https://doi.org/10.1177/2472555219879644

SLAS Discovery 1 –10© 2019 Society for LaboratoryAutomation and ScreeningDOI: 10.1177/2472555219879644journals.sagepub.com/home/jbx

Original Research

Introduction

NAD and its phosphorylated form, NADP, are essential molecules to all organisms, acting as redox factors and sub-strates in many biological pathways. NAD biosynthesis fuels bioenergetic processes and maintains balanced cell regulation. Nicotinamide mononucleotide adenylyltransfer-ase (NMNAT; EC 2.7.7.1) catalyzes the reversible reaction of nicotinamide mononucleotide (NMN+) or nicotinic acid mononucleotide (NaMN+) and ATP to form NAD+ or NaAD+ and pyrophosphate (Fig. 1A).1 Targeting human NMNAT has potential utility in drug discovery for various types of cancers,1,2 neurodegenerative diseases,2,3 and aging.3 For infectious diseases, bacterial NMNAT is a potential antibiotic target,2,4 and recently reported cloning and expression of NMNATs from parasitic protozoa5–11 sug-gest NMNAT as a target for eukaryotic pathogens.

The human genome encodes for three isoforms of NMNAT, termed NMNAT1, NMNAT2 and NMNAT3, which have distinct subcellular localizations: NMNAT1 in the nucleus, NMNAT2 in the cytosol and Golgi, and

NMNAT3 in the cytosol and mitochondria.2,12 NMNAT has potential as a drug target for cancer if potency and selectiv-ity for cancer cells can be achieved.13,14 NMNAT also has potential as a drug target for neurodegenerative diseases and aging if activators can be identified. Guarente states that “a model emerges in which aging is associated with PARP [poly (ADP-ribose) polymerase] activation, NAD+ depletion, sirtuin inactivation, mitochondrial dysfunction,

879644 JBXXXX10.1177/2472555219879644SLAS DISCOVERY: Advancing Life Sciences R&DHaubrich et al.research-article2019

1Institute for Rare and Neglected Diseases Drug Discovery, Mountain View, CA, USA2Department of Chemistry, University of Nevada, Reno, Reno, NV, USA3DCSwinney Consulting, Belmont, CA, USA

Received Aug 6, 2019, and in revised form Sept 3, 2019. Accepted for publication Sept 9, 2019.

Supplemental material is available online with this article.

Corresponding Author:Brad A. Haubrich, Department of Chemistry, University of Nevada, Reno, 1664 N Virginia St., Reno NV 89557, USA. Email: bhaubrich@unr.edu

Development of a Bioluminescent High-Throughput Screening Assay for Nicotinamide Mononucleotide Adenylyltransferase (NMNAT)

Brad A. Haubrich1,2 , Chakk Ramesha1, and David C. Swinney1,3

AbstractNicotinamide mononucleotide adenylyltransferase (NMNAT; EC 2.7.7.1) catalyzes the reversible production of NAD+ from NMN+ and ATP and is a potential drug target for cancer and neurodegenerative diseases. A sensitive bioluminescent assay format suitable to high-throughput screening (HTS) and mechanistic follow-up has not been reported and is of value to identify new modulators of NMNATs. To this end, we report the development of a bioluminescent assay using Photinus pyralis ATP-dependent luciferase and luciferin for NMNAT1 in a 384-well plate format. We also report a mechanistic follow-up paradigm using this format to determine time dependence and competition with substrates. The assay and follow-up paradigm were used to screen 912 compounds from the National Cancer Institute (NCI) Mechanistic Diversity Set II and the Approved Oncology Set VI against NMNAT1. Twenty inhibitors with greater than 35% inhibition at 20 µM were identified. The follow-up studies showed that seven actives were time-dependent inhibitors of NMNAT1. 2,3-Dibromo-1,4-naphthoquinone was the most potent, time-dependent inhibitor with IC50 values of 0.76 and 0.26 µM for inhibition of the forward and reverse reactions of the enzyme, respectively, and was shown to be NMN and ATP competitive. The bioluminescent NMNAT assay and mechanistic-follow-up will be of use to identify new modulators of NAD biosynthesis.

