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Small Molecule Therapeutics

Discovery and Characterization of NovelNonsubstrate and Substrate NAMPT InhibitorsJulie L.Wilsbacher1, Min Cheng1, Dong Cheng1, Samuel A.J. Trammell2, Yan Shi1,Jun Guo1, Stormy L. Koeniger1, Peter J. Kovar1, Yupeng He1, Sujatha Selvaraju1,H. Robin Heyman1, Bryan K. Sorensen1, Richard F. Clark1, T. Matthew Hansen1,Kenton L. Longenecker1, Diana Raich1, Alla V. Korepanova1, Steven Cepa1, Danli L. Towne1,VivekC.Abraham1,HuaTang1, Paul L.Richardson1, ShaunM.McLoughlin1, IlariaBadagnani1,Michael L. Curtin1, Michael R. Michaelides1, David Maag1, F. Gregory Buchanan1,Gary G. Chiang1,Wenqing Gao1, Saul H. Rosenberg1, Charles Brenner2, and Chris Tse1

Abstract

Cancer cells are highly reliant on NADþ-dependent processes,including glucose metabolism, calcium signaling, DNA repair,and regulation of gene expression. Nicotinamide phosphoribo-syltransferase (NAMPT), the rate-limiting enzyme for NADþ

salvage from nicotinamide, has been investigated as a target foranticancer therapy. Known NAMPT inhibitors with potent cellactivity are composed of a nitrogen-containing aromatic group,which is phosphoribosylated by the enzyme. Here, we identifiedtwonovel types ofNAM-competitiveNAMPT inhibitors, only oneof which contains amodifiable, aromatic nitrogen that could be a

phosphoribosyl acceptor. Both types of compound effectivelydeplete cellular NADþ, and subsequently ATP, and produce celldeathwhenNAMPT is inhibited in cultured cells formore than 48hours. Careful characterization of the kinetics of NAMPT inhibi-tion in vivo allowed us to optimize dosing to produce sufficientNADþ depletion over time that resulted in efficacy in an HCT116xenograft model. Our data demonstrate that direct phosphori-bosylation of competitive inhibitors by theNAMPT enzyme is notrequired for potent in vitro cellular activity or in vivo antitumorefficacy. Mol Cancer Ther; 16(7); 1236–45. �2017 AACR.

IntroductionIn addition to activation of oncogenes and loss of tumor

suppressor genes, cancer cells depend on normal, nononcogenicprocesses to copewith the stresses resulting fromdysregulated cellsignals. This heightened dependence on normal processes, alsoknown as nononcogene addiction, represents an opportunity fortargeting of cancer cells (1). Cancer metabolism, often summa-rized as the Warburg effect, is characterized by high utilization ofglucose for glycolysis, which depends on NADþ. The glycolyticintermediates that are produced enter the pentose phosphatepathway, resulting in conversion of NADPþ to NADPH (2). Inaddition to the roles of NADþ and NADPþ in metabolic redoxcycles, NADþ is a consumed substrate of enzymes such as PARPs

that are involved in DNA damage recognition and repair, cyclicADP ribose synthetases that generates calcium-mobilizing secondmessengers, and sirtuins that regulate transcription andmetabolicprocesses (2, 3). The latter process of NADþ consumption thatresults in nicotinamide production represents a form of nonon-cogene addiction, makingmany cancer cells highly dependent onsalvage synthesis of NADþ (4).

Depending onwhich genes are expressed and the availability ofsubstrates,mammalian cells can utilize tryptophan through the denovo NADþ synthesis pathway or salvage nicotinamide, nicotinicacid (NA), or nicotinamide riboside (NR) to maintain NADþ

levels (Supplementary Fig. S1; refs. 2, 3, 5, 6). As long as it is notmethylated (7), the nicotinamide produced byNADþ-consumingenzymes can be recycled back to NADþ in a pathway initiated bynicotinamide phosphoribosyltransferase (NAMPT; ref. 8–10).This enzyme utilizes phosphoribosyl pyrophosphate (PRPP) asa phosphoribose donor to convert nicotinamide to nicotinamidemononucleotide (NMN). NA, which is obtained from the diet orproduced by bacterial hydrolysis of NADþ metabolites in the gut(11), is utilized in a pathway initiated by NA phosphoribosyl-transferase (NAPRT1). In a significant percentage of cancers, theNAPRT1 gene is epigenetically silenced, providing an opportunityto select patients whose cancers depend on the NAMPT-mediatedpathway to regenerate NADþ (12, 13). In addition, several cancercell lines lack expression of key enzymes within the de novopathway and are unable to generate NADþ from tryptophan(14). Given the increased reliance on regenerating NADþ fromnicotinamide, inhibition of NAMPT represents a potential ther-apeutic intervention point to preferentially target cancer cells.Indeed, coadministration of an NAMPT inhibitor plus NA might

1AbbVie Inc., North Chicago, Illinois. 2Department of Biochemistry CarverCollege of Medicine, University of Iowa, Iowa City, Iowa.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for S.A.J. Trammell: The Novo Nordisk Foundation Center forBasic Metabolic Research, Section for Metabolic Imaging and Liver Metabolism,University of Copenhagen, Copenhagen, Denmark; current address for D. Raich,MPI Research, Mattawan, MI; and current address for G.G. Chiang, eFFECTORTherapeutics, San Diego, CA.

