NAMPT, an enzyme essential for NAD+ biosynthesis, has been extensively studied as an anticancer target for developing potential novel therapeutics. Several NAMPT inhibitors have been discovered, some of which have been subjected to clinical investigations. Yet, the on-target hematological and retinal toxicities have hampered their clinical development. In this study, we report the discovery of a unique NAMPT inhibitor, LSN3154567. This molecule is highly selective and has a potent and broad spectrum of anticancer activity. Its inhibitory activity can be rescued with nicotinic acid (NA) against the cell lines proficient, but not those deficient in NAPRT1, essential for converting NA to NAD+. LSN3154567 also exhibits robust efficacy in multiple tumor models deficient in NAPRT1. Importantly, this molecule when coadministered with NA does not cause observable retinal and hematological toxicities in the rodents, yet still retains robust efficacy. Thus, LSN3154567 has the potential to be further developed clinically into a novel cancer therapeutic. Mol Cancer Ther; 16(12); 2677–88. ©2017 AACR.

Nicotinamide adenine dinucleotide (NAD+) is a cofactor essential for a number of cellular processes. NAD+ is synthesized from nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and tryptophan (1–6). In mammals, the synthesis of NAD+ from NAM first involves the conversion of NAM to NAM mononucleotide (NMN), which is catalyzed by NAM phosphoribosyl transferase (NAMPT), a rate-limiting enzyme (7, 8), followed by the conversion of NMN to NAD+ by NMN adenyltransferase (NMNAT). Similarly, the synthesis of NAD+ from NA first involves the conversion of NA to NA mononucleotide catalyzed by NA phosphoribosyl transferase 1 (NAPRT1) followed by a two-step conversion to NAD+. The in vivo concentration of NA is extremely low due to its rapid excretion and metabolism, so the contribution of NA to NAD+ biosynthesis is limited in mammals (3). More recently, NR can also be converted to NAD+ in a two-step reaction catalyzed by NRK and NMNAT (5, 6). The de novo biosynthesis of NAD+ from tryptophan mainly occurs in the liver and under certain stressed conditions (4). Therefore, the NAM salvage pathway represents the major route to NAD+ biosynthesis in the mammals (9–11).

In cancer cells, NAMPT plays a crucial role in several physiological processes including energy generation, reductive biosynthesis, mitochondrial function, and the response to oxidative stress (1, 12–14). It is also overexpressed in several types of tumors (8, 15–19) and its expression appears to be associated with tumor progression (15, 20). The inhibition of NAMPT leads to the attenuation of tumor growth and induction of apoptosis (11; 21–24). Thus, NAMPT represents a promising therapeutic target for developing potential novel cancer drugs.

As a promising anticancer target, NAMPT has been the subject of intensive drug discovery research (25–33). Several NAMPT small molecule inhibitors, for example, CHS 828/GMX-1778 and its prodrug GMX1777, and FK866/APO866, have been investigated in the clinic (30, 33). In these clinical studies, thrombocytopenia was demonstrated to be the dose-limiting toxicity (30, 33). Retinopathy, degeneration and loss of the photoreceptor and outer nuclear layers of the retina, was reportedly associated with FK866/APO866 and CHS 828/GMX-1778 in the rat (30, 34). Similar toxicity findings were also reported for other NAMPT inhibitors (35, 36). One potential effective approach to mitigate these toxicities is by coadministration of NAMPT inhibitors with NA. It is encouraging that thrombocytopenia was significantly reduced in a murine model when FK866 was coadministered with NA (9), thus indicating that NA coadministration could be a viable approach. However, more recent studies reported that NA coadministration failed to rescue the retinal toxicity and also abolished the efficacy of NAMPT inhibitors in tumor xenograft models derived from NAPRT1-deficient cancer cell lines (34–36). On the basis of these findings, it remained unclear whether NA coadministration could be used to mitigate the toxicities associated with NAMPT inhibition, especially the retinal toxicity.

To mitigate the toxicities associated with NAMPT inhibition, we sought to develop an orally bioavailable NAMPT inhibitor that demonstrates much improved tolerability and yet still retains robust efficacy when coadministered with NA. To this end, we identified such a unique NAMPT inhibitor, LSN3154567. In this study, we report the results of our in vitro and in vivo characterizations of this molecule with respect to its activity, selectivity, and NA rescue. We also report the potential patient tailoring markers that could be used to identify tumors more sensitive to NAMPT inhibition. On the basis of these findings, we believe that with NA coadministration, LSN3154567 has the potential to be developed into a novel cancer therapeutic for treating various forms of human malignancies.

Alternatives and animal use

Animals were only used where no reasonable or viable in vitro or ex vivo alternative was available. All animal experiments complied with local and national regulations and guidelines on animal use and were approved by the Institutional Animal Care and Use Committee.

Assay for NAD+ levels in cancer cells

Cancer cells were cultured as described before (37, 38). Cells were seeded into a 96-well culture plate at a density of 8 × 104 cells per well and incubated at 37°C in 5% CO2 for 4 hours, and then treated with LSN3154567 at various concentrations (0.03-1,000 nmol/L) for 24 hours. All treatments were conducted in duplicates. NAD+ levels were determined by measuring fluorescence at 360 (excitation) and 420 nm (emission; CytoFluor reader). NAD+ levels were also measured by LC-MS as described previously (37, 38). For NA rescue time course studies, cells were grown and treated with LSN3154567 (100 nmol/L) ± NA (10 μmol/L) for different periods of times prior to measuring NAD+ levels.

Antiproliferation assay

The cell lines used were obtained from the following: A2780 and KM-12 (NCI DCTD), KMS-11 and MKN-74 (JCRB), OPM-2 and Kelly (DSMZ); and all other cancer cells (ATCC). The identity of these cell lines was not tested or verified prior to use in this study. All the cells were cultured according to the provider's recommendations, except SW872, which was grown in RPMI-1640 supplemented with 10% FBS with 5% CO2. Cells were seeded in 96-well plates, cultured overnight, and treated with LSN3154567 (0.03 to 1,000 nmol/L) ± NA (10 μmol/L) in duplicate at 37°C in 5% CO2 for 72 hours. Staurosporine (10 μmol/L) was used as positive control. Cell viability was determined by a CytoTox-Glo Cytotoxicity assay kit (Promega Corporation) per the manufacturer's instructions.

