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Title: Discovery and Characterization of Novel Non-substrate and Substrate NAMPT Inhibitors

Authors: Julie L. Wilsbacher1,*, Min Cheng1, Dong Cheng1, Samuel A. J. Trammell2,3, 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 Raich1,4, Alla V. Korepanova1, Steven Cepa1, Danli L. Towne1, Vivek C. Abraham1, Hua Tang1, Paul L. Richardson1, Shaun M. McLoughlin1, Ilaria Badagnani1, Michael L. Curtin1, Michael R. Michaelides1, David Maag1, F. Greg Buchanan1, Gary G. Chiang1, 5, Wenqing Gao1, Saul H. Rosenberg1, Charles Brenner2 and Chris Tse1

Author affiliations: 1AbbVie Inc., 1 North Waukegan Rd., North Chicago, IL 60064, USA

2Department of Biochemistry Carver College of Medicine, University of Iowa, Iowa City, IA, USA

3Current address: The Novo Nordisk Foundation Center for Basic Metabolic Research, Section for Metabolic Imaging and Liver Metabolism, University of Copenhagen, 2200 Copenhagen, Denmark

4Current address: MPI Research, 54943 North Main Street, Mattawan, MI 49071, USA

5Current address: eFFECTOR Therapeutics, San Diego, CA 92121, USA

Running title: Novel non-substrate NAMPT inhibitors

Keywords: NAMPT, NAMPT inhibitor, tumor metabolism, NAD, ATP

Abbreviations: Nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), nicotinamide phosphoribosyltransferase (NAMPT), phosphoribosyl pyrophosphate (PRPP), nicotinamide mononucleotide (NMN), nicotinic acid phosphoribosyltransferase (NAPRT1), internal

on February 22, 2020. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 3, 2017; DOI: 10.1158/1535-7163.MCT-16-0819

http://mct.aacrjournals.org/

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standard (IS), structure-activity relationship (SAR), adenosine triphosphate (ATP), adenosine diphosphate (ADP), ADP ribose (ADPr), dimethylsulfoxide (DMSO), fructose-6-phosphate (F6P), glucose-6-phosphate (G6P), fructose-1,6-bisphosphate (F16BP), glycedraldehyde-3-phosphate (G3P), dihydroxyacetone phosphate (DHAP), oral/by mouth (po), once a day (qd), mg/kg per day (mkd), dose for 3 days and no dose for 4 days for 3 cycles [(3on,4off)*3].

Financial support: This work was financially supported by AbbVie, Inc.

Corresponding author: Julie Wilsbacher, Oncology Discovery, AbbVie, Inc., 1 N. Waukegan Road, North Chicago, Illinois, USA 60064. Phone: 847-937-9635; E-mail: [email protected].




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ABSTRACT

Cancer cells are highly reliant on nicotinamide adenine dinucleotide (NAD+)-dependent

processes including glucose metabolism, calcium signaling, DNA repair, and regulation of gene

expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for

NAD+ salvage from nicotinamide (NAM), has been investigated as a target for anti-cancer

therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-

containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two

novel types of NAM competitive NAMPT inhibitors, only one of which contains a modifiable,

aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively

deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited

in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT

inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time

that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct

phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent

in vitro cellular activity or in vivo anti-tumor efficacy.




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INTRODUCTION

In addition to activation of oncogenes and loss of tumor suppressor genes, cancer cells

depend on normal, non-oncogenic processes to cope with the stresses resulting from

dysregulated cell signals. This heightened dependence on normal processes, also known as non-

oncogene addiction, represents an opportunity for targeting of cancer cells (1). Cancer

metabolism—often summarized as the Warburg effect—is characterized by high utilization of

glucose for glycolysis, which depends on nicotinamide adenine dinucleotide (NAD+). The

glycolytic intermediates that are produced enter the pentose phosphate pathway, resulting in

conversion of nicotinamide adenine dinucleotide phosphate (NADP+) to NADPH (2). In addition

to the roles of NAD+ and NADP+ in metabolic redox cycles, NAD+ is a consumed substrate of

enzymes such as poly(ADP ribose) polymerases that are involved in DNA damage recognition

and repair, cyclic ADP ribose synthetase that generates calcium-mobilizing second messengers,

and sirtuins that regulate transcription and metabolic processes (2,3). The latter process of NAD+

consumption which results in nicotinamide (NAM) production represents a form of non-

oncogene addiction, making many cancer cells highly dependent on salvage synthesis of NAD+

(4).