KeywordsNAD+, bioluminescence, NMN, inhibition, time dependence, MMOA, screening, assay

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and degeneration of cells and tissues, and where this calam-ity can be corrected or forestalled by supplementation with NAD+ precursors.”15 Activation of NMNAT may provide an additional means to increase the concentrations of NAD formed from the precursors. Epigallocatechin gallate was discovered to activate NAD production by NMNATs, in an isoform-specific manner, nearly doubling NMNAT2 activ-ity, while affecting NMNAT1 by 10%.2 However, potent and selective inhibitors of NMNATs have yet to be described.

Our goal in this work was to develop an assay format and follow-up paradigm suitable to screen for new modulators of NMNAT and efficiently characterize the molecular mechanism of action (MMOA) for time dependence and competition with substrates. The assays reported in litera-ture used to characterize NMNAT activity have identified compounds with modest activity against NMNAT.2 These assays used low-throughput screens of substrate analogs and inhibitors chosen for activity against other NAD-related enzymes. The assay formats used for the forward reaction included discontinuous and continuous tandem reactions with alcohol dehydrogenase, high-performance liquid chro-matography (HPLC),16 or inorganic pyrophosphatase.4 For the reverse reaction (NAD+ + PPi → NMN+ + ATP) (Fig. 1A), assay formats have largely relied on radioactivity and/or time-consuming chromatography. Due to these unideal methods, some recent NMNAT and NMN+ quantitation assays have favored colorimetric5,9,17,18 or fluorescent6,19 techniques. We were interested in devising an assay with sensitive detection and formats applicable to high-through-put screening (HTS) for measuring the forward and reverse reactions of NMNAT to identify and characterize potential modulators of NMNAT. Such assay formats could help serve in confirming actives in the forward as well as reverse reactions of NAD biosynthesis.

As noted above, our goal also included developing a paradigm to enable early characterization of the MMOA of the inhibitors. Early understanding of the MMOA of modu-lators identified in target-based screens will facilitate opti-mization of molecules with the most suitable mechanistic properties. For example, it is important when starting with a target-based screen to differentiate molecules as competi-tive inhibitors, noncompetitive inhibitors, or activators, and whether they are time dependent or not. Establishing assays to identify compounds for time dependence also may iden-tify potent compounds that may be missed and that have properties that could lead to increased selectivity. We have hypothesized that time-dependent inhibition in a nonequi-librium system can increase the selectivity of an inhibitor, due to kinetic selectivity, and thereby increase the therapeu-tic index and potential utility of a medicine.20–23

The goals of this work were achieved with the develop-ment a bioluminescent assay using Photinus pyralis ATP-dependent luciferase (PpLuc) and luciferin for NMNAT1 in a 384-well plate format that measures both the forward (Fig. 1B) and reverse (Fig. 1C) directions of its reversible enzymatic reaction. The mechanistic paradigm identified the most potent inhibitors as time dependent and substrate competitive. These inhibitors had less activity without pre-incubation. The assay and mechanistic follow-up paradigm will be of use to identify new modulators of NMNATs to treat unmet medical needs in neurodegenerative diseases, cancers, and infectious diseases.

Materials and Methods

Reagents

The cyclic alkylaminoluciferin CycLuc1 and reduced coen-zyme A (CoASH) were purchased from EMD Millipore

Figure 1. NMNAT1 reaction scheme and assay paradigm. (A) NMNAT catalyzes the reversible adenylyl transfer reaction of NMN+ and ATP to NAD+ and PPi. The forward and reverse directions of the NMNAT-catalyzed reaction are defined as NMN+ + ATP → NAD+ + PPi and NAD+ + PPi → NMN+ + ATP, respectively. (B) Screening assay paradigm of NMNAT1 in the forward direction. (C) Screening assay paradigm of NMNAT1 in the reverse direction.