Construction of NAPRT1 knockdown and expression cell lines

NAPRT1 shRNA lentiviruses were produced using HEK293T cells and Fugene 6 based on the manufacturer's instructions (Promega). Briefly, HEK293T cells when reaching a confluence of 80% to 90% were transfected with a DNA plasmid solution containing: Fugene 6 (Promega); and pCMV-VSV-g, Delta8.9, and shRNA vector-TRC2 (Sigma-Aldrich). After growing for 72 hours, the culture supernatant was collected, concentrated using a Lenti-X concentrator (Clontech), and then titered for shRNA TRC2-NAPRT1 lentiviruses. Calu-6 cells were then infected with no-target or shRNA TRC2-NAPRT1 lentiviruses in the presence of polybrene (Millipore-Sigma) and then selected with blastcidin for stable cell lines.

A2780 cells were infected with no-target pLenti6.3-vector or pLenti6.3-NAPRT lentivirus in the presence of polybrene and then selected with puromycin for stable cell lines. The efficiency of infection was assessed by Western blotting analysis of NARPT1 expression levels. Briefly, a total amount of cellular proteins (20 μg each) was resolved by 4% to 15% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with an anti-NAPRT1 antibody (HPA024017, Sigma) and an anti-β-actin antibody (Sigma), respectively. The detection and quantitation of NAPRT1 protein were carried out using a luminescent image analyzer LAS-4000 (Fujifilm).

Cancer cell sensitivity profiling (CCSP)

To assess the antiproliferative activity of LSN3154567 against a large number of cancer cell lines across many different tumor types (325 cell lines), a novel cancer cell sensitivity profiling (CCSP) assay format was developed (treating cells for 2 population doublings). All cell lines were obtained from commercial sources and cultured in conditions recommended by vendors. Cell line histology and site of origin annotation were derived from the vendor or the COSMIC cancer cell line database. Cell line authenticity was confirmed by STR-based DNA finger printing and multiplex PCR (IDEXX-Radil). Growth curves were used to determine average population doubling time for each cell line. Cell density was optimized to ensure robust, logarithmic cell growth for the duration of compound treatment.

To test LSN3154567, cells were recovered from frozen stocks. The day before testing, cells were seeded in complete media in 384-well white-walled clear bottom microtiter plates at the predetermined optimal density for each cell line. LSN3154567 (2.0 to 0.0001 μmol/L) was added 16 hours after plating. Antiproliferative activity was determined using an assay kit (CellTiter-Glo, Promega) according to the manufacturer's protocol.

Gene mutation, copy number and expression data was compiled from public domain data sets from COSMIC (http://cancer.sanger.ac.uk/cosmic) and CCLE (https://portals.broadinstitute.org/ccle) and from internal sources.

In vivo studies

For efficacy studies, athymic nude mice (female) and NOD SCID mice (female) were purchased from Harlan Laboratories and Jackson Laboratories, respectively. The following cancer cells were used: A2780 (2–5 × 106 cells/animal), NCI-H1155 (5 × 106 cells/animal), Namalwa (2 × 105 cells/animal), HT1080 (5 × 106 cells/animal), and SK-N-SH (5 × 106 cells/animal). Cells were grown as described before, mixed with matrigel (1:1), and implanted subcutaneously into the rear flank of the mice. For A2780, NCI-H1155, HT1080, and SK-N-SH tumor models, female CD-1 nu/nu CB17 athymic nude mice (7/group) were used, whereas for Namalwa tumor model, female CB17 NOD, SCID mice (7/group) were used. The animals were orally dosed twice daily (BID) with LSN3154567 formulated in 1% of hydroxyethylcellulose, 0.25% Tween 80, and 0.05% of antifoam for 2 to 3 weeks after tumors reached 300 to 500 mm3. The tumor volume and body weight were measured twice weekly. Tumor growth inhibition (T/C) is calculated by the following equation: T/C = (Vt − V0) drug treated/(Vt − V0) vehicle control.

For in vivo target inhibition studies, the A2780 tumor model was used as described above. When tumor volume reached about 500 mm3, animals bearing tumors were treated with LSN3154567 for two doses as described above. Tumor samples were collected 7 hours after the second dose and processed as described before (37). NAD+ levels were quantified as described before (38).

Analysis of dihydroxyacetone phosphate and fructose 1,6,-bisphosphate

Cellular and tumor extracts were prepared and injected into an LC-triple quadrupole mass spectrometer (API 4000, AB Sciex), coupled with a pair of Shimadzu 10ADVP pumps, and a Shimadzu SIL-20A autosampler (Shimadzu) as described before (38).

PK and exposure studies

For bioavailability studies, male CD-1 mice (Charles River Lab) were dosed with LSN3154567 at 2 mg/kg intravenously in 20% Captisol (w/v), 25 mmol/L NaPO4, pH 2, q.s. formulation or 2 mg/kg orally in the same vehicle as described above for in vivo studies in a cross over design with 3-d wash period in between two arms. Blood samples were obtained through retro orbital at 0, 0.08 (IV only), 0.25, 0.75, 2, 4, 8, and 24 hours and frozen until analysis. For exposure analysis, blood samples (≈20 μL) were obtained through tail clipping at 0.5, 1, 2, 4, 8, and 24 hours. The samples were collected into EDTA-coated capillary tubes (Fisher Scientific) and spotted onto Whatman DMPK-C DBS collection cards (GE Healthcare Bio-Sciences). The cards were allowed to dry for 2 hours, placed in Ziploc bags (Johnson & Son). A slice (3 mm) of each card was extracted with methanol/acetonitrile (1:1, v/v; 180 μL) containing a Lilly proprietary internal standard (20 ng/mL) in 96-well plates and analyzed by LC-MSMS (AB Sciex API 4000 triple-quadrupole mass spectrometer, Life Technologies Corporation). PK parameters were calculated using Watson LIMS (version 7.4; Thermo Scientific).

Toxicity studies

For toxicity assessments, female rats (Charles River Laboratories) or male and female dogs (Covance Research Products) were dosed with LSN3154567 orally in the same vehicle described above. For NA rescue studies, NA was formulated in phosphate buffered saline (pH 7.4). Rats were given LSN3154567 orally once daily at 20, 40, or 80 mg/kg for two cycles of 4 days with 3 days between the first and second cycles. Dogs were also dosed orally at 1 or 2.5 mg/kg (without NA) or at 5 mg/kg (with NA; BID) for 4 days. Exposure was obtained and analyzed as described above.