Depending on which genes are expressed and the availability of substrates, mammalian

cells can utilize tryptophan through the de novo NAD+ synthesis pathway or salvage NAM,

nicotinic acid (NA) or nicotinamide riboside (NR) in order to maintain NAD+ levels

(Supplementary figure 1) (2,3,5,6). As long as it is not methylated (7), the nicotinamide

produced by NAD+ consuming enzymes can be recycled back to NAD+ in a pathway initiated by

NAM phosphoribosyltransferase (NAMPT) (8-10). This enzyme utilizes phosphoribosyl

pyrophosphate (PRPP) as a phosphoribose donor to convert NAM to nicotinamide

mononucleotide (NMN). NA, which is obtained from the diet or produced by bacterial

hydrolysis of NAD+ metabolites in the gut (11), is utilized in a pathway initiated by nicotinic

acid phosphoribosyltransferase (NAPRT1). In a significant percentage of cancers, the NAPRT1

gene is epigenetically silenced, providing an opportunity to select patients whose cancers depend

on the NAMPT-mediated pathway to regenerate NAD+ (12,13). Additionally, several cancer cell

lines lack expression of key enzymes within the de novo pathway and are unable to generate

NAD+ from tryptophan (14). Given the increased reliance on regenerating NAD+ from NAM,




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inhibition of NAMPT represents a potential therapeutic intervention point to preferentially target

cancer cells. Indeed, co-administration of a NAMPT inhibitor plus NA might constitute a way to

target the heightened NAD+ requirements of tumor while protecting patients from toxicities in

non-tumor tissues (15). In addition, provision of NR has been shown to protect rats from

paclitaxel-induced peripheral neuropathy (16).

The concept of inhibiting NAMPT as an anti-cancer strategy was first evaluated clinically

in 2007 with FK866 (17) and GMX1778 (18). These first generation NAMPT inhibitors

exhibited potent anti-tumor activity in murine xenograft models but experienced limitations

when evaluated clinically. Poor and variable oral bioavailability coupled with short plasma half-

life necessitated the administration of FK866 and GMX1777 (19), a soluble prodrug of

GMX1778, by 24-96 hour IV infusion (17). The lack of efficacy observed with first generation

NAMPT inhibitors was hypothesized to result from insufficient target engagement prior to

reaching doses that caused toxicity (10). As such, there has been a resurgence of activities to

develop better NAMPT inhibitors with improved pharmaceutical properties.

Several second generation NAMPT inhibitors have been described in the literature and in

patent applications (10,20-24). Similar to FK866 and GMX1778, GNE-617 and other second

generation NAMPT inhibitors with cellular activity have an aromatic nitrogen positioned for

phosphoribosylation by NAMPT (Figure 1A) (10,25,26). Phosphoribosylation of NAMPT

inhibitors has been confirmed by mass spectrometry or co-crystal structures (25,27-29).

According to published work, the substrate activity of NAMPT inhibitors is required for potent

cellular activity in vitro and preclinical efficacy in vivo (27).

Here we identified novel non-substrate NAMPT inhibitors with robust preclinical

efficacy and pharmacokinetics properties that enable oral dosing. The lead molecule in this

series, A-1293201, contains an isoindoline ‘head group’ which lacks the aromatic nitrogen of the

NAM and NAM-mimetic compounds. Work presented establishes the degree of NAMPT

inhibition required to kill cancer cells with A-1293201 in vitro and in vivo. Our data demonstrate

that formation of a phosphoribose adduct by NAMPT is not a requirement for activity within

tumor cells.




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MATERIALS AND METHODS

Cell culture

PC3, HCT116, and NCI-H1975 cells were obtained from ATCC in 2001, 1999, and 2006,

respectively. Cells were cultured in RPMI 1640 containing 50 mM HEPES (Gibco 22400)

supplemented with 10% fetal bovine serum for HCT116 and NCI-H1975 cells and with 1%

sodium pyruvate, 1% non-essential amino acids, and 10% fetal bovine serum for PC3 cells. Cells

were incubated at 37°C in 5% CO2 and 80% relative humidity. Cells were authenticated by STR

analysis in July 2016 for PC3 and HCT116 cells and in August 2013 for NCI-H1975 cells.