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(Darmstadt, Germany). d-Luciferin was purchased from Fisher (Pittsburgh, PA). NMN+, NAD+, ATP, sodium pyro-phosphate, and 2,3-dihalo-1,4-naphthoquinones were pur-chased from Sigma (St. Louis, MO). The Mechanistic Diversity Set II and Approved Oncology Drugs Set VI com-pound libraries, 1 and 10 mM DMSO, respectively, were obtained from the NCI.

Human NMNAT1 and PpLuc were prepared by Biozilla as detailed below.

All other reagents, unless otherwise noted, were pur-chased from Sigma.

Expression and Purification of Recombinant NMNAT1

Full-length NMNAT1 (accession no. UniProtKB–Q9HAN9) was cloned into the pET21d expression vector in-frame with a C-terminal 6× His tag. BL21 (DE3) Escherichia coli were transformed with the construct and cultured in lysogeny broth (LB) to an optical density (OD; at 590 nm) of 0.9. Expression of NMNAT1 was induced with isopropyl-β-D-1-thiogalactopyranoside (0.5 mM) for 17 h at 24 °C. Cells were harvested and resuspended in lysis buffer (50 mM HEPES [pH 7.5], 300 mM NaCl, 10% glycerol, 20 mM imidazole, 10 µM phenylmethylsulfonyl fluoride, and 2 mM β-mercaptoethanol). The bacteria were lysed by sonication and freeze/thaw cycles. Insoluble protein was removed by centrifugation (12,000g, 30 min, 4 °C) and the soluble fraction loaded onto a nickel immo-bilized metal affinity chromatography (Ni-IMAC) col-umn. The column was washed with 50 mM HEPES (pH 7.5), 300 mM NaCl, 10% glycerol, 40 mM imidazole, 0.02% Triton X-100, and 2 mM β-mercaptoethanol fol-lowed by 50 mM HEPES (pH 7.5), 300 mM NaCl, 10% glycerol, 80 mM imidazole, and 2 mM β-mercaptoethanol. Recombinant NMNAT1 was eluted from the IMAC col-umn with 4.5 mL of 25 mM HEPES (pH 7.5), 300 mM NaCl, 10% glycerol, 500 mM imidazole, and 2 mM β-mercaptoethanol. Eluted NMNAT1 was further fraction-ated by size-exclusion chromatography (SEC) using a Superdex-200, 10/300 GL column preequilibrated with SEC running buffer, 50 mM Tris-HCl (pH 7.5), and 100 mM NaCl. Fractions were collected, and a portion of each was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to identify the fractions containing NMNAT1. A single band corresponding to the molecular weight of NMNAT1 was eluted in fractions 13–15 and confirmed to be active NMNAT enzyme by activity measurement (Suppl. Fig. S1A). From 2 × 1 L of culture, 2.6 mg of protein was recovered at 1.3 mg/mL. Purified NMNAT1 was stored at –80 °C in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 20% glycerol until used.

Expression and Purification of Recombinant PpLuc

Full-length PpLuc (accession no. UniProtKB–Q27758) was cloned into the pET21d expression vector in-frame fused to myelin basic protein to its N-terminal side and to a 6× His tag to its a C-terminal side. BL21 (DE3) E. coli were transformed with the construct and cultured in LB to an OD (at 590 nm) of 0.9. PpLuc was purified through a Ni-IMAC as described above for NMNAT. The eluate from the nickel column con-tained one major band on SDS-PAGE and was dialyzed against 20 mM Tris-Cl (pH 8.0), 20% glycerol, and 1 mM DTT, and used without further purification (Suppl. Fig. S1B).

