Tracey C. Fleischer

Chemical Proteomics Identifies Nampt as the Target of CB30865, An Orphan Cytotoxic Compound

Drug discovery based on cellular phenotypes is impeded by the challenge of identifying the molecular target. To alleviate this problem, we developed a chemical proteomic process to identify cellular proteins that bind to small molecules. CB30865 is a potent (subnanomolar) and selective cytotoxic compound of previously unknown mechanism of action. By combining chemical proteomics with biochemical and cellular pharmacology we have determined that CB30865 cytotoxicity is due to subnanomolar inhibition of nicotinamide phosphoribosyltransferase (Nampt), an enzyme present in the NAD biosynthetic pathway. Cancer cells develop dependence on Nampt due to increased energy requirements and the elevated activity of NAD consuming enzymes such as sirtuins and mono and poly(ADP-ribose) polymerases (PARPs). These findings suggest new chemical starting points for Nampt inhibitors and further implicate this enzyme as a target in cancer.

Analogues of 4-[(7-Bromo-2-methyl-4-oxo-3H-quinazolin-6-yl)methylprop-2-ynylamino]-N-(3-pyridylmethyl)benzamide (CB-30865) as Potent Inhibitors of Nicotinamide Phosphoribosyltransferase (Nampt)

We have shown previously that the target of the potent cytotoxic agent 4-[(7-bromo-2-methyl-4-oxo-3H-quinazolin-6-yl)methyl-prop-2-ynylamino]-N-(3-pyridylmethyl)benzamide (CB38065, 1) is nicotinamide phosphoribosyltransferase (Nampt). With its cellular target known we sought to optimize the biochemical and cellular Nampt activity of 1 as well as its cytotoxicity. It was found that a 3-pyridylmethylamide substituent in the A region was critical to cellular Nampt activity and cytotoxicity, although other aromatic substitution did yield compounds with submicromolar enzymatic inhibition. Small unsaturated groups worked best in the D-region of the molecule, with 3,3-dimethylallyl providing optimal potency. The E region required a quinazolin-4-one or 1,2,3-benzotriazin-4-one group for activity, and many substituents were tolerated at C² of the quinazolin-4-one. The best compounds showed subnanomolar inhibition of Nampt and low nanomolar cytotoxicity in cellular assays.

Development and validation of a spontaneous preterm delivery predictor in asymptomatic women.

BACKGROUND Preterm delivery remains the leading cause of perinatal mortality. Risk factors and biomarkers have traditionally failed to identify the majority of preterm deliveries. OBJECTIVE To develop and validate a mass spectrometry-based serum test to predict spontaneous preterm delivery in asymptomatic pregnant women. STUDY DESIGN A total of 5501 pregnant women were enrolled between 17(0/7) and 28(6/7) weeks gestational age in the prospective Proteomic Assessment of Preterm Risk study at 11 sites in the United States between 2011 and 2013. Maternal blood was collected at enrollment and outcomes collected following delivery. Maternal serum was processed by a proteomic workflow, and proteins were quantified by multiple reaction monitoring mass spectrometry. The discovery and verification process identified 2 serum proteins, insulin-like growth factor-binding protein 4 (IBP4) and sex hormone-binding globulin (SHBG), as predictors of spontaneous preterm delivery. We evaluated a predictor using the log ratio of the measures of IBP4 and SHBG (IBP4/SHBG) in a clinical validation study to classify spontaneous preterm delivery cases (<37(0/7) weeks gestational age) in a nested case-control cohort different from subjects used in discovery and verification. Strict blinding and independent statistical analyses were employed. RESULTS The predictor had an area under the receiver operating characteristic curve value of 0.75 and sensitivity and specificity of 0.75 and 0.74, respectively. The IBP4/SHBG predictor at this sensitivity and specificity had an odds ratio of 5.04 for spontaneous preterm delivery. Accuracy of the IBP4/SHBG predictor increased using earlier case-vs-control gestational age cutoffs (eg, <35(0/7) vs ≥35(0/7) weeks gestational age). Importantly, higher-risk subjects defined by the IBP4/SHBG predictor score generally gave birth earlier than lower-risk subjects. CONCLUSION A serum-based molecular predictor identifies asymptomatic pregnant women at risk of spontaneous preterm delivery, which may provide utility in identifying women at risk at an early stage of pregnancy to allow for clinical intervention. This early detection would guide enhanced levels of care and accelerate development of clinical strategies to prevent preterm delivery.

Abstract 3526: Coadministration of nicotinic acid with the Nampt inhibitor MPC-9528 enhances antitumor activity in Naprt deficient cancer cells in culture and in xenografts