High throughput cellular screen and target identification

Cytotoxicity was determined using the CellTiter Glo cell viability assay that detects total

intracellular ATP (Promega). Inhibitor affinity capture was performed as described in (30) using

molecular probes based on the FK866 or A-933414 structure. Captured proteins were separated

by SDS-PAGE and Sypro staining and identified by LC-MS/MS. FK866 and GMX1778 were

synthesized at AbbVie. Additional compounds were synthesized using methods described in U.S.

Patent 2016/9302989 B2 (31).

NAMPT protein purification and enzyme assay

The 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

synthesized and cloned into the pLVX-IRES-puro expression vector. Full length NAMPT-FLAG

was transiently expressed in HEK 293-6E cells (NRC-Canada) and purified in a two-step process

using anti-FLAG affinity chromatography and size exclusion chromatography. Enzyme assays

were performed using the direct NMN detection method (32).

X-ray crystallography

Purified NAMPT at 10-13 mg/mL was incubated in the presence of 10 mM nicotinamide

for 2 hours on ice. Crystals grew at 4°C in hanging drop vapor diffusion setup containing one to

one ratio of the protein and reservoir solution (25% PEG 3350, 0.2 M ammonium sulfate, and 0.1

M HEPES, pH 7.5). Inhibitor complexes were formed by soaking the crystals in the presence of




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the compound, and prepared for X-ray studies by brief transfer into reservoir solution with 20%

(v/v) glycerol and rapid plunge into liquid nitrogen. Diffraction data for the complex with A-

1293201 were collected under gaseous nitrogen at 100k at the Canadian Light Source

(Saskatoon, Saskatchewan, Canada) Beamline 08ID-113, while data for the complex with A-

1326133 were similarly collected at the Advanced Photon Source (Argonne, IL, USA) Beamline

17-ID. Diffraction intensities were processed using autoPROC (33) and the structure was solved

by sequential molecular replacement with coordinates from pdb code 2GVJ using MOLREP (34)

within the CCP4 program suite (35). The model was rebuilt using COOT (36) and refined against

structure factors using the programs REFMAC5 (37) and autoBUSTER (38). Figures were

prepared using the program PyMOL (Schroedinger, LLC, New York, NY). Atomic coordinates

for the complexes with A-1293201 and A-1326133 have been deposited in the public wwPDB

with accession codes 5U2M and 5U2N, respectively. NAMPT crystal structure data and

refinement statistics are listed in Supplementary Table S1.

Cellular assays

For PC3 cell viability assays, cells were treated in 96 well plates with compounds -/+ 0.3

mM NMN for 5 days followed by a Cell Titer Glo assay (Promega). At the 5 day time point ATP

was already depleted and the signal correlated with cell viability as measured by other methods.

For shorter time course assays, HCT116, PC3 or NCI-H1975 cells were treated for the indicated

times, then cellular levels of NAD+ + 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 with Vybrant Green (Invitrogen) and imaged in an

IncuCyte FLR (Essen Bioscience).

NAD+ metabolome analysis

NAD+ and related metabolites were analyzed as described previously (39,40). Briefly, 1

μL of one of two internal standard solutions (Solution A and Solution B) were added to each

sample (NCI-H1975 pellet containing 1 x 106 cells) before extraction. Solution A was composed

of extract from Fleischmann’s yeast grown in the presence of U-13C glucose diluted to produce a

final concentration of 1:80 heavy extract to sample solution. Solution B contained 60 μM 18O

nicotinamide riboside and 18O nicotinamide. Samples were deproteinized using buffered ethanol




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(75%/25% (v/v) ethanol/10 mM HEPES pH 7.1) heated to 80°C for 3 minutes with vortexing.

Particulate was pelleted with centrifugation (16,100g, 10 minutes, 4°C). The supernatant was

transferred to fresh tubes and then dried overnight via speed vacuum. Reconstituted extracts were

separated and analyzed via LC MS/MS as previously described (39,40). Metabolites were

detected using a Waters TQD operated in positive ion mode by multiple reaction monitoring

(MRM). Metabolite peak areas were converted to mole amounts by regression to internal

standard (IS) peak areas. Intracellular concentrations were calculated by dividing the mole

amount by the total intracellular volume, assuming an intracellular volume of 2.5 pL per cell.

One-way ANOVA followed by Dunnett’s multiple comparisons test was performed using

GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla California USA,

www.graphpad.com.

