signaling efficacy.Finally, we investigated whether ML380 and ML375 medi-

ate their PAM and NAM effects, respectively, from a commonsite on the M5 receptor. Using the IP accumulation assay, wedetermined the ability of ML375 or the orthosteric antagonist,atropine, to inhibit the agonist response to a single, fixedconcentration of ML380 (10 mM). Atropine and ML375 bothfully inhibited the response to ML380 (pIC50 values 8.13 60.10 and 5.796 0.21, respectively; Fig. 9). As ML380 is clearlyallosteric with respect to ACh and [3H]-NMS (vide supra), theinhibition mediated by atropine is almost certainly allosteric,but characterized by a high degree of negative cooperativity.Moreover, three-way binding studies monitoring the competi-tion between atropine, a [3H]-NMS, in the absence or presenceof ML380 (Supplemental Fig. 1) revealed that atropine hasneutral affinity cooperativity with respect to modulator (Log a5 0.00 6 0.12), suggesting that the inhibition seen in thefunctional assay must be mediated by high negative efficacycooperativity.

Fig. 5. Effect of ML380 (A, C, E) or ML375 (B, D, F) on CCh, oxo-M, orpilocarpine-stimulated IP accumulation in CHO-hM5 cells. Data areexpressed as a percentage of maximal ACh response and represent themean 6 S.E.M. of at least three independent experiments performed intriplicate. Fitted curves are from global analysis of datasets according toeq. 2 (or eq. 3 for pilocarpine; in Materials and Methods) with parameterestimates shown in Table 2 and Results.

Fig. 6. Three-way radioligand-binding studies to demon-strate the effect of ML380 (0.3–30 mM) on (A) ACh, (B) CCh,(C) oxo-M, and (D) pilocarpine-mediated inhibition of [3H]-NMS binding in whole CHO-hM5 cells. Cells were incubatedwith radioligand for 2 h at 37°C; data are expressed as apercentage of control specific binding and represent themean6S.E.M. of at least three independent experiments performed intriplicate. Fitted curves are from global analysis of datasetsaccording to eq. 4 (in Materials and Methods) with parameterestimates shown in Table 3.

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Unfortunately, the data forML375 provide less resolution—the full inhibition of the ML380 response could indicate acompetitive interaction for a common allosteric site, or a highdegree of negative cooperativity with respect to a secondallosteric binding site. Assuming the former, analysis of theML375 antagonist inhibition curve using a modified compet-itive Cheng-Prusoff equation (Leff et al., 1993) yielded anestimated pKB of 6.14 6 0.10, which is similar to the affinityestimates of the modulator obtained in our other assays basedon allosteric model analyses (vide supra). The most parsimo-nious explanation, thus, is that both modulators interact witha common site.

DiscussionRecent screening and medicinal chemistry efforts have

resulted in the first subtype-selective small-molecule positiveand negative allosteric modulators of muscarinic M5 mAChRs(Bridges et al., 2010; Gentry et al., 2013, 2014). ML380 hasbeen described as a PAM of ACh-stimulated calcium mobili-zation at both human and rat muscarinic M5 mAChRs, withmoderate selectivity versus the M1 and M3 mAChR subtypes.Radioligand-binding studies also suggested that ML380 couldincrease the affinity of ACh for the M5 mAChR (Gentryet al., 2014). ML375 is a highly subtype-selective NAM of

ACh-stimulated calcium mobilization at both human and ratmuscarinic M5 mAChRs, which was shown to be allosteric byits ability to slow [3H]-NMS dissociation from the receptor(Gentry et al., 2013). Despite these data, relatively little isknown about the mechanisms of modulation by these alloste-ric ligands. The aim of the current studies was to fullycharacterize the pharmacology of the PAM, ML380, andthe NAM, ML375, at the muscarinic M5 mAChR usingradioligand-binding and canonical Gq/11-linked signaling as-says (IP accumulation and Ca21mobilization). By doing so, wereveal that these allosteric ligands have minimal effects onantagonists, but have the potential to modulate both orthos-teric agonist affinity and intrinsic efficacy of agonists in aprobe-dependent manner.Our studies confirm that ML380 and ML375 display PAM

agonist and NAM behavior, respectively, when interactingwith a range of orthosteric agonists in assays of IP accumu-lation and Ca21 mobilization, with ML375 possessing sub-micromolar affinity for the M5 mAChR and ML380displaying similar affinity as CCh and oxo-M. Three-wayradioligand-binding studies with the inverse agonist radio-ligand, [3H]-NMS and ACh, CCh, or oxo-M suggest thatML380 and ML375 exert their effects in a manner broadlyconsistent with a two-state mechanism (Fig. 2; Table 2), aspreviously described for PAMs of the muscarinic M1 receptor,