Corresponding Author: Julie L. Wilsbacher, AbbVie, Inc., 1 N. Waukegan Road,North Chicago, IL 60064. Phone: 847-937-9635; Fax: 847-938-2365; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-16-0819

�2017 American Association for Cancer Research.

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constitute a way to target the heightened NADþ requirements oftumor while protecting patients from toxicities in nontumortissues (15). In addition, provision of NR has been shown toprotect rats from paclitaxel-induced peripheral neuropathy (16).

The concept of inhibiting NAMPT as an anticancer strategy wasfirst evaluated clinically in 2007 with FK866 (17) and GMX1778(18). These first-generation NAMPT inhibitors exhibited potentantitumor activity in murine xenograft models but experiencedlimitations when evaluated clinically. Poor and variable oralbioavailability coupled with short plasma half-life necessitatedthe administration of FK866 and GMX1777 (19), a solubleprodrug of GMX1778, by 24- to 96-hour intravenous infusion(17). The lack of efficacy observed with first-generation NAMPTinhibitors was hypothesized to result from insufficient targetengagement prior to reaching doses that caused toxicity (10). Assuch, there has been a resurgence of activities to develop betterNAMPT inhibitors with improved pharmaceutical properties.

Several second-generation NAMPT inhibitors have beendescribed in the literature and in patent applications (10, 20–24). Similar to FK866 andGMX1778,GNE-617 andother second-generation NAMPT inhibitors with cellular activity have an aro-matic nitrogen positioned for phosphoribosylation by NAMPT(Fig. 1A; refs. 10, 25, 26). Phosphoribosylation of NAMPT inhi-bitors has been confirmed by mass spectrometry or cocrystalstructures (25, 27–29). According to published work, the sub-strate activity of NAMPT inhibitors is required for potent cellularactivity in vitro and preclinical efficacy in vivo (27).

Here, we identified novel nonsubstrate NAMPT inhibitorswith robust preclinical efficacy and pharmacokinetics proper-

ties that enable oral dosing. The lead molecule in this series, A-1293201, contains an isoindoline "head group," which lacksthe aromatic nitrogen of the nicotinamide and nicotinamide-mimetic compounds. Work presented establishes the degree ofNAMPT inhibition required to kill cancer cells with A-1293201in vitro and in vivo. Our data demonstrate that formation of aphosphoribose adduct by NAMPT is not a requirement foractivity within tumor cells.

Materials and MethodsCell culture

PC3, HCT116, and NCI-H1975 cells were obtained from theATCC in 2001, 1999, and 2006, respectively. Cells were culturedin RPMI1640 containing 50 mmol/L HEPES (Gibco 22400)supplemented with 10% FBS for HCT116 and NCI-H1975 cellsandwith 1% sodiumpyruvate, 1%nonessential amino acids, and10% FBS for PC3 cells. Cells were incubated at 37�C in 5% CO2

and 80% relative humidity. Cells were authenticated by STRanalysis in July 2016 for PC3 and HCT116 cells and in August2013 for NCI-H1975 cells.

High-throughput cellular screen and target identificationCytotoxicity was determined using the CellTiter Glo cell

viability assay that detects total intracellular ATP (Promega).Inhibitor affinity capture was performed as described in ref. 30using molecular probes based on the FK866 or A-933414structures. Captured proteins were separated by SDS-PAGE andSYPRO staining and identified by LC/MS-MS. FK866 and

Figure 1.

A-1293201 binds to NAMPT and inhibits its enzymatic activity. A, Structures of nicotinamide (NAM), FK866, GMX1778, GNE-617, and A-1293201. The aromaticnitrogen that is phosphoribosylated by NAMPT is colored red. B, NAMPT enzyme inhibition dose–response curve for A-1293201. Data are mean � SEM from twoindependent experiments performed with duplicates. C, Overlaid crystal structures of A-1293201 (green) and FK866 (yellow) in NAMPT. The PRPP-bindingregion (subsite A), nicotinamide-binding region (subsite B), tether region (subsite C), and the distal opening of the active site (subsite D) are indicated.

Novel Nonsubstrate NAMPT Inhibitors

www.aacrjournals.org Mol Cancer Ther; 16(7) July 2017 1237




GMX1778 were synthesized at AbbVie. Additional compoundswere synthesized using methods described in U.S. Patent 2016/9302989 B2 (31).

NAMPT protein purification and enzyme assayThe full-length DNA encoding human NAMPT, residues 1 to

491 (GenBank accession number NM_005746.2) with the FLAG-tag (DYKDDDDK) introduced at the C-terminus, was synthesizedand cloned into the pLVX-IRES-puro expression vector. Full-length NAMPT-FLAG was transiently expressed in HEK 293-6Ecells (NRC-Canada) and purified in a two-step process using anti-FLAG affinity chromatography and size exclusion chromatogra-phy. Enzyme assays were performed using the direct NMN detec-tion method (32).