Glycolytic metabolite analysis

NCI-H1975 cells were harvested and aliquoted into 1 x 106 cells in duplicate. Samples

were spun and washed with cold PBS followed by metabolite extraction in 500 µL of 80% (v/v)

methanol in water containing 2.5 µM U-13C glucose-6-phosphate as the IS. Samples were

vortexed and incubated at -80°C for 20 minutes then spun for 30 minutes at 13,000g and 4°C.

Supernatants were transferred into cold Eppendorf tubes, lyophilized to dryness, and stored at -

80°C until analysis. Lyophilized samples were reconstituted with 50 µL of water, agitated for 15

minutes at 4°C, then diluted with 50 µL of acetonitrile, and agitated for 15 minutes at 4°C.

Extracts were separated and analyzed as described previously (41); however, only positive mode

MRM transitions for select metabolites were included in the method. Linear dynamic range for

each metabolite was defined with standards and samples were diluted and re-injected when

necessary. Changes in metabolite concentrations are reported as fold change to control.

In vivo pharmacology

All experiments were conducted in accordance with IACUC guidelines. Compounds

were formulated in the following vehicle 2% (vol) EtOH, 5% (vol) Tween 80, 20% (vol) PEG-

400, 73% (vol) 0.2% HPMC, and were administered orally in the pharmacology studies.

HCT116 cells were grown to passage 2 in vitro. A total of 0.5x106 cells were inoculated into the

right flank of female C.B.-17 SCID mice on day 0 in a volume of 0.1 mL. Tumors were size




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matched on day 11 post-inoculation with a mean tumor volume of 231 ± 28 (SD) mm3 with

dosing beginning on the next day. Tumor volume was calculated twice weekly. Measurements of

the length (L) and width (W) of the tumor were taken via electronic caliper and the volume was

calculated according to the following equation: V = L x W2/2 using Study Director version 3.1

(Studylog Systems, Inc., South San Francisco). For pharmacodynamic studies, HCT116 tumor

bearing mice were size matched and given oral dose(s) of A-1307138. Tumor samples were

collected at various time points following dosing and were flash-frozen in liquid nitrogen.

Tumors were homogenized and NADt was measured using the NAD+/NADH Quantification Kit

from MBL International Corporation.

RESULTS

Discovery of novel NAMPT inhibitors

To identify potential novel anti-cancer targets, a cell proliferation screen was performed

to simultaneously identify oncology-relevant biologic activity (i.e., antiproliferative activity) and

small molecules responsible for the activity of interest. The compounds could then be used to

identify the target through affinity capture of the inhibitor/target complex. One cluster of

compounds exhibited activity in five of the eight cell lines tested (Supplementary figure 2A).

Inhibitor affinity capture studies of a novel compound, the isoindoline urea A-933414, from the

hit cluster revealed NAMPT as the major biochemical target (Supplementary figure 2B and 2C).

Inhibition of PC3 cell viability by A-933414 was rescued by co-incubation of cells with the

product of NAMPT, NMN, which overcomes intracellular NAMPT inhibition by virtue of

extracellular dephosphorylation to nicotinamide riboside and intracellular conversion to NAD+

via the nicotinamide riboside kinase pathway (42). Rescue by NMN indicated that NAMPT was

solely responsible for the antiproliferative activity of this chemotype (Supplementary figure 2D

and Supplementary Table S2). These experiments also suggest that these compounds are

exquisitely specific for NAMPT as little to no antiproliferative activity was observed at

concentrations 1,000-fold higher in the presence of NMN.

Extensive SAR work on this series indicated that structural modifications to several

portions of the molecule provide improved potency, physical properties, and reduced clearance,




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resulting in significant murine exposure after oral dosing (Supplementary Table S3) without

compromising selectivity toward NAMPT ((31), manuscript in preparation). A lead compound

from this series, A-1293201 (Figure 1A), was found to potently inhibit recombinant, human

NAMPT in an enzyme activity assay (Figure 1B). Examination of the interactions between A-

1293201 and NAMPT by X-ray crystallography revealed several distinct sites for cooperative

interactions (Figure 1C). The sites include the PRPP binding subsite A, which is hydrophilic in

nature and unoccupied by NAM; the NAM binding subsite B, where important pi-stacking and

hydrogen bond interactions with NAM are made; the tether subsite C, which is narrow,

lipophilic, and not occupied by bound NAM; and the distal opening subsite D, which widens and

provides several bound waters and hydrophobic surfaces. Similar to FK866, the isoindoline urea

portion of A-1293201 is engaged in important pi-stacking interactions in subsite B as well as

hydrogen bonds and hydrophobic interaction in subsite C. In contrast to FK866 as well as recent

second generation NAMPT inhibitors, the isoindoline urea compounds are the first known potent

non-phosphoribosylated NAMPT inhibitors.