NMNAT1 Enzyme Assay

Preparation of Detection Reagent Mixture. The ATP detec-tion reagent was prepared by mixing PpLuc (26 µg/mL), d-luciferin or CycLuc124 (59.3 µM), DTT (2.67 mM), bovine serum albumin (BSA; 0.167 mg/mL), CoASH (1.65 mM), and MgSO4 (16.67 mM) in NMNAT buffer (see below) supplemented with an extra 37.6 mM HEPES to pH 7.4. The detection mixture was incubated at the ambient room temperature for at least 30 min.

NMNAT1 Assay. Unless otherwise noted, in a final volume of 20 µL the reaction consisted of either 6–10 nM (forward reaction) or 0.5–1 nM (reverse reaction) NMNAT1 and an appropriate substrate in NMNAT buffer (15 mM HEPES adjusted to pH 7.4, 20 mM NaCl, 10 mM MgCl2, 1 mM EGTA, and 0.02% [v/]) Tween-20). The substrates used for the forward reaction were ATP (20 µM) and NMN+ (100 µM), and substrates used for the reverse reaction were NAD+ (100 µM) and pyrophosphate (100 µM). The reac-tion was allowed to proceed at ambient room temperature (about 23 °C) and was stopped by the addition of 45 µL of the detection reagent. Following 20 min of additional incu-bation, the luminescent signal coming from the amount of ATP either remaining (forward reaction) or generated (reverse reaction) was measured using a Luminoskan Ascent plate reader (Thermo Labsystems, Waltham, MA).

High-Throughput Screening. HTS of NMNAT1 inhibition was performed in the forward direction (NMN+ → NAD+), by measuring depletion of ATP-proportional light. Inhibi-tors (5 µL of 80 µM 8% DMSO [v/v]) and NMNAT1 (5 µL of 40 nM) were added to 384-well plates followed by sub-strates (10 µL of 40 µM ATP and 200 µM NMN+). All liq-uid transfers were made using a Biomek 2000 automated liquid handling system (Beckman Coulter, Indianapolis, IN). The final concentrations of the reagents were 10 nM NMNAT1, 20 µM ATP, 100 µM NMN+, 20 µM inhibitor,

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and 2% (v/v) DMSO. Each plate contained multiple wells for control and ATP standards that had 2% DMSO and no inhibitor. All compounds were tested in duplicate. The reac-tion was initiated by the addition of the substrate and was allowed to proceed for 55 min at ambient room tempera-ture. The reaction was terminated by adding 45 µL/well of the detection reagent, and the resulting luminescence was measured after 20 min of additional incubation using a Luminoskan Ascent plate reader. The NMNAT-catalyzed metabolism was calculated by eq 1A:

ATP depleted [ ] =

−−

×[ ]RLUATP RLU

RLUATP bkgdATPstd (1A)

where RLU and RLUATP are relative light signals for the sample and ATP standard, respectively, bkgd is a back-ground light signal of 100 µM NMN+ and luciferase reagent in the absence of NMNAT1 and ATP, and [ATPstd] is the concentration of ATP used with no NMNAT1.

Follow-Up Screen. Follow-up assays were run exactly as described above, but in addition to the forward reaction, inhibition of the reverse reaction (NAD+ → ATP) by com-pounds was also measured using 0.5 nM NMNAT, 100 µM NAD+, and 100 µM PPi as substrates, and the NMNAT-catalyzed ATP generation was calculated by eq 1B:

ATP generated [ ] = ×[ ]RLU

RLUATPATPstd (1B)

where RLU and RLUATP are relative light signals for the sample and ATP standard, respectively, and [ATPstd] is the concentration of ATP used with no enzyme.

Counterscreen. Compounds selected from HTS for follow-up were tested for their effect on the PpLuc assay in the absence of the NMNAT1. NMN+ or NAD+/PPi were included in background and normalization data points to account for any loss of signal due to these entities in the luciferase reaction mixture.