Clinical observations were recorded daily during each study. Blood samples for clinical pathology assessments were obtained as described above. Extensive hematology assessments were performed using whole blood and clinical biochemistry using serum, which included total (WBC) and differential leucocyte count, erythrocyte count (RBC), hematocrit (HCT), hemoglobin concentration (Hb), reticulocyte (RET) and platelet counts (PLAT); blood cell morphology was assessed via blood smears and red cell indices (MCV, MCHC, and MCH) were derived. Coagulation assessments included prothrombin (PT) and activated partial thromboplastin times (APTT). Serum biochemistry analyses included alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), creatine kinase (CK), blood urea nitrogen (BUN), creatinine (CREA), total bilirubin (TBIL), cholesterol (CHOL), triglycerides (TRIG), total protein (TPROT), albumin (ALB), globulin (GLOB), albumin–globulin ratio (A:G), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and inorganic phosphorus (P).

Animals were euthanized 24 hours after the last dose, and a full necropsy was performed on the following tissues that were fixed in 10% neutral buffered formalin (except eyes and testes which were fixed in Davidson's or Bouin's substitute fixatives, respectively): adrenals, bone (sternum), brain (cerebellum, cerebrum and brain stem), colon (dog only), epididymides, eyes, heart, ileum, jejunum, kidney, liver, lungs, muscle (skeletal), sciatic nerve, ovaries, pancreas, prostate, spleen, stomach, testes, thymus, thyroid, uterus, and vagina. Sections were prepared, stained with hematoxylin and eosin, and examined by a board-certified pathologist; pathology findings were peer reviewed.

Discovery of LSN3154567

Given the fact that the NAMPT inhibitors reported are all known to be associated with hematological and or retinal toxicities, the discovery of safer and better tolerated NAMPT inhibitors may lead to new treatment options for cancer patients. To identify such NAMPT inhibitors, we screened a more focused set of compounds selected based on the virtual screening of our database using computational models built from published X-Ray crystal structures (32). This effort led to the identification of several actives. Further structure-based design efforts led to the discovery of several series of potent and selective NAMPT inhibitors, which exhibited robust antitumor activity in vivo. One of the initial series to be advanced had excellent drug like properties and was highly permeable, but the characterization of a leading compound from this series in rat and dog toxicity studies revealed retinal and hematological toxicities. The hematological toxicities, but not the retinal toxicity, could be fully mitigated by using the NA co-administration strategy. To overcome this retinal toxicity issue, we reasoned that an NAMPT inhibitor with decreased permeability and increased polarity could have a reduced exposure in the retina. We thought that a molecule with such properties would have improved tolerability with respect to retinopathy while still maintaining its robust efficacy. To this end, we identified such as a potent and specific NAMPT inhibitor, LSN3154567 (Fig. 1A), after carrying out intensive in vitro, in vivo, and toxicology studies. This molecule makes key interactions with the residues composed of part of the nicotinamide binding pocket (Fig. 1B). On the basis of the X-ray crystal structures obtained, LSN3154567 is a competitive inhibitor with respect to nicotinamide. In this study, we show for the first time that the retinopathy associated with this molecule could be mitigated via NA coadministration (see later).

Figure 1.

A, The chemical structure of LSN3154567, 2-hydoxy-2-methyl-N-{2-[(pyridin-3-yloxy)acetyl]-1,2,3,4-tetrahydroisoquinolin-6-YL}propane-1-sulfonamide. B, High resolution x-ray crystal structure (2.03Å) of LSN3154567 in NAMPT.

Figure 1.

A, The chemical structure of LSN3154567, 2-hydoxy-2-methyl-N-{2-[(pyridin-3-yloxy)acetyl]-1,2,3,4-tetrahydroisoquinolin-6-YL}propane-1-sulfonamide. B, High resolution x-ray crystal structure (2.03Å) of LSN3154567 in NAMPT.

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LSN3154567 is a potent and selective NAMPT inhibitor

To assess its selectivity, specificity, and effects on cellular NAD+ levels, we characterized LSN3154567 in various biochemical and cellular assays. This molecule inhibited purified NAMPT with an IC50 of 3.1 nmol/L. When tested against a panel of human kinases (>100; CEREP Kinase panel), it did not exhibit any significant activity (i.e.: IC50 ≥ 1 μmol/L) against the kinases tested except CSF1R (IC50 ≈ 0.84 μmol/L). Because LSN3154567 exhibits a more much potent activity against NAMPT as demonstrated by biochemical and cellular assays (see below), we believe that its activity against CSF1R is physiologically irrelevant. To further assess its selectivity, we tested whether this molecule inhibited glucose 6-phosphate dehydrogenase, alcohol dehydrogenase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which represent different types of dehydrogenases requiring NADP(H) or NAD(H) as cofactors. We showed that it did not exhibit any significant inhibitory activity against these dehydrogenases when tested at ≤ 50 μmol/L.

To further characterize LSN3154567, we treated A2780 and HCT-116 cells with the molecule for 24 and 72 hours, and assessed its effects on NAD+ formation and cell proliferation. We showed that it potently inhibited NAD+ formation in and proliferation of both cell lines with the IC50 values of 4.95 and 11.5 nmol/L (for A2780), and 1.8 and 8.9 nmol/L (for HCT-116), respectively. It is known that some cancer cells, such as NCI-H1155, only use NAM for NAD+ biosynthesis, but others, like HCT-116, can also utilize NA via the NAPRT1-mediated NA pathway to synthesize NAD+ (11). If LSN3154567 is a specific NAMPT inhibitor, addition of NA to the growth media should abolish its inhibitory activity against HCT-116, but not NCI-H1155. This is indeed the case. The addition of NA abolished its inhibitory activities against HCT-116 with regard to NAD+ formation and proliferation, but not against NCI-H1155 cells (Fig. 2A and B). Similar results were obtained with other cell lines tested (Supplementary Fig. S1 and S2). Furthermore, the addition of NAM to the growth media also abolished its inhibitory activities against different cell lines (Supplementary Fig. S3). This is expected as LSN3154567 is a competitive inhibitor of NAMPT with respect to NAM. Finally, to further confirm the selectivity of LSN3154567, we generated NAPRT1-regulated cell lines. One such cell line was derived from Calu6, an NAPRT1-proficient cell line, in which the NAPRT1 gene was downregulated via shRNA and another cell line was derived from A2780, an NAPRT-deficient cell line, in which the NAPRT1 gene was reintroduced and expressed (Materials and Methods). We show that NA was unable to rescue the antiproliferative activity of LSN3154567 against Calu6 when NAPRT1 expression was ablated in the cell (Fig. 2C–E). On the other hand, we also show that NA was able to completely rescue the antiproliferative activity of the molecule against A2780 when the NAPRT1 gene was expressed in the cell (Fig. 2F–H). Taken together, the results of these studies show that LSN3154567 is a selective inhibitor of NAMPT.