Biological comparison of substrate and non-substrate NAMPT inhibitors in vitro

To further characterize the ramifications of having inhibitors that do not serve as

substrates to NAMPT, a nucleophilic nitrogen was added to the NAM-mimetic portion of these

compounds that occupied subsite B, producing potent and selective azaisoindoline urea inhibitors

that could be phosphoribosylated by NAMPT (Supplementary Table S2). A-1267211, the

azaisoindoline analog of A-933414, and a racemic mixture of A-1331597, the azaisoindoline

analog of A-1293201 were both shown to be phosphoribosylated after incubation with NAMPT

enzyme under conditions in which NAM is also phosphoribosylated (Supplementary figure 3A).

Phosphoribosylation of A-1267211 and another azaisoindoline, A-1307138, also occurred

following treatment of PC3 cells with the compounds for 24 hours (Supplementary figure 3B).

A set of matched pairs of inhibitors that differ only in the presence or absence of the

nitrogen at position 5 of the isoindoline ring were used for further analysis (Supplementary Table

S4). The pairs of isoindoline and azaisoindoline ureas were tested in PC3 cells for inhibition of

cell viability (Figure 2). In most cases the azaisoindoline version of each compound pair

exhibited greater cellular potency than the isoindoline counterpart, ranging from 1.2- to 48-fold

improvement in activity (Figure 2). Although the isoindolines tended to be less potent in cells,




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many inhibited NAMPT and caused cytotoxicity with IC50s below 50 nM, and about a quarter of

the isoindolines had similar cellular potencies as their azaisoindoline counterpart. In contrast,

removal of the nitrogen from the pyridyl ring of FK866 completely abolished cellular potency

(Figure 2 and Supplementary Tables S2 and S4).

Representative compounds from the isoindoline and azaisoindoline series were further

characterized in biochemical and cellular assays to identify other distinguishing features.

Compounds tested included the azaisoindoline/isoindoline pairs of A-1326133/A-1292945 and

A-1331597/A-1293201 (Supplementary Table S2). The isoindoline of each pair showed 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, the substrate compound A-

1326133 was more potent than its corresponding isoindoline in the viability assays. Inhibition of

PC3 cell viability by all of the compounds was rescued by co-incubation of cells with NMN

(Supplementary Table S2).

Effects of A-1293201, A-1331597, A-1292945 and A-1326133 on depletion of

NAD+/NADH and ATP in HCT116 colorectal cancer cells over time were also measured.

Inhibition of NAMPT by all four compounds resulted in maximal NADt (levels of NADH plus

NAD+) depletion within 24 hours and ATP depletion within 48-72 hours (Figure 3A-D).

Following NADt and ATP depletion, HCT116 cell viability was severely compromised within

72 hours. ATP depletion and inhibition of cell viability only occurred at compound

concentrations that resulted in greater than 90% reduction in NADt levels at 48 hours. If there is

less than 90% depletion of NADt, the NADt levels recover and cells survive. These data suggest

that cytotoxicity elicited by NAMPT inhibition is dependent on the kinetics of ATP depletion,

which occurs subsequent to NADt depletion.

Comparison of NADt depletion dose-response curves after 24 hour treatment for the

azaisoindoline/isoindoline pairs is shown in Figure 3E. While the IC50 for NADt depletion for A-

1293201 was 1.8x lower than for A-1331597, both compounds depleted NADt by >90% at 37

nM. In contrast, the IC50 and the maximal NADt depletion for A-1326133 were both lower than

for A-1292945. The maximal NADt depletion occurred at 4.1 nM for A-1326133, but for A-

1293201 NADt was not depleted >90% until 12.3 nM. NADt recovery following washout of the

compounds was also determined by treating HCT116 cells with the NAMPT inhibitors for 24




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hours, removing the compound and washing the cells, and measuring NADt levels over time.