X-ray crystallographyPurified NAMPT at 10 to 13 mg/mL was incubated in the

presence of 10 mmol/L nicotinamide for 2 hours on ice.Crystals grew at 4�C in hanging drop vapor diffusion setupcontaining 1:1 ratio of the protein and reservoir solution (25%PEG 3350, 0.2 mol/L ammonium sulfate, and 0.1 mol/LHEPES, pH 7.5). Inhibitor complexes were formed by soakingthe crystals in the presence of the compound and prepared forX-ray studies by brief transfer into reservoir solution with 20%(v/v) glycerol and rapid plunge into liquid nitrogen. Diffractiondata for the complex with A-1293201 were collected undergaseous nitrogen at 100 k at the Canadian Light Source Beam-line 08ID-113, while data for the complex with A-1326133were similarly collected at the Advanced Photon Source Beam-line 17-ID. Diffraction intensities were processed using autoP-ROC (33), and the structure was solved by sequential molecularreplacement with coordinates from pdb code 2GVJ usingMOLREP (34) within the CCP4 program suite (35). The modelwas rebuilt using COOT (36) and refined against structurefactors using the programs REFMAC5 (37) and autoBUSTER(38). Figures were prepared using the program PyMOL (Schroe-dinger, LLC). Atomic coordinates for the complexes with A-1293201 and A-1326133 have been deposited in the publicwwPDB with accession codes 5U2M and 5U2N, respectively.NAMPT crystal structure data and refinement statistics are listedin Supplementary Table S1.

Cellular assaysFor PC3 cell viability assays, cells were treated in 96-well

plates with compounds � 0.3 mmol/L nicotinamide for 5 days,followed by a CellTiter Glo assay (Promega). At the 5-day timepoint, ATP was already depleted and the signal correlated withcell viability as measured by other methods. For shorter timecourse assays, HCT116, PC3, or NCI-H1975 cells were treatedfor the indicated times, then cellular levels of NADþ þ NADH(NADt) were measured using the NADH-Glo assay (Promega),and ATP levels were measured using the CellTiter Glo assay.Cell numbers were measured by quantifying cells stained withVybrant Green (Invitrogen) and imaged in an IncuCyte FLR(Essen Bioscience).

NADþ metabolome analysisNADþ and related metabolites were analyzed as described

previously (39, 40). Briefly, 1 mL of one of two internal standardsolutions (solution A and solution B) were added to each sample(NCI-H1975 pellet containing 1 � 106 cells) before extraction.

Solution A was composed of extract from Fleischmann yeastgrown in the presence of U-13C glucose diluted to produce a finalconcentration of 1:80heavy extract to sample solution. Solution Bcontained 60 mmol/L 18O nicotinamide riboside and 18O nico-tinamide. Samples were deproteinized using buffered ethanol[75%/25% (v/v) ethanol/10 mmol/L HEPES pH 7.1] heated to80�C for 3 minutes with vortexing. Particulate was pelleted withcentrifugation (16,100 � g, 10 minutes, 4�C). The supernatantwas transferred to fresh tubes and then dried overnight via speedvacuum. Reconstituted extracts were separated and analyzed viaLC/MS-MS as described previously (39, 40). Metabolites weredetected using a Waters TQD operated in positive ion mode bymultiple reactionmonitoring (MRM).Metabolite peak areas wereconverted tomole amounts by regression to internal standard (IS)peak areas. Intracellular concentrations were calculated by divid-ing the mole amount by the total intracellular volume, assumingan intracellular volume of 2.5 pL per cell. One-way ANOVAfollowed by Dunnett multiple comparisons test was performedusing GraphPad Prism version 7.00 for Windows, GraphPadSoftware (www.graphpad.com).

Glycolytic metabolite analysisNCI-H1975 cells were harvested and aliquoted into 1 � 106

cells in duplicate. Samples were spun and washed with cold PBS,followed by metabolite extraction in 500 mL of 80% (v/v) meth-anol in water containing 2.5 mmol/L U-13C glucose-6-phosphateas the IS. Samples were vortexed and incubated at �80�C for 20minutes then spun for 30 minutes at 13,000 � g and 4�C.Supernatants were transferred into cold Eppendorf tubes, lyoph-ilized to dryness, and stored at�80�C until analysis. Lyophilizedsamples were reconstituted with 50 mL of water, agitated for 15minutes at 4�C, then diluted with 50 mL of acetonitrile, andagitated for 15 minutes at 4�C. Extracts were separated andanalyzed as described previously (41); however, only positivemodeMRM transitions for selectmetabolites were included in themethod. Linear dynamic range for each metabolite was definedwith standards, and samples were diluted and reinjected whennecessary. Changes in metabolite concentrations are reported asfold change to control.