None of the active compounds selected for follow-up inhibited PpLuc activity. 2,3-Dibromo-1,4-naphthoquinone (DBNQ) was also tested for degradation of ATP upon prein-cubation; without enzyme, no change in ATP quantitation was noted relative to ATP signals without preincubation with DBNQ.

Molecular Mechanism of Action Assays

Time Dependence. A time dependence screen was per-formed with selected inhibitors from the primary screen (HTS). Time-dependent studies were performed by pre-incubating 2 and 20 µM test compounds with NMNAT1

for 45 min at ambient room temperature using the reac-tion parameters described above, NMNAT1-catalyzed reactions were initiated with the addition of the substrate pair (100 µM NMN+ and 20 µM ATP) in 20% volume (4 µL), and the reaction was allowed to proceed for an addi-tional 10 min. Time dependence was noted by an increase of inhibition at the 20 µM inhibitor with preincubation and a dose response between 2 and 20 µM compound concentrations.

Dose–response studies for time dependence were con-ducted with dihalo-naphthoquinones (DHNQs) by generat-ing IC50 plots. Following 45 min of preincubation, NMNAT1-catalyzed forward and reverse reactions were initiated and measured as described above.

DBNQ association experiments were conducted by varying the preincubation time of DBNQ and NMNAT1 from 0 to 45 min with 1 µM DBNQ and 6 nM NMNAT1 (forward reaction) and 1 nM NMNAT1 (reverse reaction), followed by the addition of substrate pairs and 10 min of incubation.

Evaluation of Mechanism of Action. Inhibition can be com-petitive, noncompetitive, or uncompetitive in relationship to substrates. The mechanism of action was evaluated by preincubating with and without substrates. The activity of competitive inhibitors will decrease in the presence of com-peting substrate concentrations, will be unchanged for non-competitive inhibition, and will increase with uncompetitive inhibition. To evaluate the mechanism of action of the inhibitors, test compounds at IC50 and NMNAT were prein-cubated for 45 min without and with substrates (ATP and NMN) and then enzymatic activity was measured. A loss of NMNAT1 inhibitory activity upon co-preincubation with one substrate was interpreted as competitive behavior of the inhibitor with respect to that substrate.

Data Analysis

For dose and time dependence experiments, Michaelis–Menten plots, IC50 values, and association experiments, data were fit to respective graphs using GraphPad Prism 6 (La Jolla, CA). Statistical values were derived from the BEST test.25 Results with p < 0.05 were considered statisti-cally significant. HTS data and corresponding histograms were analyzed using built-in software from Collaborative Drug Discovery (CDD; Burlingame, CA).

Results

Development of Bioluminescent NMNAT1 Assay

Discontinuous enzymatic assays involving a coupled ATP-dependent luciferase to measure light-proportional ATP have been successful for kinases and other classes of enzymes.26 We adapted a tandem PpLuc step to quantitate ATP in the

Haubrich et al. 5

reaction mixture. Using sulfate and CoASH to slow the light signal decay of PpLuc,27,28 we stabilized light signals to achieve linearity of signal and optimized to it our chosen ATP concentration of 20 µM, approximating 0.5 Km. Standard curves were generated to simulate NMNAT-catalyzed metab-olism of ATP, including NMN+ at assay concentrations, and NAD+ and PPi concentrations consistent with consumption of ATP and NMN+ during the enzymatic assay (Suppl. Table S1 and Suppl. Fig. S2). The curves showed good linearity. For the follow-up IC50 and association studies, d-luciferin in the detection reagent was replaced with CycLuc, which gave a higher signal-to-noise ratio at lower enzyme concentrations but did not affect the IC50 (data not shown).