The NADt recovery was plotted for all four compounds at the concentrations at which NADt was

depleted by >90% at 24 hours before washout (Figure 3F). The kinetics of NADt recovery was

similar for A-1293201 and its partner, A-1331597, as well as for the isoindoline A-1292945. For

these compounds, NADt had recovered by 50% around 4 hours after washout. However,

recovery of NADt following washout of A-1326133 was delayed and did not reach 50% until 8

hours. By 16 hours following washout NADt levels had fully recovered for all four of the

compounds.

Effects of lead isoindoline and azaisoindoline NAMPT inhibitors on the NAD metabolome

To determine the effects of A-1293201 and A-1326133 on the NAD+ metabolome and

glycolysis, NCI-H1975 cells were treated with either vehicle (0.1% DMSO) or 3 concentrations

(IC20, IC50, or IC>90) of the compounds or FK866 for 8, 24, or 48 hours. The compound

concentrations and incubation times were chosen based on the kinetics of NADt and ATP

depletion in NCI-H1975 cells (Supplementary figure 4). After 24 hours and at concentrations

matching the respective IC50 and IC>90, each compound significantly depressed NMN and NAD+

(Figure 4). At 24 hours, ADP ribose (ADPr) was decreased significantly by treatment with each

compound at IC>90, while NADP+ tended to be lowered (Figure 4). However, the data indicate

that NMN, NADP+ and ADPr all strongly correlated with NAD+ in each experiment and all

trended down together. The pattern observed at 24 hours continued at 48 hours (Figure 4). The

effects of FK866, A-1293201 and A-1326133 on glycolytic metabolites was also measured, and

all three compounds caused similarly increased levels of fructose-6-phosphate/glucose-6-

phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate/glycedraldehyde-3-

phosphate (Figure 5), which is consistent with inhibition of glycolysis via NAD+ depletion

previously reported for FK866 (43). Together these data confirm that A-1293201 and A-1326133

target NAD+ metabolism, which subsequently depresses glycolysis.

In vivo characterization of lead isoindoline and azaisoindoline NAMPT inhibitors

The evaluation of the time of inhibition requirement for cytotoxicity identified by the in

vitro time course studies was extended to an in vivo setting using A-1307138, an analog of A-




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1326133. Following a single oral dose of 15 mg/kg in an HCT116 xenograft tumor model, A-

1307138 treatment reduced tumor NADt levels by greater than 90% after 24 hours, but NADt

concentrations quickly recovered to ~50% of pre-treatment levels 72 hours post dose (Figure

6A). Notably, transient depletion of NADt levels by >90% for less than 24 hours once a week

did not elicit any suppression of tumor growth (Figure 6C). Extending the duration of NADt

depletion by two to three consecutive days of dosing resulted in substantial inhibition of tumor

growth in vivo (Figure 6C), which corresponds to sustained NADt depletion, with >90%

depletion maintained for over 72 hours (Figure 6B). These data are consistent with the in vitro

observations that inhibition of NAMPT for at least 48 hours is required to elicit NADt depletion-

dependent cytotoxicity. It was determined that maintaining the time-over-threshold (IC90) for 48

to 70 hours was required for robust anti-tumor efficacy. This is exemplified by the potent anti-

tumor efficacy of A-1293201 and A-1326133 in the HCT116 xenograft model (Figure 6D). Body

weight loss did not exceed 10% for any of the compounds and no deaths occurred during the in

vivo studies (Supplementary figure 5).

DISCUSSION

We have identified and characterized novel potent and selective non-substrate and

substrate NAMPT inhibitors. Both types of compounds exhibit potent in vitro and in vivo anti-

proliferative activity following oral administration and display similar kinetics of NADt and ATP

depletion and induction of cytotoxicity, which is rescued by NMN. Based on mechanistic

studies, inhibition of NAMPT by >90% for at least 48 hours is required for anti-proliferative

effects in HCT116 cells, in vitro and in vivo. As has been shown for previously published

NAMPT inhibitors, within hours after treatment, NAD+ levels are decreased, but 24 to 48 hours

are required for complete NAD+ depletion within the mitochondria and blockade of ATP

generation. Once ATP is depleted, cell death occurs through apoptosis, autophagy, or oncosis

(10,44). The kinetics of NAD+ depletion can differ between cell types, possibly due to

differences in NAD+ consumption rates by PARPs or other enzymes that utilize NAD+ as a

substrate. The kinetics of NAD+ and ATP depletion in NCI-H1975 cells are slower than in

HCT116 cells. As a result, NAD+ levels are not depleted >90% until between 36 and 48 hours

and inhibition of glycolysis and ATP depletion are also delayed. A-1293201 and A-1326133




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produce similar effects on the NAD+ metabolome and glycolytic metabolites as FK866. All of

these properties indicate that the isoindoline and azaisoindoline compounds cause cell death by

inhibiting NAMPT in cells and xenografts.