In vivo pharmacologyAll experiments were conducted in accordance with IACUC

guidelines. Compounds were formulated in the following vehicle2% (vol) EtOH, 5% (vol) Tween 80, 20% (vol) PEG-400, 73%(vol) 0.2% HPMC and were administered orally in the pharma-cology studies. HCT116 cells were grown to passage 2 in vitro. Atotal of 0.5 � 106 cells were inoculated into the right flank offemaleC.B.-17 SCIDmice onday 0 in a volumeof 0.1mL. Tumorswere size matched on day 11 postinoculation with a mean tumorvolume of 231� 28 (SD)mm3with dosing beginning on the nextday. Tumor volume was calculated twice weekly. Measurementsof the length (L) and width (W) of the tumor were taken viaelectronic caliper, and the volume was calculated according to thefollowing equation: V ¼ L � W2/2 using Study Director version3.1 (Studylog Systems, Inc.). For pharmacodynamic studies,HCT116 tumor–bearing mice were size matched and given oraldose(s) of A-1307138. Tumor samples were collected at varioustime points following dosing and were flash frozen in liquidnitrogen. Tumors were homogenized and NADt was measuredusing the NADþ/NADH Quantification Kit from MBL Interna-tional Corporation.

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ResultsDiscovery of novel NAMPT inhibitors

To identify potential novel anticancer targets, a cell prolifera-tion screen was performed to simultaneously identify oncology-relevant biologic activity (i.e., antiproliferative activity) and smallmolecules responsible for the activity of interest. The compoundscould thenbeused to identify the target through affinity capture ofthe inhibitor/target complex. One cluster of compounds exhib-ited activity infiveof the eight cell lines tested (Supplementary Fig.S2A). Inhibitor affinity capture studies of a novel compound, theisoindoline urea A-933414, from the hit cluster revealed NAMPTas the major biochemical target (Supplementary Fig. S2B andS2C). Inhibition of PC3 cell viability by A-933414 was rescued bycoincubation of cells with the product of NAMPT, NMN, whichovercomes intracellular NAMPT inhibition by virtue of extracel-lular dephosphorylation to nicotinamide riboside and intracel-lular conversion to NADþ via the nicotinamide riboside kinasepathway (42). Rescue by NMN indicated that NAMPT was solelyresponsible for the antiproliferative activity of this chemotype(Supplementary Fig. 2D; Supplementary Table S2). These experi-ments also suggest that these compounds are exquisitely specificforNAMPT, as little to no antiproliferative activitywas observed atconcentrations 1,000-fold higher in the presence of NMN.

Extensive SAR work on this series indicated that structuralmodifications to several portions of the molecule provideimproved potency, physical properties, and reduced clearance,resulting in significant murine exposure after oral dosing (Sup-plementary Table S3) without compromising selectivity towardNAMPT (manuscript in preparation; ref. 31). A lead compoundfrom this series, A-1293201 (Fig. 1A), was found to potentlyinhibit recombinant, human NAMPT in an enzyme activity assay(Fig. 1B). Examination of the interactions between A-1293201andNAMPTbyX-ray crystallography revealed several distinct sitesfor cooperative interactions (Fig. 1C). The sites include the PRPP-binding subsite A, which is hydrophilic in nature and unoccupiedby nicotinamide; the nicotinamide binding subsite B, whereimportant pi-stacking and hydrogen bond interactions with nic-otinamide are made; the tether subsite C, which is narrow,lipophilic, and not occupied by bound nicotinamide; and thedistal opening subsite D, which widens and provides several

bound waters and hydrophobic surfaces. Similar to FK866, theisoindoline urea portion of A-1293201 is engaged in importantpi-stacking interactions in subsite B aswell ashydrogenbonds andhydrophobic interaction in subsite C. In contrast to FK866 as wellas recent second-generation NAMPT inhibitors, the isoindolineurea compounds are the first known potent nonphosphoribosy-lated NAMPT inhibitors.

Biological comparison of substrate and nonsubstrate NAMPTinhibitors in vitro

To further characterize the ramifications of having inhibitorsthat do not serve as substrates to NAMPT, a nucleophilic nitrogenwas added to the nicotinamide-mimetic portion of these com-pounds that occupied subsite B, producing potent and selectiveazaisoindoline urea inhibitors that could be phosphoribosylatedby NAMPT (Supplementary Table S2). A-1267211, the azaisoin-doline analogue of A-933414, and a racemic mixture of A-1331597, the azaisoindoline analogue of A-1293201, were bothshown to be phosphoribosylated after incubation with NAMPTenzyme under conditions in which nicotinamide is also phos-phoribosylated (Supplementary Fig. S3A). Phosphoribosylationof A-1267211 and another azaisoindoline, A-1307138, alsooccurred following treatment of PC3 cells with the compoundsfor 24 hours (Supplementary Fig. S3B).