Activity of NMNAT1

Human NMNAT1 was expressed in E. coli with an N-terminal His tag and purified to homogeneity as described in Materials and Methods. A discontinuous in vitro biolumi-nescent assay was used to quantify ATP from light gener-ated by PpLuc.26 The assay was optimized to achieve linearity of activity with respect to NMNAT1 concentration and time in the forward (Fig. 2A) and reverse (Fig. 2B) directions. The activity showed saturable dependence on substrate concentrations as shown for the reaction in the forward direction. NMNAT1 had apparent Km values of 43 and 16 µM for ATP and NMN+, respectively (Fig. 2C,D), in agreement with the published values of 58.5 and 22.3 µM, respectively, using alternative assay methods.12

HTS of Oncology Libraries with NMNAT1

The conditions for screening compounds in the 384-well format were chosen to ensure linearity with time and protein as well as provide a sufficient signal-to-noise ratio. The compounds were tested at a single concentration (20 µM) in duplicate. Figure 3A shows the distribution of activity with the majority of compounds being inactive. Of the 912 com-pounds tested, only 9 inhibited NMNAT1 activity >75%; this subset consisted of bis-sulfanylsulfonyl (NSC-624158), cisplatin (NSC-119875), chloro(triethylphosphine) gold (NSC-313981), and several organo-plumbane, -stannane, and -mercury compounds. An additional 11 compounds were added to the list of hits after changing the hit criteria to >35% inhibition at 20 µM.

Time-Dependent Assay Follow-Up on HTS Actives

Of the 20 hits from the HTS, 13 were metalorganic com-pounds and were excluded from follow-up. To access time-dependent inhibition of NMNAT1 by the remaining seven molecules, a secondary screen that included a 45 min prein-cubation with 20 and 2 µM compounds was performed. From this screen, all of these compounds were identified as time-dependent inhibitors by the increased inhibition fol-lowing preincubation (Table 1A). These compounds included NSC-624158 and four naphthoquinones, of which DBNQ is the most potent molecule. It should be pointed out here that the Mechanistic Diversity Set II library contained

Figure 2. Enzyme kinetics of NMNAT1. Linearity of NMNAT1-catalyzed reaction with respect to time and protein concentration for (A) forward direction (NMN+ + ATP → NAD+ + PPi) and (B) reverse direction (NAD+ + PPi → NMN+ + ATP). (C) Michaelis–Menten plots of variable ATP, Km = 43 µM, and (D) NMN+, Km = 16 µM, for the forward direction.

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>19 naphthoquinones; none of them showed any significant activity in the HTS. Even menadione (NSC-4170) and phthi-ocol (NSC-11897), the two close analogs of DBNQ, showed <35% inhibition in HTS. To make sure that these compounds are not false negatives, they were tested at 20 µM without and with preincubations and found to be inactive (Table 1B). On the other hand, 2,3-dichloro-1,4-naphthoquinone (DCNQ) a commercially available analog of DBNQ, showed time-dependent inhibition of NMNAT1 (Table 1C), thus suggest-ing selectivity of the DHNQs for NMNAT1.

IC50 Values of the DHNQs without and with Preincubation

The IC50 values were determined in reactions without and with preincubation and initiated by the addition of sub-strates NMN+ and ATP for the forward reaction and NAD and PPi for the reverse reaction. Following preincubation with NMNAT, DBNQ and DCNQ had IC50 values of 0.76 and 1.17 µM, respectively, while their IC50 values were >20 µM without preincubation (Fig. 4A,B). For the reverse reaction, IC50 with preincubation with DBNQ was 0.26 µM (Fig. 4C). The time-dependent profile of 1 µM DBNQ for NMNAT1 inhibition was investigated by varying preincu-bation times (Fig. 5A,B). The increase in inhibition potency with preincubation time was noted in both forward and reverse directions for DBNQ.

Substrate Competition by DBNQ

Enzyme activity of DBNQ-preincubated NMNAT1 was partially recovered when co-preincubated with each NMN+ (p = 0.0536) and ATP, at 5 and 0.5 Km, respectively, indi-cating the competitive behavior of DBNQ with both sub-strates (Fig. 5C). Preincubation in the absence of substrates had a p value of 0.019.