Figure 2.

NA rescue of inhibitory activity of LSN3154567 in cancer cells. A and B, NCI-H1155 and HCT116 cells were grown and treated in duplicates with LSN3154567 (100 nmol/L) and ±NA (10 μmol/L) as described (Materials and Methods). NAD+ formation and cell death were quantified as described (Materials and Methods). Calu6 (C to E) and A2780 (F to H) derived cells were grown and treated in duplicates with LSN3154567 at various concentrations and ±NA (10 μmol/L) as described (Materials and Methods). Cell death was quantified as described (Materials and Methods). C, Western blotting analysis of NAPRT1 levels in Calu6 cells transfected with vector (V) or shRNA (KD); D and E, NA rescue of the antiproliferative activity of LSN3154567 against Calu6 cells transfected with vector and shRNA, respectively; F, Western blotting analysis of NAPRT1 levels in A2780 cells transfected with vector (V) or NAPRT1 expression system (OE); G and H, NA rescue of the antiproliferative activity of LSN3154567 against A780 cells transfected with vector and NAPRT1 expression system, respectively. *, P ≤ 0.001 vs. control.

Figure 2.

NA rescue of inhibitory activity of LSN3154567 in cancer cells. A and B, NCI-H1155 and HCT116 cells were grown and treated in duplicates with LSN3154567 (100 nmol/L) and ±NA (10 μmol/L) as described (Materials and Methods). NAD+ formation and cell death were quantified as described (Materials and Methods). Calu6 (C to E) and A2780 (F to H) derived cells were grown and treated in duplicates with LSN3154567 at various concentrations and ±NA (10 μmol/L) as described (Materials and Methods). Cell death was quantified as described (Materials and Methods). C, Western blotting analysis of NAPRT1 levels in Calu6 cells transfected with vector (V) or shRNA (KD); D and E, NA rescue of the antiproliferative activity of LSN3154567 against Calu6 cells transfected with vector and shRNA, respectively; F, Western blotting analysis of NAPRT1 levels in A2780 cells transfected with vector (V) or NAPRT1 expression system (OE); G and H, NA rescue of the antiproliferative activity of LSN3154567 against A780 cells transfected with vector and NAPRT1 expression system, respectively. *, P ≤ 0.001 vs. control.

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LSN3154567 exhibits a broad spectrum of anticancer activity

To assess its anticancer activity, we tested LSN3154567 against a number of different types of cancer cell lines cultured in the absence or presence of NA (10 μmol/L). As shown, LSN3154567 exhibited a potent antiproliferative activity against many cell lines in the absence of NA (Supplementary Table S1). The addition of NA rescued the antiproliferative activity of the molecule against several cancer cell lines expressing a high level of NAPRT1, but not those expressing a low level of NAPRT1 (Supplementary Table S1). Similar results were obtained with many more cell lines tested (see Fig. 7A). These results further confirmed that LSN3154567 is a specific NAMPT inhibitor. This molecule also exhibited a significant proapoptotic activity in NCI-H1155 and HCT116 (Supplementary Fig. S4). Taken together, LSN3154567 has a broad spectrum of anticancer activity with the potential for treating a variety of different types of tumors.

LSN3154567 alone or coadministered with NA exhibits a potent antitumor activity in tumor xenograft models

On the basis of its potent biochemical and cellular activities, we wanted to further establish that LSN3154567 inhibits the target in an in vivo setting. First, we characterized its pharmacokinetic (PK) properties. LSN3154567 exhibits good physical chemical properties that allow oral dosing. When dosed orally with 2 mg/kg in mice, it had an exposure of 195 nmol/L*hour in the plasma with a peak concentration of 57 nmol/L (at 0.25 hour) and an oral bioavailability of 39%. When dosed intravenously with 2 mg/kg, it had a hepatic clearance of 158.73 mL/min/kg and a volume of distribution at 7.1 L/kg. The half-life of terminal elimination was estimated to be 2.76 hours. Thus, LSN3154567 has appropriate PK properties for in vivo studies. We then developed an A2780 tumor xenograft model and tested its target inhibition activity. We showed that LSN3154567 exhibited a dose-dependent inhibition of NAD+ formation with estimated TED50 value of 2.0 mg/kg. Thus, LSN3154567 inhibits NAMPT in vivo.

One of the major consequences of NAMPT inhibition is the attenuation of glycolysis at the GAPDH step because this enzyme requires NAD+ for activity (37, 38). As a result, metabolites such as fructose 1, 6-bisphosphate (FBP) and dihydroxyacetone phosphate (DHAP) before or at the GAPDH step are increased. Therefore, the changes in these metabolites may be used as potential pharmacodynamic (PD) markers. Based on this, we assessed the in vivo consequence of target inhibitory activity of LSN3154567 in tumors derived from NCI-H1155, an NAPRT1-deficient NSCLC cell line. The treatment with 10 mg/kg led to a significant decrease in NAD+ levels (Fig. 3A and B): ≈94 and 99% at 96 and 120 hours, respectively, and also a time-dependent increase in FBP and DHAP levels from 24 to 72 hours (Fig. 3C and D). Both metabolites reached the maximal levels by 72 hours, but started to decrease by 96 hours, indicating tumor cell death. So, this suggests that in order to achieve robust efficacy, LSN3154567 has to be dosed for at least 4 days. Based on this, we developed a 4-day on and 3-day off dosing schedule (see below).

Figure 3.

Inhibition of NAMPT by LSN3154567 led to decreased NAD+ levels and increased glycolytic metabolite levels in tumor xenografts. Tumor xenografts derived from NCI-H1155 were grown and treated with LSN3154567 (10 mg/kg BID) for 24, 48, 96, and 120 hours; and processed for the analysis of NAD+ and glycolytic metabolite levels by LC-MS (Materials and Methods). A, NAD+ levels in tumors; B, inhibition of NAD+ formation (%); C, fructose 1,6-bisphosphate (FBP) levels in tumors; and D, dihydroxyacetone phosphate (DHAP). Error bars represent standard deviation. *, P ≤ 0.001 vs. control.