TABLE 3Allosteric ternary complex model parameters for the interaction between ML380 or ML375 with the orthosteric antagonist/inverse agonist, [3H]-NMS,and the indicated agonists

Agonist apKIbpKB

ML380 ML375

cLog a (a) dLog a9 (a9) bpKBcLog a (a) dLog a9 (a9) bpKB

ACh 4.64 6 0.07 4.79 0.87 6 0.12 (7.4) 20.17 6 0.06 (0.67) 4.94 6 0.09 21.37 6 0.12 (0.04) 0.08 6 0.03 (1.2) 6.87 6 0.14CCh 4.32 6 0.06 4.79 0.74 6 0.11 (5.5) 20.36 6 0.06 (0.44) 4.43 6 0.13 21.52 6 0.18 (0.03) 0.06 6 0.03 (1.1) 6.82 6 0.20Oxo-M 4.86 6 0.06 4.79 1.06 6 0.11 (12) 20.40 6 0.06 (0.40) 4.94 6 0.09 20.85 6 0.13 (0.14) 0.16 6 0.03 (1.4) 6.52 6 0.15Pilocarpine 4.83 6 0.05 4.79 0.00 6 0.18 (1.0) 20.37 6 0.05 (0.43) 4.88 6 0.09 20.57 6 0.11 (0.27) 20.01 6 0.02 (0.98) 6.72 6 0.30

Estimated parameters represent the mean 6 S.E. of three experiments performed in duplicate. Three-way binding interactions were analyzed according to eq. 4 (Materialsand Methods).

aNegative logarithm of the orthosteric agonist equilibrium dissociation constant.bNegative logarithm of the allosteric modulator equilibrium dissociation constant.cLogarithm of affinity cooperativity between orthosteric agonist and allosteric modulator (antilog values shown in parentheses).dLogarithm of affinity cooperativity between [3H]-NMS and allosteric modulator (antilog values shown in parentheses).

Fig. 7. Three-way radioligand-binding studies to demonstratethe effect of ML375 (0.3–30 mM) on (A) ACh-, (B) CCh-, (C) oxo-M–, and (D) pilocarpine-mediated inhibition of [3H]-NMSbinding in whole CHO-hM5 cells. Cells were incubated withradioligand for 2 h at 37°C; data are expressed as a percentageof control specific binding and represent the mean 6 S.E.M. ofat least three independent experiments performed in tripli-cate. Fitted curves are from global analysis of datasets accord-ing to eq. 4 (in Materials and Methods) with parameterestimates shown in Table 3.

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BQCA and BQZ12 (Canals et al., 2012; Abdul-Ridha et al.,2014a). However, the modulation of the partial agonist,pilocarpine, by both ML380 and ML375 indicated thatadditional mechanisms might contribute to their pharmacol-ogy. ML380 displayed neutral affinity cooperativity withrespect to pilocarpine, yet potentiated its function in IPaccumulation assays by approximately 60-fold (Tables2 and 3). In addition, the PAM agonist displayed a steeperconcentration–response relationship when mediating IP ac-cumulation in both the absence and presence of orthostericagonists (Figs. 3 and 5), suggesting that it may bind co-operatively with respect to itself or that it has the potential tochange the sensitivity of the coupling of the receptor to theGq/11 pathway. Finally, ML375 displayed only weak negativeaffinity cooperativity with pilocarpine, yet ablates the func-tional response to this agonist (Tables 2 and 3). These effectscannot be accommodated within a simple model allowing onlyfor affinity modulation and indicate that both ML380 andML375 might additionally modulate the signaling efficacy ofthe M5 mAChR.As effects of allosteric modulators on relative efficacy are

often difficult to detect in functional assays (due to the use offull agonists that reach the maximal system response and/orassays with high degrees of stimulus–response coupling), weused the receptor-alkylating agent, PBZ, to reduce levels ofreceptor reserve. Pretreatment with PBZ reduced the potency

andmaximal response of ACh to stimulate IP accumulation inCHO-hM5 cells and opened a window to visualize bothnegative and positive effects on the maximal response to theagonist. Under these conditions, ML380 clearly increasedthe potency of both ACh and oxo-M, but did not increase themaximal response (Fig. 8), suggesting that it modulates onlythe affinity of full agonists. Similar studies with the M1