A set of matched pairs of inhibitors that differ only in thepresence or absence of thenitrogen at position5of the isoindolinering was used for further analysis (Supplementary Table S4). Thepairs of isoindoline and azaisoindoline ureas were tested in PC3cells for inhibition of cell viability (Fig. 2). In most cases, theazaisoindoline version of each compound pair exhibited greatercellular potency than the isoindoline counterpart, ranging from1.2- to 48-fold improvement in activity (Fig. 2). Although theisoindolines tended to be less potent in cells, many inhibitedNAMPT and caused cytotoxicity with IC50s below 50 nmol/L, andabout a quarter of the isoindolines had similar cellular potenciesas their azaisoindoline counterpart. In contrast, removal of thenitrogen from the pyridyl ring of FK866 completely abolishedcellular potency (Fig. 2; Supplementary Tables S2 and S4).

Representative compounds from the isoindoline and azai-soindoline series were further characterized in biochemical andcellular assays to identify other distinguishing features.

Figure 2.

Cellular comparison of azaisoindoline/isoindolinepairs. Matched structural pairs of isoindoline andazaisoindoline NAMPT inhibitors or FK866 and itsanalogue that lacked an aromatic nitrogen weretested in 5-day cell viability assays in PC3 cells.The IC50 in PC3 cells is plotted on the y-axis andpair number is plottedon the x-axis.~ azaisoindoline;*, isoindoline;^, FK866. Magenta indicates that thearomatic ring contains N, and cyan indicates that thearomatic ring contains C.






Compounds tested included the azaisoindoline/isoindolinepairs of A-1326133/A-1292945 and A-1331597/A-1293201(Supplementary Table S2). The isoindoline of each pairshowed similar potency in NAMPT enzyme assays, and A-1331597 and A-1293201 also had comparable IC50s in PC3,HCT116, and NCI-H1975 cell viability assays. In contrast, thesubstrate compound A-1326133 was more potent than itscorresponding isoindoline in the viability assays. Inhibitionof PC3 cell viability by all of the compounds was rescued bycoincubation of cells with NMN (Supplementary Table S2).

Effects of A-1293201, A-1331597, A-1292945, and A-1326133on depletion of NADþ/NADH and ATP in HCT116 colorectalcancer cells over time were also measured. Inhibition of NAMPTby all four compounds resulted in maximal NADt (levels ofNADHplusNADþ) depletion within 24 hours and ATP depletion

within 48 to 72 hours (Fig. 3A–D). Following NADt and ATPdepletion, HCT116 cell viability was severely compromised with-in 72 hours. ATP depletion and inhibition of cell viability onlyoccurred at compound concentrations that resulted in greaterthan 90% reduction inNADt levels at 48 hours. If there is less than90%depletion of NADt, theNADt levels recover and cells survive.These data suggest that cytotoxicity elicited by NAMPT inhibitionis dependent on the kinetics of ATP depletion, which occurssubsequent to NADt depletion.

Comparison of NADt depletion dose–response curves after 24-hour treatment for the azaisoindoline/isoindoline pairs is shownin Fig. 3E. Although the IC50 for NADt depletion for A-1293201was 1.8 times lower than for A-1331597, both compoundsdepleted NADt by >90% at 37 nmol/L. In contrast, the IC50 andthemaximalNADtdepletion forA-1326133were both lower than

Figure 3.

NADt depletion following A-1293201 and A-1326133 treatment results in cell death. A and B, HCT116 cells were incubated with the indicated concentrationsof A-1293201 (A), A-1331597 (B), A-1292945 (C), or A-1326133 (D) for 24, 48, or 72 hours. Total NAD (NADt¼NADHþNADþ), ATP, and cell number were assessed ateach time point. Dashed line, 90% depletion of NADt. E, Depletion of NADt levels in HCT116 cells following 24-hour treatment with azaisoindoline/isoindolinepairs. F, Time course of recovery of NADt after washout in HCT116 cells treated with azaisoindoline/isoindoline pairs for 24 hours. Paired compounds in E and F arecolored the same with closed symbols and solid lines for azaisoindolines and open symbols and dashed lines for isoindolines. Data in A–E are the mean � SEMfrom 3 to 5 independent experiments. Data in F are mean � SEM from 2 to 4 independent experiments.

Wilsbacher et al.





for A-1292945. The maximal NADt depletion occurred at 4.1nmol/L for A-1326133, but for A-1292945, NADt was not deplet-ed >90% until 12.3 nmol/L. NADt recovery following washout ofthe compounds was also determined by treating HCT116 cellswith theNAMPT inhibitors for 24hours, removing the compoundand washing the cells, and measuring NADt levels over time. The

NADt recovery was plotted for all four compounds at the con-centrations at which NADt was depleted by >90% at 24 hoursbefore washout (Fig. 3F). The kinetics of NADt recovery wassimilar for A-1293201 and its partner, A-1331597, as well as forthe isoindoline A-1292945. For these compounds, NADt hadrecovered by 50% around 4 hours after washout. However,recovery of NADt following washout of A-1326133 was delayedand did not reach 50% until 8 hours. By 16 hours followingwashout, NADt levels had fully recovered for all four of thecompounds.