Discussion

NAD biosynthesis is an important contributor to cellular homeostasis. It is hypothesized that selective inhibition is a potential target for cancers and increased biosynthesis for neurodegenerative diseases. Targeting the energetics of the cell, including NAD+ and ATP, has been hypothesized as therapy for cancer since the 1980s.29

To our knowledge, a plate-based HTS assay method for screening NMNAT has not previously been reported. NMNAT inhibitors described from other laboratories require high concentrations to effect NMNAT activity. Gallotannin, the strongest inhibitor of NMNAT1 reported before this study, has an IC50 of 10 µM for NMNAT1 and shows slight selectivity for NMNAT3, with an IC50 of 2 µM.2 Product analogs with polyphosphate insertions to NAD+ demonstrate mediocre inhibition toward NMNATs.2 Most recently, a ureyl NMN+ analog, Vacor adenine dinu-cleotide, was found to have IC50 values for NMNAT2 and NMNAT3 of 20 and 463 µM, respectively, with no detectible effect on NMNAT1 activity.30 Not surprisingly, our screen of the NCI mechanistic set, which contains several adenos-ine analogs and other ATP-competitive inhibitors for kinases, reflected this poor inhibition by substrate analogs with little activity against NMNAT1 at 20 µM (Fig. 3A). Thus, while an attractive drug target, NMNATs remain a challenging enzyme to inhibit, illustrating the need for inex-pensive, efficient, HTS-compatible formats and follow-up paradigms. The bioluminescent assay reported here is the first plate-based, HTS-capable assay format. In addition, the DHNQs identified and reported here are to our knowl-edge the first near-submicromolar inhibitors known for NMNATs.

The screening method also included a mechanistic follow-up. The inhibitors of NMNAT were evaluated for time dependence by preincubation of enzyme with inhibitor

Figure 3. NMNAT1 inhibitor screen paradigm and results. (A) Relative abundance of compounds at percent luminescent signal relative to control signal. Compounds (912 total) from NCI Mechanistic Diversity Set II and the Approved Oncology Set VI compound libraries were screened at a single concentration (20 µM), in duplicate, against NMNAT1. HTS was performed as described under Materials and Methods. Each plate contained 4 ATP standards (no enzyme) and 12 control standards (no inhibitor) on either side of the compound wells. (B) Screening flowchart and distribution of relative activities detected during HTS.

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Table 1. Activity of HTS Hits and Analogs without and with Preincubation.

Molecule Name NCI IdentifierCommon

Name Structure

Percent Control Activity Time Dependent

20 µM20 µM, with

Preincubation2 µM, with

Preincubation

A. Mechanistic Set Actives IR-0002456 NSC-618332 DBNQ

60.1 4.2 15.3 Yes

IR-0002506 NSC-631529

58.4 3.5 116.8 Yes

IR-0002631 NSC-631521

64.4 18.5 42.7 Yes

IR-0002675 NSC-624158 21.4 2.1 9.9 Yes

IR-0002715 NSC-634503 59.1 –18.6 40.6 Yes

IR-0002887 NSC-219734 60.8 9.2 113.2 Yes

IR-0002912 NSC-166454 Decamine 64.0 31.3 114.0 Yes

B. Naphthoquinone Inactives IR-0002175 NSC-4170 Menadione 107.0 93.7 NT Inactive

IR-0002195 NSC-11897 Phthiocol 104.0 70.5 NT Inactive

C. Naphthoquinone Analogs IR-0003759 N/A DCNQ 71.8 –3.5 17.1 Yes

N/A = not applicable; NT = not tested.

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Figure 4. DHNQ dose–response on NMNAT1-catalzyed metabolism without (gray) and with (black) a 45 min preincubation prior to addition of substrate pair. (A) Forward reaction (NMN+ + ATP → NAD+ + PPi) with DBNQ; preincubated IC50 = 0.76 µM. (B) Forward reaction with DCNQ; preincubated IC50 = 1.17 µM. (C) Reverse reaction (NAD+ + PPi → NMN+ + ATP) with DBNQ; preincubated IC50 = 0.26 µM. Each data point is an average of two independent determinations.