Figure 3.

Inhibition of NAMPT by LSN3154567 led to decreased NAD+ levels and increased glycolytic metabolite levels in tumor xenografts. Tumor xenografts derived from NCI-H1155 were grown and treated with LSN3154567 (10 mg/kg BID) for 24, 48, 96, and 120 hours; and processed for the analysis of NAD+ and glycolytic metabolite levels by LC-MS (Materials and Methods). A, NAD+ levels in tumors; B, inhibition of NAD+ formation (%); C, fructose 1,6-bisphosphate (FBP) levels in tumors; and D, dihydroxyacetone phosphate (DHAP). Error bars represent standard deviation. *, P ≤ 0.001 vs. control.

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To assess its potential clinical utility, we developed several different tumor models in which LSN3154567 was evaluated. By testing in these relevant tumor models, we also hope to identify the specific types of tumor histologies that can be effectively targeted by the molecule, thereby used as a basis for patient selection in the clinic. To this end, we identified and characterized several cancer cell lines low in NAMPT and deficient in NAPRT1 expression, which may represent potential target patient populations (see later): NCI-H1155, HT1080, Namalwa, A2780, and SK-N-SH; and subsequently used these cell lines to establish tumor xenograft models for efficacy studies. Using the 4-day on and 3-day off dosing schedule developed, we showed that LSN3154567 exhibited a rapid dose-dependent inhibition of tumor growth in the NCI-H1155 model (Fig. 4A). At 10 mg/kg (exposure ≈ 2701 nmol/L*hour), it exhibited significant tumor growth inhibition (≈103%) with regression. Similar results were obtained with HT1080 and Namalwa tumor models (Fig. 4B and C). Importantly, this molecule caused a significant amount of tumor regression in the Namalwa and HT1080 tumor models (−55 and −56%), especially when dosed at 10 and 20 mg/kg twice a day, respectively (Fig. 4B and C). These studies have established a PK/PD relationship for LSN3154567. Taken together, these studies show that LSN3154567 exhibits significant efficacy in several tumor models that represent the major types of tumor histologies in the clinic.

Figure 4.

Inhibition of NAMPT by LSN3154567 led to significant tumor growth inhibition in different tumor xenograft models. Animals bearing tumor xenografts derived from NCI-H1155 (A and D–F), Namalwa (B), and HT-1080 (C) were grown and treated with LSN3154567 (Materials and Methods). A, Treated with LSN3154567 (2.5, 5, 10, and 20 mg/kg) twice a day (BID) on a 4-day on and 3-day off schedule for 17 days; B, treated with LSN3154567 (2.5, 5, and 10 mg/kg; BID) on a 4-day on and 3-day off schedule for 17 days; C, treated with LSN3154567 (5, 10, and 20 mg/kg; BID) on a 4-day on and 3-day off schedule for 21 days; D, treated with LSN3154567 (20 mg/kg; BID) and NA (25-400 mg/kg; BID) on a 4-day on and 3-day off for 3 cycles; E, treated with LSN3154567 (20 mg/kg; BID) and NA (75 mg/kg; BID) on a 4-day on and 17-day off (1 cycle) or 4-day on and 3-day off followed by 4-day on 10-day off schedule for 21 days; and F, after the treatment (E), plasma samples were collected at days 1 and 3; and the concentrations of LSN3154567, NA, and NAM were determined as described (Materials and Methods).

Figure 4.

Inhibition of NAMPT by LSN3154567 led to significant tumor growth inhibition in different tumor xenograft models. Animals bearing tumor xenografts derived from NCI-H1155 (A and D–F), Namalwa (B), and HT-1080 (C) were grown and treated with LSN3154567 (Materials and Methods). A, Treated with LSN3154567 (2.5, 5, 10, and 20 mg/kg) twice a day (BID) on a 4-day on and 3-day off schedule for 17 days; B, treated with LSN3154567 (2.5, 5, and 10 mg/kg; BID) on a 4-day on and 3-day off schedule for 17 days; C, treated with LSN3154567 (5, 10, and 20 mg/kg; BID) on a 4-day on and 3-day off schedule for 21 days; D, treated with LSN3154567 (20 mg/kg; BID) and NA (25-400 mg/kg; BID) on a 4-day on and 3-day off for 3 cycles; E, treated with LSN3154567 (20 mg/kg; BID) and NA (75 mg/kg; BID) on a 4-day on and 17-day off (1 cycle) or 4-day on and 3-day off followed by 4-day on 10-day off schedule for 21 days; and F, after the treatment (E), plasma samples were collected at days 1 and 3; and the concentrations of LSN3154567, NA, and NAM were determined as described (Materials and Methods).

Close modal

Given the fact that NAMPT inhibition is associated with the hematological and retinal toxicities, we reasoned that one approach to mitigate these toxicities is to coadminister LSN3154567 with NA. By doing so, NA is only converted to NAD+ in the normal cells, but not in NAPRT1-deficient tumor cells. As a result, NA coadministration should rescue the toxicities associated with NAMPT inhibition in the normal cells, but not tumor efficacy. To test the feasibility of this approach, we first showed that at least 3 μmol/L of NA was required for complete rescue of the antiproliferative activity of LSN3154567 against NAPRT1-proficient cancer cell lines (also see Fig. 2). This suggests that >3 μmol/L of NA may be required to generate a sufficient amount of NAD+ required by the normal cells when the NAMPT-mediated pathway is inhibited. Once entering the circulation, NA can be further metabolized to NAM (2–4). As shown early, LSN3154567 is a competitive inhibitor with respect to NAM. Thus, increasing NAM concentrations could decrease the inhibitory activity of LSN3154567. To this end, we also assessed the effects of NAM on the inhibitory activity of LSN3154567 (Supplementary Table S2). As shown, it would require 325 to 489 μmol/L of NAM to rescue 50% of the inhibitory activity of 0.041 μmol/L of LSN3154567 (Supplementary Table S2). To further confirm these in an in vivo setting, we then carried out an efficacy study in which LSN3154567 was coadministered with NA. As shown in Fig. 4D, when dosed at 25 to 400 mg/kg (BID), neither NA nor NAM derived from NA significantly affected the efficacy of the molecule. The results of this study suggest that NA coadministration is feasible.