Effects of lead isoindoline and azaisoindoline NAMPTinhibitors on the NAD metabolome

To determine the effects of A-1293201 and A-1326133 onthe NADþ metabolome and glycolysis, NCI-H1975 cells weretreated with either vehicle (0.1% DMSO) or three concentra-tions (IC20, IC50, or IC>90) of the compounds or FK866 for 8,24, or 48 hours. The compound concentrations and incubationtimes were chosen based on the kinetics of NADt and ATPdepletion in NCI-H1975 cells (Supplementary Fig. S4). After 24hours and at concentrations matching the respective IC50 andIC>90, each compound significantly depressed NMN and NADþ

(Fig. 4). At 24 hours, ADP ribose (ADPr) was decreased signif-icantly by treatment with each compound at IC>90, whileNADPþ tended to be lowered (Fig. 4). However, the dataindicate that NMN, NADPþ, and ADPr all strongly correlatedwith NADþ in each experiment and all trended down together.The pattern observed at 24 hours continued at 48 hours (Fig. 4).The effects of FK866, A-1293201, and A-1326133 on glycolyticmetabolites were also measured, and all three compoundscaused similarly increased levels of fructose-6-phosphate/glu-cose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxy-acetone phosphate/glycedraldehyde-3-phosphate (Fig. 5),which is consistent with inhibition of glycolysis via NADþ

depletion previously reported for FK866 (43). Together, thesedata confirm that A-1293201 and A-1326133 target NADþ

metabolism, which subsequently depresses glycolysis.

In vivo characterization of lead isoindoline and azaisoindolineNAMPT inhibitors

The evaluation of the time of inhibition requirement forcytotoxicity identified by the in vitro time course studies wasextended to an in vivo setting using A-1307138, an analogue ofA-1326133 (Supplementary Table S2). Following a single oraldose of 15 mg/kg in an HCT116 xenograft tumor model, A-1307138 treatment reduced tumor NADt levels by greater than90%after 24hours, butNADt concentrations quickly recovered toapproximately 50%ofpretreatment levels 72hours postdose (Fig.6A). Notably, transient depletion of NADt levels by >90% for lessthan 24 hours once aweek did not elicit any suppression of tumorgrowth (Fig. 6C). Extending the duration of NADt depletion by 2to 3 consecutive days of dosing resulted in substantial inhibitionof tumor growth in vivo (Fig. 6C), which corresponds to sustainedNADt depletion, with >90% depletion maintained for over 72hours (Fig. 6B). These data are consistent with the in vitro observa-tions that inhibition of NAMPT for at least 48 hours is required toelicit NADt depletion–dependent cytotoxicity. It was determinedthat maintaining the time-over-threshold (IC90) for 48 to 70hours was required for robust antitumor efficacy. This is exem-plified by the potent antitumor efficacy of A-1293201 andA-1326133 in the HCT116 xenograft model (Fig. 6D). Body

Figure 4.

A-1293201, A-1326133, and FK866 have similar effects on theNADþmetabolome.NCI-H1975 cellswere treatedwith either vehicle (0.1%DMSO) or 3 dosages (IC20,IC50, or IC>90) of A-1293201, A-1326133, or FK866 for 24 or 48 hours. Left,levels of NMN, NADþ, NADP, and ADPr after treatment of cells with compoundsfor 24 hours; right, levels ofNMN,NADþ, NADP, andADPr after treatment of cellswith compounds for 48 hours. Data, mean þ SEM of duplicate samples.� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






weight loss did not exceed 10% for any of the compounds, and nodeaths occurredduring the in vivo studies (Supplementary Fig. S5).

DiscussionWehave identified and characterized novel potent and selective

nonsubstrate and substrate NAMPT inhibitors. Both types ofcompounds exhibit potent in vitro and in vivo antiproliferativeactivity following oral administration and display similar kineticsofNADt andATPdepletion and inductionof cytotoxicity, which isrescued by NMN. On the basis of mechanistic studies, inhibitionof NAMPT by >90% for at least 48 hours is required for anti-proliferative effects in HCT116 cells, in vitro and in vivo. As hasbeen shown for previously published NAMPT inhibitors, within

hours after treatment, NADþ levels are decreased, but 24 to 48hours are required for complete NADþ depletion within themitochondria and blockade of ATP generation. Once ATP isdepleted, cell death occurs through apoptosis, autophagy, oroncosis (10, 44). The kinetics of NADþ depletion can differbetween cell types, possibly due to differences in NADþ con-sumption rates by PARPs or other enzymes that utilize NADþ as asubstrate. The kinetics ofNADþ andATP depletion inNCI-H1975cells are slower than in HCT116 cells. As a result, NADþ levels arenot depleted >90%until between 36 and 48 hours and inhibitionof glycolysis and ATP depletion are also delayed. A-1293201 andA-1326133produce similar effects on theNADþmetabolome andglycolytic metabolites as FK866. All of these properties indicatethat the isoindoline and azaisoindoline compounds cause celldeath by inhibiting NAMPT in cells and xenografts.