In contrast to previously described NAMPT inhibitors, including FK866, GMX1778 and

GNE-617, A-1293201 does not contain an aromatic nitrogen in its NAM-mimetic group (Figure

1A). Crystallographic studies indicate that A-1293201 participates in similar interactions with

NAMPT as the other inhibitors (45,46). A-1293201 and A-1326133 occupy the NAM binding

site and engage in a critical pi-stacking interaction with tyrosine 18 in the same manner as NAM

and FK866 (Figure 1C and Supplementary figure 6). The central regions of all the compounds

traverse a narrow, lipophilic tunnel out to an opening distal to the active site. The NAM-mimetic

moiety, where the presence of the aromatic nitrogen allows for phosphoribosyl adduct formation,

is the area of divergence between A-1293201 and the other compounds. Our data indicate that

phosphoribosylation of inhibitors is not required for potent inhibition of NAMPT and induction

of cell death in cells or in xenograft models.

It is notable that most azaisoindolines display improved potency in cellular assays

compared to their isoindoline analogs, even though their permeability and protein binding

properties are similar. One hypothesis is that the aromatic nitrogen in the azaisoindoline results

in improved pi-stacking interactions with NAMPT and/or serves as a substrate, leading to

increased target potency. However, similar IC50s in enzyme assays for the azaisoindoline/

isoindoline pairs of A-1326133/A-1292945 and A-1331597/A-1293201 suggests that improved

pi stacking interactions are not occurring, and these data are corroborated by the overlaid crystal

structures of A-1293201 and A-1326133 (Supplementary figure 6). In addition, there does not

appear to be a difference in dissociation rates between A-1293201 and A-1331597 or between A-

1326133 and A-1292945 in TR-FRET assays (Supplementary figure 7), even when the

compounds are pre-incubated with the enzyme and PRPP for 24 hours to allow

phosphoribosylation. These results suggest that for this set of compounds, phosphoribosylation

does not lead to increased residence time on the enzyme. In these assays, both substrate and non-

substrate compounds have slower off rates when PRPP is bound, indicating that both classes of

compounds bind cooperatively with PRPP. These data suggest that the increased cellular potency




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of azaisoindolines relative to isoindolines is not the result of increased target affinity upon

phosphoribosylation.

Another potential reason for the increased cellular potency of the azaisoindolines is that

phosphoribosylated compounds are retained longer in cells due to decreased transit of the

modified compound through the plasma membrane. Increased cellular retention was reported to

contribute to cellular potency for GMX1778, which undergoes phosphoribosylation (28). In our

washout studies recovery of NADt levels is similar between A-1293201 and A-1331597,

suggesting that A-1331597 is not retained in cells. Thus, while phosphoribosylation can improve

the cellular potency for some compounds within the isoindoline series, in general there is not a

requirement for modification by NAMPT for potent inhibition, and enhanced binding can be

attained through additional interactions distal to the NAM binding site.

Our discovery of potent and selective NAMPT inhibitors that do not contain an aromatic

nitrogen in the NAM moiety challenges the current dogma that this feature is required for

cellular efficacy. A-1293201 and other isoindolines are efficacious despite the lack of a

phosphoribosylation site which could lead to cellular retention or other unexpected effects in

cells. As a result, treatment with A-1293201 or other isoindolines may allow better control of

NAMPT inhibition in normal cells.

ACKNOWLEDGEMENTS

The 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 authors would also like to acknowledge Stella Doktor, Patricia

Stuart, Amanda Olson, Donald Osterling, and DeAnne Stolarik from the DMPK-BA Department

at AbbVie for the in vitro ADME and in vivo pharmacokinetics measurements.

Research described in this paper was performed using beamline 08ID-1 at the Canadian Light

Source, which is supported by the Canada Foundation for Innovation, Natural Sciences and

Engineering Research Council of Canada, the University of Saskatchewan, the Government of




16

Saskatchewan, Western Economic Diversification Canada, the National Research Council

Canada, and the Canadian Institutes of Health Research.

Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the

companies of the Industrial Macromolecular Crystallography Association through a contract with

Hauptman-Woodward Medical Research Institute.