Because significant efficacy was obtained using the 4-day on and 3-day off dosing schedule for 3 cycles or 21 days, we wondered whether codosing with NA for just 1 or 2 cycles might be sufficient to achieve robust efficacy. In this study, we treated animals bearing NCI-H1155 tumors with LSN3154567 (20 mg/kg; BID) and NA (75 mg/kg; BID) for 1 cycle (4 day treatment and 17 day off), or 2 cycles (4 day treatment, 3 day off, and then 4 day treatment and 10 day off). As shown in Fig. 4E, codosing the molecule with NA for 2 cycles is sufficient to achieve significant efficacy (tumor growth inhibition: >99%). Under this condition, the exposure of LSN3154567 was estimated to be 3,138 nmol/L*h, whereas the exposures of NA and NAM formed were 5 to 460 and 2 to 30 μmol/L, respectively (Fig. 4F). These results indicate that the concentrations of NA were sufficient to rescue the normal cells from LSN3154567 inhibition, but the concentrations of NAM were not adequate to affect the activity of the molecule.

To further confirm this finding, we carried out an efficacy study in which LSN3154567 was coadministered with NAM (Fig. 5). We showed that when NAM was dosed at 12.5 to 50 mg/kg (exposures ≈ 14–50 μmol/L), it did not affect the efficacy of LSN3154567 (exposures ≈ 0.14–0.23 μmol/L; Fig. 4F). But, when dosed at 100 to 200 mg/kg (exposure ≈ 106–294 μmol/L), it started to attenuate the efficacy of LSN3154567 (exposure ≈ 0.064–0.079 μmol/L) as the tumor growth inhibition was affected (Fig. 5) even though the molecule still maintained ≈70% to 80% of tumor growth inhibition. Thus, to minimize the effects of NAM, its concentrations should not exceed ≈1,700 to 3,700 (106/0.064–294/0.079) times over the exposure of LSN3154567 at which the significant efficacy is observed.

Figure 5.

Effects of NAM on efficacy of LSN3154567 in the tumor model. Animals bearing tumor xenografts derived from NCI-H1155 were grown and treated with LSN3154567 (20 mg/kg; BID) and NAM (12.5-400 mg/kg; BID) on a 4-day on and 3-day off schedule for 3 cycles (Materials and Methods).

Figure 5.

Effects of NAM on efficacy of LSN3154567 in the tumor model. Animals bearing tumor xenografts derived from NCI-H1155 were grown and treated with LSN3154567 (20 mg/kg; BID) and NAM (12.5-400 mg/kg; BID) on a 4-day on and 3-day off schedule for 3 cycles (Materials and Methods).

Close modal

The retinopathy associated with LSN3154567 could be mitigated with NA coadministration

To assess whether LSN3154567 caused retinopathy, we first treated rats with this molecule at 20, 40, and 80 mg/kg for 4 days. To our surprise, no apparent retinopathy was observed. As expected, the hematological toxicities were observed. When the molecule was dosed at 20, 40, and 80 mg/kg, the plasma exposures obtained were 8,974, 18,061, and 38,327 nmol/L*h, respectively. Thus, the molecule exhibits exposure multiples of respective 3-, 7-, and 14-fold over the exposure (2,701 nmol/L*h) required for robust efficacy (≈103%) without NA coadministration. These findings suggest that LSN3154567 is the first NAMPT inhibitor to be reported that does not cause retinopathy in the rat (30, 35).

To further test LSN3154567, we treated dogs with the molecule at 1 and 2.5 mg/kg (Materials and Methods). At these dose levels, the retinal toxicity was observed (Fig. 6 A–F). Degeneration of the outer nuclear layer occurred in all four animals (Fig. 6A–F), but was less pronounced in the animals treated with 1 mg/kg. The retinal change was characterized by disorganization and necrosis of cells in the outer nuclear layer, slight vacuolation of the photoreceptors adjacent to the pigmented epithelium and outer limiting membrane, and loss of photoreceptor detail in affected areas (Fig. 6A–F). At the 1 and 2.5 mg/kg dose levels, the plasma exposures were determined to be 1,483 and 2,468 nmol/L*h, respectively. On the basis of these findings, the molecule appears to cause retinopathy even at an exposure (1,483 nmol/L*h) that is ≈50% of the exposure (2,701 nmol/L*h) required for robust efficacy. This finding suggests that the canine retinal cells might be more sensitive to NAMPT inhibition.

Figure 6.

Administration of LSN3154567 alone, but not coadministered with NA, resulted in histopathological changes in the retina of dogs. Histopathological changes in the retina of dogs treated with 1 mg/kg (A–C); and 2.5 mg/kg (D–F) once daily for 4 days. Disorganization and necrosis of cells in the outer nuclear layer (), slight vacuolation of the photoreceptors adjacent to the pigmented epithelium and outer limiting membrane, and loss of photoreceptor detail in affected areas (). No histopathological changes in the retina of dogs cotreated with LSN3154567 (5 mg/kg) and NA 200 mg/kg BID) for 4 days (G–I).

Figure 6.

Administration of LSN3154567 alone, but not coadministered with NA, resulted in histopathological changes in the retina of dogs. Histopathological changes in the retina of dogs treated with 1 mg/kg (A–C); and 2.5 mg/kg (D–F) once daily for 4 days. Disorganization and necrosis of cells in the outer nuclear layer (), slight vacuolation of the photoreceptors adjacent to the pigmented epithelium and outer limiting membrane, and loss of photoreceptor detail in affected areas (). No histopathological changes in the retina of dogs cotreated with LSN3154567 (5 mg/kg) and NA 200 mg/kg BID) for 4 days (G–I).

Close modal

To test whether NA coadministration could mitigate the retinal toxicity, we treated animals with LSN3154567 at a higher dose (5 mg/kg) along with NA (200 mg/kg). At this dosing regimen, the molecule was well tolerated. Neither retinopathy (Fig. 6G–I) nor hematological toxicities were observed. At this dose level, a plasma exposure of 11,831 nmol/L*h was obtained (Supplementary Fig. S5), thus indicating a margin of safety > ≈4-fold (11,831/3,138 nmol/L*h, an exposure required for robust efficacy (>99% of tumor growth inhibition) when the molecule is coadministered with NA. At this dose level, the molecule has an exposure multiple of ≈8-fold (11,831/1,483 nmol/L*h) over the lowest exposure tested at which retinopathy was observed. Finally, the concentration of LSN3154567 when dosed at 5 mg/kg was determined to be ≈0.49 μmol/L during the study. On the basis of this, it would require on average >830 to 1,800 μmol/L (1,700-3,700 × 0.49) of NAM to affect LSN3154567 activity. When dosed with 100 mg/kg of NA, the maximal plasma concentration (Cmax) of NAM in the dog was determined to be ≈67 to 130 μmol/L. If the dog was dosed with 200 mg/kg of NA, the Cmax of NAM was extrapolated to be ≈134 to 260 μmol/L. Even at this highest level, NAM was not expected to cause any significant rescue of LSN3154567 inhibitory activity. Taken together, these results suggest that NA coadministration improves the retinal and hematological toxicities associated with LSN3154567 without significantly affecting efficacy. Thus, NA coadministration is a viable approach for the development of LSN3154567 in the clinic.