In contrast to previously described NAMPT inhibitors, includ-ing FK866, GMX1778, and GNE-617, A-1293201 does not con-tain an aromatic nitrogen in its nicotinamide-mimetic group (Fig.1A). Crystallographic studies indicate that A-1293201 partici-pates in similar interactions with NAMPT as the other inhibitors(45, 46). A-1293201 and A-1326133 occupy the nicotinamide-binding site and engage in a critical pi-stacking interaction withtyrosine 18 in the same manner as nicotinamide and FK866 (Fig.1C; Supplementary Fig. S6). The central regions of all the com-pounds traverse a narrow, lipophilic tunnel out to an openingdistal to the active site. The nicotinamide-mimetic moiety, wherethe presence of the aromatic nitrogen allows for phosphoribosyladduct formation, is the area of divergence between A-1293201and the other compounds. Our data indicate that phosphoribo-sylation of inhibitors is not required for potent inhibition ofNAMPT and induction of cell death in cells or in xenograftmodels.

It is notable thatmost azaisoindolines display improved poten-cy in cellular assays compared with their isoindoline analogues,even though their permeability and protein-binding propertiesare similar. One hypothesis is that the aromatic nitrogen in theazaisoindoline results in improved pi-stacking interactions withNAMPT and/or serves as a substrate, leading to increased targetpotency. However, similar IC50s in enzyme assays for the azai-soindoline/isoindoline pairs of A-1326133/A-1292945 and A-1331597/A-1293201 suggests that improved pi-stacking interac-tions are not occurring, and these data are corroborated by theoverlaid crystal structures of A-1293201 and A-1326133 (Sup-plementary Fig. S6). In addition, there does not appear to be adifference in dissociation rates between A-1293201 and A-1331597 or between A-1326133 and A-1292945 in TR-FRETassays (Supplementary Fig S7), even when the compounds arepreincubated with the enzyme and PRPP for 24 hours to allowphosphoribosylation. These results suggest that for this set ofcompounds, phosphoribosylation does not lead to increasedresidence time on the enzyme. In these assays, both substrateand nonsubstrate compounds have slower off rates when PRPP isbound, indicating that both classes of compounds bind cooper-atively with PRPP. These data suggest that the increased cellularpotencyof azaisoindolines relative to isoindolines is not the resultof increased target affinity upon phosphoribosylation.

Another potential reason for the increased cellular potency ofthe azaisoindolines is that phosphoribosylated compounds areretained longer in cells due to decreased transit of the modifiedcompound through the plasma membrane. Increased cellularretention was reported to contribute to cellular potency for

Figure 5.

A-1293201, A-1326133, and FK866 have similar effects on glycolytic metabolites.NCI-H1975 cellswere treatedwith vehicle (0.1%DMSO), A-1293201, A-1326133, orFK866 at three dosages (IC20, IC50, or IC>90) for 8, 24, or 48 hours. For each timepoint, fold changes from control were determined for fructose-6-phosphate(F6P) þ glucose-6-phosphate (A; G6P); fructose-1,6-bisphosphate (B; F16BP),and glycedraldehyde-3-phosphate (G3P) þ dihydroxyacetone phosphate (C;DHAP) and are reported as fold change to control. Note that only IC>90 data areshown. Fold change to control for IC20 and IC50 dosages across all compoundsand time points were 1.3� 0.22, 1.6� 0.29, and 1.2� 0.30 for F6PþG6P, F16BP,and G3P þ DHAP, respectively. Data, mean þ SD of duplicate samples.

Wilsbacher et al.





GMX1778, which undergoes phosphoribosylation (28). In ourwashout studies, recovery of NADt levels is similar between A-1293201 and A-1331597, suggesting that A-1331597 is notretained in cells. Thus, although phosphoribosylation canimprove the cellular potency for some compounds within theisoindoline series, in general, there is not a requirement formodification by NAMPT for potent inhibition, and enhancedbinding can be attained through additional interactions distal tothe nicotinamide-binding site.

Our discovery of potent and selective NAMPT inhibitors thatdo not contain an aromatic nitrogen in the nicotinamidemoiety challenges the current dogma that this feature is

required for cellular efficacy. A-1293201 and other isoindolinesare efficacious despite the lack of a phosphoribosylation site,which could lead to cellular retention or other unexpectedeffects in cells. As a result, treatment with A-1293201 or otherisoindolines may allow better control of NAMPT inhibition innormal cells.

Disclosure of Potential Conflicts of InterestJ.L. Wilsbacher has ownership interest (including patents) in AbbVie, Inc. Y.

He is an employee at AbbVie, Inc. D. Maag is a full-time employee at and hasownership interest (including patents) in AbbVie. G.G. Chiang is the seniorgroup leader/principal research scientist at AbbVie, is a director at eFFECTOR

Figure 6.