mAChR verified that this PAM effect was conserved acrossmAChRs; under identical assay conditions, the M1 mAChRPAM, BQZ12, caused only a leftward shift in theconcentration–response curve to ACh, with no change inmaximal agonist response (Fig. 8). However, the NAM,ML375, clearly decreased the maximal response to ACh,respectively (Fig. 8), suggesting that it engenders changes inthe ability of theM5mAChR to couple to G protein(s) as well asmodulating the affinity of agonist binding. The effect ofML380on pilocarpine signaling efficacy is also a clear indicator thatthe PAM can exert probe-dependent effects on receptor–effector coupling as well.Although the location of allosteric sites on the M5 mAChR

has not been extensively studied, mutagenesis studies, as wellas computational and structural insights, support the notionthat there is at least one common allosteric binding site in theextracellular vestibule of mAChRs (Huang et al., 2005; Droret al., 2013; Kruse et al., 2013). Thus, despite topographicalsimilarities, these data raise the potential of differentialmolecular modes of allosteric modulation within the samereceptor family. Across the wider GPCR superfamily, differentmodes of allosteric modulation have already been observed: atthe family C mGlu1 and mGlu5 receptors, NAMs such asCPCCOEt and MPEP do not alter the ability of orthostericagonists to bind to the receptor, but exclusively negativelymodulate agonist efficacy (Litschig et al., 1999; Bradley et al.,2011). At the other end of the spectrum are the prototypicalmAChRNAMs, gallamine and C7/3-phth, and theM1mAChR-selective PAMs, BQCA and BQZ12, which appear to modulateonly ACh affinity (Canals et al., 2012; Abdul-Ridha et al.,2014a). Many allosteric modulators of GPCRs exert a mixedmechanism with respect to agonist affinity and efficacy,including muscarinic M2 and M4 receptor PAMs (Valantet al., 2012; Croy et al., 2014) and mGlu4 and mGlu5 receptor

Fig. 8. Effect of ML380 (10 mM) on (A) ACh- and (B) oxo-M–

stimulated IP accumulation in CHO-hM5 cells; (C) BQZ12(10–30 nM) on ACh-stimulated IP accumulation in CHO-hM1 cells; and (D) ML375 (10–30 mM) on ACh-stimulated IPaccumulation in CHO-hM5 cells, all after 30-minute pre-treatment with 3 mM PBZ, followed by three phosphate-buffered saline washes. Dashed line indicates the control(untreated) response to ACh. Data points represent themean 6 S.E.M. of at least three independent experimentsperformed in duplicate or triplicate. Fitted curves to ACh (6PBZ) are from a global fit to eq. 1; curves of ACh in thepresence of allosteric modulator are fitted according to athree-parameter logistic equation.

Fig. 9. Effect of atropine or ML375 on IP accumulation in CHO-hM5 cellsstimulated by a single, fixed concentration of ML380 (10 mM). Data pointsrepresent the mean 6 S.E.M. of at least three independent experimentsperformed in triplicate. Fitted curves are from a four-parameter logisticequation.

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PAMs (Mathiesen et al., 2003; Bradley et al., 2011). Thestudies described in this work indicate that positive modula-tion of the M5 mAChR can occur via regulation of eitheraffinity (for ACh and oxo-M) or efficacy (as for pilocarpine) andthat negative modulation is mediated by changes in bothproperties.If, as our findings suggest, this is achieved via interaction

with a common allosteric site, it would be interesting forfuture studies to determine whether this site represents theprototypical allosteric site proposed for other mAChRs in theextracellular vestibule (Kruse et al., 2014), or whether the M5

mAChR modulators interact with a hitherto unidentified siteon this receptor. More importantly, this may have implica-tions to the ultimate use of such compounds and how theireffects translate into native tissue or in vivo studies. Forinstance, a recent investigation by Foster et al. (2014) using anearlier generation M5 mAChR PAM (ML129) (Bridges et al.,2009) found that somatodendritic M5 mAChRs increase sub-stantia nigra pars compacta dopamine neuron excitability,whereas terminal M5 mAChRs inhibit dopamine release. It isenvisaged that the availability of newer generation PAMs andNAMs with different modes of allosteric modulator effects canalso be used to shed further insights into the physiology andpotential therapeutic targeting of the M5 mAChR.

Authorship Contributions

Participated in research design: Berizzi, Gentry, Langmead,Christopoulos.

Conducted experiments: Berizzi, Gentry, Rueda, Den Hoedt.Performed data analysis: Berizzi, Gentry, Rueda, Den Hoedt,

Langmead, Christopoulos.Wrote or contributed to the writing of the manuscript: Berizzi,

Gentry, Rueda, Sexton, Langmead, Christopoulos.

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Address correspondence to: Dr. Christopher J. Langmead or Prof. ArthurChristopoulos, Monash Institute of Pharmaceutical Sciences, Monash Univer-sity, 381 Royal Parade, Parkville, VI 3052, Australia. E-mail: [email protected] or [email protected]

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