This research used resources of the Advanced Photon Source, a U.S. Department of Energy

(DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne

National Laboratory under Contract No. DE-AC02-06CH11357.

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FIGURE LEGENDS

Figure 1. A-1293201 binds to NAMPT and inhibits its enzymatic activity. (A) Structures of

NAM, FK866, GMX1778, GNE-617, and A-1293201. The aromatic nitrogen that is

phosphoribosylated by NAMPT is colored red. (B) NAMPT enzyme inhibition dose-response

curve for A-1293201. Data are mean ± SEM from 2 independent experiments performed with

duplicates. (B) Overlaid crystal structures of A-1293201 (green) and FK866 (yellow) in

NAMPT. The PRPP binding region (subsite A), NAM binding region (subsite B), tether region

(subsite C) and the distal opening of the active site (subsite D) are indicated.

Figure 2. Cellular comparison of azaisoindoline/isoindoline pairs. Matched structural pairs of

isoindoline and azaisoindoline NAMPT inhibitors or FK866 and its analog that lacked an

aromatic nitrogen were tested in 5 day cell viability assays in PC3 cells. The IC50 in PC3 cells is

plotted on the y axis and pair number is plotted on the x axis. azaisoindoline, isoindoline, ♦ FK866. Magenta indicates that the aromatic ring contains N and cyan indicates that the aromatic

ring contains C.

Figure 3. NADt depletion following A-1293201 and A-1326133 treatment results in cell death. (A, B) HCT116 cells were incubated with the indicated concentrations of A-1293201 (A)




22

A-1331597 (B), A-1292945 (C) or A-1326133 (D) for 24, 48, or 72 hours. Total NAD (NADt =

NADH + NAD+), ATP and cell number were assessed at each time point. Dashed line indicates

90% depletion of NADt. (E) Depletion of NADt levels in HCT116 cells following 24 hour

treatment with azaisoindoline/ isoindoline pairs. (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 are colored 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

± SEM from 3-5 independent experiments. Data in (F) are mean ± SEM from 2-4 independent

experiments.

Figure 4. A-1293201, A-1326133, and FK866 have similar effects on the NAD+ metabolome. NCI-H1975 cells were treated with 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, NAD+,

NADP, and ADPr after treatment of cells with compounds for 24 hours. (Right) Levels of NMN,

NAD+, NADP, and ADPr after treatment of cells with compounds for 48 hours. Data are the

mean + SEM of duplicate samples. *p<0.05, **p<0.01, ***p<0.001

Figure 5. A-1293201, A-1326133, and FK866 have similar effects on glycolytic metabolites. NCI-H1975 cells were treated with vehicle (0.1% DMSO), A-1293201, A-1326133, or FK866 at

3 dosages (IC20, IC50, or IC>90) for 8, 24 or 48 hours. For each time point, fold changes from

control were determined for (A) fructose-6-phosphate (F6P) + glucose-6-phosphate (G6P), (B)

fructose-1,6-bisphosphate (F16BP), and (C) glycedraldehyde-3-phosphate (G3P) +

dihydroxyacetone phosphate (DHAP) and are reported as fold change to control. Note only IC>90

data are shown. Fold change to control for IC20 and IC50 dosages across all compounds and time

points were 1.3±0.22, 1.6±0.29, and 1.2±0.30 for F6P+G6P, F16BP, and G3P+DHAP,

respectively. Data are the mean + SD of duplicate samples.

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 hours after treating mice

with a single with 15 mg/kg oral dose of A-1307138. (B) NADt levels in HCT116 xenografts in

samples harvested 6 or 24 hours after treating mice orally with 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 are average ± SD from xenografts harvested from 4 mice. (C) Efficacy




23

plots for HCT116 xenografts in mice dosed orally (po) 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 vs. dashed line. (D) Efficacy plots for HCT116 xenografts in mice

treated with A-1326133 and A-1293201 at the indicated doses on a po qd (3on,4off) x3 schedule.

For efficacy studies, dosing schedules are indicated by triangles under the x axis, and tumor

volumes are graphed as mean ± SEM for 10 mice/treatment group.




Published OnlineFirst May 3, 2017.Mol Cancer Ther Julie L. Wilsbacher, Min Cheng, Dong Cheng, et al. Substrate NAMPT InhibitorsDiscovery and Characterization of Novel Non-substrate and

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