Potential patient stratification strategies

As shown above, NA coadministration is required for the clinical development of LSN3154567. To identify the patients who are most likely to respond to LSN3154567 and NA cotreatment, we tested the molecule against 325 different cancer cell lines cultured in the presence or absence of 10 μmol/L of NA (Materials and Methods). As shown in Fig. 7A, this molecule was active against 219 cell lines (IC50 ≤ 0.25 μmol/L) when cultured in the absence of NA. However, it remained active against 72 cell lines (IC50 ≤ 0.25 μmol/L) when cultured in the presence of NA (Fig. 7A). Thus, approximately 33% of the sensitive cell lines (72/219) deficient in NAPART1 remain sensitive to LSN3154567, representing a potentially large patient population. Based on NA rescue data obtained, relative sensitivity of each cancer type with ≥5 cell lines is shown (Fig. 7B) and the patients with those most sensitive tumors may potentially benefit from the treatment with LSN3154567 (Fig. 7B). Further analysis of >15,000 genes shows that NAPRT1 is the only indicator that is highly associated with the insensitivity of cell lines to the molecule when cultured in the presence of NA (10 μmol/L; Supplementary Fig. S6A). Consistent with this, a number of cell lines with a higher mRNA level of NAPRT1 are often insensitive to LSN3154567 in the presence of NA, indicating that NA rescued these cells from NAMPT inhibition (Supplementary Fig. S6B). However, a number of cell lines with a lower mRNA level of NAPRT1 are often sensitive to LSN3154567 even when supplemented with NA (Supplementary Fig. S6B), indicating that NA could not rescue these NAPRT1-deficient cells from NAMPT inhibition. In addition, we also found that NAMPT levels in different cancer cell lines were inversely correlated with their sensitivity to the NAMPT inhibitor. Taken together, these results indicate that the cancer patients with tumors deficient in NAPRT1 and low in NAMPT expression levels represent the potential patient populations that may benefit from LSN3154567 and NA cotreatment.

Figure 7.

Inhibition of NAMPT by LSN3154567 led to a broad spectrum of antiproliferative activity. Different cancer cells were grown and treated with LSN3154567 at various concentrations (2.0–0.0001 μmol/L) in the presence or absence of NA (10 μmol/L). A, Antiproliferative activity of LSN3154567 against 325 cancer cell lines in the presence of NA (10 μmol/L); B, relative sensitivity of each cancer type with 5 or more cell lines to LSN3154567 in the presence of NA (10 μmol/L). The number above each histogram represents the number of cell lines tested from each cancer type.

Figure 7.

Inhibition of NAMPT by LSN3154567 led to a broad spectrum of antiproliferative activity. Different cancer cells were grown and treated with LSN3154567 at various concentrations (2.0–0.0001 μmol/L) in the presence or absence of NA (10 μmol/L). A, Antiproliferative activity of LSN3154567 against 325 cancer cell lines in the presence of NA (10 μmol/L); B, relative sensitivity of each cancer type with 5 or more cell lines to LSN3154567 in the presence of NA (10 μmol/L). The number above each histogram represents the number of cell lines tested from each cancer type.

Close modal

In this study, we report a unique NAMPT inhibitor. LSN3154567 did not cause retinopathy in the rat. More importantly, coadministration with NA completely rescued its retinal and hematological toxicities in the dog tested, but did not significantly affect the tumor efficacy. As the presence of these on-target toxicities has dampened the pharmaceutical drug discovery and clinical development efforts of NAMPT inhibitors, the discovery of LSN3154567 along with the potential approach to its clinical development may lead to uncovering the therapeutic potential of NAMPT inhibitors in the clinic.

The discovery of this unique NAMPT inhibitor, LSN3154567, was mainly based on the hypothesis that increasing the polarity and decreasing the permeability of NAMPT inhibitors would reduce their ability to cross the blood–retinal barrier, resulting in a lower exposure in the retina. This in turn would reduce the risk of retinal toxicity when coadministered with NA. Our data do not support that the overall distribution of the molecule to the eye was decreased. However, LSN3154567 did not demonstrate retinopathy in the rat, nor in the dog at the highest dose tested when coadministered with NA. The key to the successful discovery of LSN3154567 was the design and implementation of a rigorous testing paradigm for evaluating the retinal toxicity of NAMPT inhibitors in the well-validated animal toxicity models in the absence of NA coadministration. Because there were no suitable in vitro or ex vivo systems for assessing the potential of NAMPT inhibitors for the retinal toxicity, we devised an in vivo testing paradigm enabling us to screen and identify lead molecules with reduced or no retinal toxicity, and also with improved tolerability. This testing paradigm consists of the following: identify the molecules that demonstrate robust in vivo target inhibition activity; screen the molecules (TED90 ≤ 50 mg/kg) at 5- and 20-times of TED90 in a 4-day rat toxicity assay; then evaluate the molecules with reduced or no retinal toxicity in the 4-day rat toxicity assay with or without NA coadministration; and further evaluate these molecules in a 4-day dog toxicity assay with or without NA coadministration. Using this rigorous in vivo screening approach, we evaluated a number of potent molecules, the majority of which exhibited the retinal toxicity, but this extensive screening effort eventually led to the identification of LSN3154567.

As discussed earlier, LSN3154567 has unique drug-like properties. First, this molecule only causes retinopathy in the dog, but not in the rat as compared with other NAMPT inhibitors (30, 34, 35). Second, the retinal toxicity associated with the molecule could be rescued with NA coadministration. Third, this molecule is much better tolerated than the earlier lead molecules. As it stands now, we still do not understand why this molecule has such unique properties, but we do believe that a number of factors might have played a role. For example, LSN3154567 has a much lower permeability (0.31 × 10−4 cm/s) and higher polarity (clogD at pH 7.4:0.61) than the earlier lead molecules. And LSN3154567 is much better tolerated in vivo. Therefore, it is possible that the differences in their physical chemical properties might have affected their exposures in the retina and other tissues.