Figure 5. Mechanistic studies with NMNAT inhibitors. Time dependence of DBNQ. DBNQ (1 µM) and NMNAT1 were preincubated for indicated time periods and the reaction was allowed to proceed for 10 min following the addition of substrates (A) 20 µM ATP and 100 µM NMN+ with 6 nM NMNAT1 in the forward direction and (B) 100 µM NAD+ and 100 µM PPi with 1 nM NMNAT1 in the reverse direction. Each data point is a mean ± SEM from six data points from three independent experiments run in duplicate. (C) Competition of DBNQ with NMN+ and with ATP. NMNAT1 was preincubated without (sold gray) or with (solid black) 0.76 µM DBNQ for 45 min, and the reaction was allowed to proceed for an additional 10 min following substrate addition (see Materials and Methods for details). For substrate competition, DBNQ and NMNAT1 were preincubated in the presence of NMN+ (horizontal striped bar) or ATP (vertical striped bar). Loss of inhibition in co-preincubated assays reflects the competitive behavior of DBNQ with respect to co-preincubated substrate. NMN+ and ATP concentrations were 100 and 20 µM, approximating 5 and 0.5 Km, respectively. Each data point is a mean ± SEM from eight data points from four independent experiments run in duplicate. Statistical values were derived from the BEST test.25

Haubrich et al. 9

and evaluating the effect of preincubation on activity (Fig. 3B). Time-dependent inhibitors will increase their inhi-bition potency following preincubation. This is a relatively simple method to determine if a molecule has time-dependent activity and is scalable to screening large numbers of active compounds. We identified compounds that were time depen-dent following preincubation. Subsequent dose–response experiments with and without preincubation revealed an increase in inhibitor potency and IC50 values of DBNQ and DCNQ in the submicromolar range when NMNAT1 was pre-incubated with inhibitor. This increase in potency was miti-gated upon co-preincubation with each NMN+ and ATP, demonstrating a bisubstrate competitive behavior of DBNQ.

As part of our ongoing efforts to identify new treatment molecules for rare diseases, including cancers, we have been investigating approaches to identify new MMOAs early in the discovery process. Much of our appreciation for the importance of MMOA to a therapeutic index is anecdotally based on the observations that many approved drugs have MMOAs that allow for efficient coupling, including slow dissociation kinetics and irreversible bind-ing.22 Transitioning from reversible equilibrium competi-tion binding to nonequilibrium competition binding will lead to noncompetitive behavior that will lower the con-centration of drug required for efficacy in a system with high levels of competing substrate. The significance of this is that the lower drug concentrations will lead to better selectivity and an increased therapeutic index. Early recog-nition of mechanistic differentiation of new drugs would be useful to inform optimization and development.31 In this report, we demonstrate that the methods to mechanistically evaluate compounds are straightforward and can be readily incorporated into a follow-up paradigm.

In summary, we report a bioluminescent assay to mea-sure the activity of NMNAT. This versatile assay is useful in plate format and is suitable for screening for new modula-tors of this enzyme. In screening a small library, time-dependent inhibitors that are competitive with NMN+ and ATP were identified. These are the most potent NMNAT1 inhibitors reported to date.

Acknowledgments

We thank the National Cancer Institute for compound libraries. The authors wish to acknowledge Biozilla led by Dr. David Chereau for providing NMNAT1 and PpLuc. The authors also wish to acknowledge Dr. Shuangluo Xia for helping to conceptu-alize the NMNAT assay; Dr. Brian S. Blais, Bryant University, for assistance with statistical analyses; and Dr. Peter Gund, CDD, for critical reading of the manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by a grant from NIAID RO1AI103476.

ORCID iD

Brad A. Haubrich https://orcid.org/0000-0003-1055-5870

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