Sustained target inhibition drives efficacy in HCT116 xenografts. A, NADt levels in HCT116 xenografts in samples harvested at 0.5, 1, 6, 24, 48, and 72 hoursafter treatingmicewith a singlewith 15mg/kgoral doseofA-1307138.B,NADt levels inHCT116 xenografts in samples harvested6or 24 hours after treatingmice orallywith 15 mg/kg of A-1307138 on a once a day (qd) schedule for 3 days. In A and B, the blue dashed line indicates 90% depletion of NADt, and data points areaverage � SD from xenografts harvested from 4 mice. C, Efficacy plots for HCT116 xenografts in mice dosed orally (p.o.) with A-1307138 on different schedules.Tumor growth curves from two separate experiments are graphed together, and curves for each study are designated by solid versus dashed line. D, Efficacyplots for HCT116 xenografts inmice treatedwithA-1326133 andA-1293201 at the indicated doses on an oral once a day (3 on, 4 off) schedule for 3 rounds of dosing. Forefficacy studies, dosing schedules are indicated by triangles under the x-axis, and tumor volumes are graphed as mean � SEM for 10 mice/treatment group.






Therapeutics, reports receiving a commercial research grant from AbbVie, andhas ownership interest (including patents) in AbbVie. S.H. Rosenberg is anemployee at and has ownership interest (including patents) in AbbVie. C.Brenner is in the scientific advisory board at ChromaDex, Inc., is the co-founderof ProHealthspan, andhas ownership interest (including patents) in intellectualproperty around uses of nicotinamide. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: J.L. Wilsbacher, M. Cheng, D. Cheng, S.A.J. Trammell,Y. Shi, J. Guo, B.K. Sorensen, R.F. Clark, T.M. Hansen, M.L. Curtin,M.R. Michaelides, D. Maag, F.G. Buchanan, G.G. Chiang, C. TseDevelopment of methodology: J.L. Wilsbacher, M. Cheng, D. Cheng, Y. Shi,J. Guo, S.L. Koeniger, P.J. Kovar, Y. He, A.V. Korepanova, P.L. Richardson,S.M. McLoughlin, D. Maag, C. Brenner, C. TseAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): J.L. Wilsbacher, M. Cheng, D. Cheng, S.A.J. Trammell,Y. Shi, S.L. Koeniger, P.J. Kovar, Y. He, S. Selvaraju, K.L. Longenecker,A.V. Korepanova, S. Cepa, D.L. Towne, V.C. Abraham,H. Tang, S.M.McLoughlin,I. Badagnani, D. Maag, C. BrennerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.L. Wilsbacher, M. Cheng, D. Cheng, S.A.J. Trammell,Y. Shi, J. Guo, S.L. Koeniger, P.J. Kovar, Y. He, K.L. Longenecker, A.V. Korepanova,S. Cepa, V.C. Abraham, S.M. McLoughlin, I. Badagnani, D. Maag, G.G. Chiang,W. Gao, C. BrennerWriting, review, and/or revision of themanuscript: J.L. Wilsbacher, M. Cheng,D. Cheng, S.A.J. Trammell, Y. Shi, J. Guo, S.L. Koeniger, P.J. Kovar, Y. He,R.F. Clark, K.L. Longenecker,D. Raich, A.V. Korepanova, V.C. Abraham,H. Tang,P.L. Richardson, I. Badagnani, M.L. Curtin, M.R. Michaelides, D. Maag,G.G. Chiang, W. Gao, S.H. Rosenberg, C. Brenner, C. TseAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.L. Wilsbacher, M. Cheng, D. Cheng, Y. Shi,P.J. Kovar, H. Tang, G.G. Chiang

Study supervision: G.G. Chiang, C. TseOther (chemical design and synthesis of NAMPT inhibitors): H.R. Heyman

AcknowledgmentsThe authors would like to thank Jameel Shah for his contributions to the

initial screen design and validation of hits, Keith Glaser for experimental advice,and Niru Soni and Junling Li for their expert technical assistance. The authorswould also like to acknowledge Stella Doktor, Patricia Stuart, Amanda Olson,Donald Osterling, and DeAnne Stolarik from the DMPK-BA Department atAbbVie for the in vitro ADME and in vivo pharmacokinetics measurements.Research described in this article was performed using beamline 08ID-1 at theCanadian Light Source, which is supported by the Canada Foundation forInnovation, Natural Sciences and Engineering Research Council of Canada, theUniversity of Saskatchewan, the Government of Saskatchewan, Western Eco-nomic Diversification Canada, the National Research Council Canada, and theCanadian Institutes ofHealthResearch.Useof the IMCA-CATbeamline 17-ID atthe Advanced Photon Source was supported by the companies of the IndustrialMacromolecular Crystallography Association through a contract with Haupt-man-Woodward Medical Research Institute. This research used resources of theAdvanced Photon Source, a U.S. Department of Energy (DOE) Office of ScienceUser Facility operated for the DOE Office of Science by Argonne NationalLaboratory under contract no. DE-AC02-06CH11357.

Grant SupportThis work was financially supported by AbbVie, Inc.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 29, 2016; revised March 17, 2017; accepted April 19,2017; published OnlineFirst May 3, 2017.

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2017;16:1236-1245. Published OnlineFirst May 3, 2017.Mol Cancer Ther Julie L. Wilsbacher, Min Cheng, Dong Cheng, et al. Substrate NAMPT InhibitorsDiscovery and Characterization of Novel Nonsubstrate and

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