The findings from this study also have significant clinical implications. First, NA coadministration improved the tolerability of LSN3154567 and rescued the retinal and hematological toxicities in the animals tested. Therefore, NA coadministration will be required for its clinical development. Niacin has been used in the clinic as an antihyperlipidemic agent (39, 40). Clinically, administration of plain NA or niacin, also known as immediate-release (IR), is associated with adverse events such as flushing (39, 40). To reduce the side effects associated with niacin IR formulation and for ease of patience compliance, sustained-release (SR) and extended-release (ER) formulations of niacin were developed (39, 40). Niacin SR formulation does reduce flushing events, but also leads to increased hepatotoxicity. Niacin ER formulation, on the other hand, clinically reduces the flushing events associated with the IR formulation and also avoids the hepatotoxicity associated with the SR formulation. Niacin ER formulation also eases patience compliance. On the basis of this, Niaspan, a niacin ER tablet, appears to be an ideal candidate for coadministration with LSN3154567. For ease of patient compliance, the molecule can also be coencapsulated with Niaspan tablets to insure that LSN3154567 and NA are always administered together. As an oral agent, Niaspan is available in three tablet strengths: 500, 750, and 1,000 mg of NA (39). Our in vitro NA rescue studies suggest that ≥3 μmol/L of NA is required for complete rescue of NAMPT inhibition in cancer cell lines proficient in NAPRT1 (see above). Our in vivo NA rescue studies suggest that 75 mg/kg of NA is sufficient to rescue the normal cells from NAMPT inhibition when dosed twice daily. At this dose, NA was found to be present at 6 to 200 μmol/L (Fig. 4F). It is also known that oral dosing with 2,000 mg of ER niacin as 2 × 1000 Niaspan tablets results in an average concentration of 17.8 μmol/L of NA in 12 hours and a Cmax of 75.5 μmol/L of NAM (41). Assuming a linear relationship between dose and exposure, dosing 500 and 1,000 mg of ER niacin would lead to concentrations of NA ranging from 4.5 to 8.9 μmol/L in 12 hours, which may be sufficient to rescue the normal cells from NAMPT inhibition. On the basis of these, LSN3154567 may be coencapsulated with Niaspan ER 500 or 1,000 tablets when dosed twice daily. Then, to determine the maximum dose to be encapsulated, we based on the PK parameters (predicted median human intravenous clearance CL of 33.8 L/h, volume of distribution of 2.8L/kg, terminal elimination half-life of 4 hours, and human oral bioavailability of 38%) and daily exposure (3,138 nmol/L*h), resulting in >99% of tumor growth inhibition, and predicted the human dose to be 54 mg (15–168 mg) and the maximum absorbable dose to be 206 mg. On the basis of these, we have developed LSN3154567 (170 mg) encapsulated with Niaspan ER 500 tablets for ease of patient compliance.

Finally, the importance of NAMPT-mediated NAD+ biosynthesis in the pathogenesis of diverse tumor types is well documented as NAMPT is overexpressed in many different types of tumors and its expression appears to be associated with cancer progression (12, 13, 16–18). Therefore, the development of cancer therapies targeting the NAMPT-mediated NAD+ biosynthetic pathway represents an attractive strategy and should have broad clinical implications. In this study, we show that LSN3154567 is effective at inhibiting the proliferation of a variety of different cancer cell lines, especially those with reduced NAMPT expression levels and deficient in NAPRT1. In light of these findings, LSN3154567 has the potential to be effective for treating a variety of different types of cancers. In addition, altered metabolite levels in tumors can be used as PD biomarkers for assessing effects of LSN3154567 on its intended target in the clinic. LC-MS methods have been successfully used to detect and quantify many metabolites derived from several metabolic pathways, including glycolytic pathway from fresh, frozen, and formalin-fixed paraffin-embedded human tumors (42, 43). Thus, the LC-MS method developed in this study may be applicable to clinical use. A successful implementation of the patient tailoring strategy and the PD biomarkers may accelerate its clinical development.

G. Zhao is a senior research advisor at Eli Lilly and Company and has ownership interest (including patents) in the same. R.L. Johnson is a research advisor for Eli Lilly and Company and has ownership interest (including patents) in the same. No potential conflicts of interest were disclosed by the other authors.

Conception and design: G. Zhao, C.F. Green, Y.-H. Hui, L. Prieto, R.L. Johnson, W. Wu, M. Del Prado, J.R. Gillig, M.-S. Kuo, S. Geeganage, T.P. Burkholder

Development of methodology: G. Zhao, S. Dong, T. Wang, B. Tan, S. Bhattachar, K.D. Roth, M.-S. Kuo

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G. Zhao, Y.-H. Hui, R. Shepard, S. Dong, T. Wang, B. Tan, L. Kays, R.L. Johnson, M. Del Prado, M.-C. Fernandez, K.D. Roth

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): G. Zhao, C.F. Green, Y.-H. Hui, L. Prieto, R. Shepard, S. Dong, T. Wang, B. Tan, X. Gong, R.L. Johnson, W. Wu, J.R. Gillig, K.D. Roth, S. Buchanan, M.-S. Kuo, T.P. Burkholder

Writing, review, and/or revision of the manuscript: G. Zhao, C.F. Green, Y.-H. Hui, L. Prieto, R. Shepard, S. Dong, B. Tan, R.L. Johnson, K.D. Roth, M.-S. Kuo, S. Geeganage, T.P. Burkholder

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): B. Tan, R.L. Johnson

Study supervision: G. Zhao, R.L. Johnson, W. Wu, S. Buchanan, S. Geeganage

Other (analysis of compound data relating to SAR and biological activity): J.R. Gillig

We thank Yue-wei Qian and He Wang for expression and purification of NAMPT; Yong Wang for high resolution x-ray crystallography and analysis of the structure of LSN3154567 in NAMPT; Yan Zhai and Karen Huss for biochemical assays; Chen Bi for help with the construction of NAPRT1 knockdown and overexpression cell lines; Debbie Young and Michele Smith for help at the early stage of the project; and Gregory D. Plowman for support. This work was supported by Eli Lilly and Company.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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