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: The tumoricidal small molecule MPC-9528 is a picomolar inhibitor of nicotinamide phosphoribosyltransferase (Nampt). Nampt catalyzes the first and rate-limiting step in NAD synthesis from nicotinamide. Nicotinic acid phosphoribosyltransferase (Naprt) catalyzes the first and rate-limiting step in an alternate pathway of NAD synthesis from nicotinic acid (NA). Cancer cells are particularly dependent on NAD and many cancer cell lines, but not most normal tissues, are deficient in Naprt activity. Therefore administration of NA could prevent MPC-9528-induced NAD depletion in normal tissues, but not in Naprt-deficient tumors, resulting in greater therapeutic index and efficacy. Methods: Cell viability was determined based on ATP levels. Naprt protein expresson was quantified by western blot and qRT-PCR. NAD was acid-extracted from cells and quantified by a coupled reaction based on fluorescent resorufin. Xenograft studies were performed in nu/nu mice. Results: In 44 out of 153 cancer cell lines surveyed, NA did not prevent MPC-9528-induced cell death, which correlated with low to undetectable levels of Naprt. MPC-9528-induced NAD depletion and cell death in HCT116 colon carcinoma cells were prevented by the addition of NA, consistent with high Naprt expression. A single dose of MPC-9528 at the maximum-tolerated dose (MTD) of 75 mg/kg caused tumor regression in HCT116 xenografts and NA coadministration completely blocked this effect. NA also completely blocked mortality in mice induced by 300 mg/kg MPC-9528, consistent with the finding that most mouse tissues have high Naprt expression. In Naprt-deficient MIA PaCa-2 xenografts, NA coadministration allowed tolerance of 200 mg/kg MPC-9528 with a substantially increased anti-tumor response relative to the MTD of 75 mg/kg MPC-9528 alone. Conclusions: Low Naprt expression correlates with the lack of effect of NA on MPC-9528 tumoricidal activity. Because Naprt deficiency is prevalent in cancer cell lines and in primary tumor specimens, but not in normal tissues, NA coadministration with MPC-9528 should increase the tolerability and efficacy of MPC-9528 in patients with Naprt-deficient tumors. A companion diagnostic designed to measure Naprt expression or activity in tumors could be used to identify tumors that would most likely benefit from such combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3526. doi:10.1158/1538-7445.AM2011-3526

Abstract 2551: The Nampt inhibitor MPC-9528 synergizes with DNA damaging agents

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: MPC-9528 reduces cellular NAD levels and causes cell death, by blocking the NAD salvage pathway through inhibition of nicotinamide phosphoribosyltransferase (Nampt). Many DNA damaging agents also reduce cellular NAD levels, by activating the NAD consuming enzyme poly(ADP-ribose) polymerase (Parp). We hypothesized that the combination of a Nampt inhibitor and a DNA damaging agent would synergize in killing cancer cells, due to a combined effect on NAD levels through two independent mechanisms. Methods: Cellular NAD was measured using a coupled enzymatic assay. Drug combination experiments were performed in HCT116 colon carcinoma cells, using measurement of ATP levels as a cell viability endpoint. Synergy, antagonism, or additivity was assessed using the MacSynergy II program. Results: In HCT116 cells, saturating doses of MPC-9528 induced depletion of NAD with a half-life of 5 hours and a decrease in ATP that was delayed approximately 14 hours relative to NAD. Lower, sublethal concentrations of MPC-9528 induced partial NAD depletion without a concomitant ATP loss. At these sublethal concentrations, MPC-9528 was found to synergize with the DNA alkylating agents temozolomide and streptozotocin, which are known to activate Parp. Additionally, MPC-9528 was found to synergize with two structurally different thymidylate synthase inhibitors, 5-fluorouracil (5-FU) and raltitrexed, neither of which have been reported to activate Parp. Individually, 5-FU and raltitrexed each caused NAD depletion in HCT116 cells, which was enhanced by combination with MPC-9528. Furthermore, both 5-FU- and raltitrexed-mediated NAD depletion and synergy with MPC-9528 were completely blocked by the Parp inhibitor olaparib. Conclusions: Parp activation induced by the alkylating agents temozolomide and streptozotocin, or by the thymidylate synthase inhibitors 5-FU and raltitrexed, is the basis for tumoricidal synergy with the Nampt inhibitor MPC-9528. This synergy is a direct consequence of the NAD depletion resulting from Parp activation coupled with the inhibition of NAD synthesis due to Nampt inhibition. These results provide a basis for clinical combination of MPC-9528 with the agents studied here or with related agents that induce Parp activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2551. doi:10.1158/1538-7445.AM2011-2551

Abstract 577: Basal NAD levels and Nampt expression correlate with in vitro and in vivo sensitivity of tumor cell lines to the Nampt inhibitor MPC-9528

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: MPC-9528 is a potent and selective inhibitor of the NAD biosynthetic enzyme nicotinamide phosphoribosyltransferase (Nampt). Inhibition of Nampt by MPC-9528 causes depletion of cellular NAD followed by a decrease in ATP and cell death. Cancer cells develop dependence on Nampt due to increased metabolic demands and the elevated activity of enzymes such as poly(ADP-ribose) polymerases (Parps) that consume NAD. MPC-9528 has shown anti-tumor activity, ranging from no response to complete regression in a variety of xenograft models. Materials and Methods: In vitro Nampt activity and cellular NAD levels were measured in coupled biochemical reactions. Cellular Parp activity was measured by immunofluorescent detection of poly(ADP-ribose) (PAR). Enzyme protein and mRNA levels were quantified by western blot and qRT-PCR, respectively. Mechanism of cell death was determined by Caspase 3/7 activity, Caspase 3 and Parp1 cleavage, and SytoxGreen staining. Cell viability was based on ATP levels. Xenografts were performed in nu/nu mice. Results: MPC-9528 inhibited Nampt activity in vitro with an average IC50 of 40 pM and suppressed cellular NAD levels and nuclear PAR levels, with potencies of 170 pM and 120 pM, respectively. In a screen of 93 cancer cell lines of diverse origin, MPC-9528 had a median TC50 of 2.8 nM with a range of 100 pM to 62 nM. Similar to cultured cells, a range of tumor responses was observed in six different xenograft models. In HCT116 colon carcinoma and HT1080 fibrosarcoma xenografts, oral administration of MPC-9528 at 75 mg/kg intermittently resulted in tumor regressions. In contrast, similar treatment of MIA PaCa-2 pancreatic cancer, N87 gastric carcinoma or HCC827 and NCI-H460 lung cancer xenografts led to partial tumor growth inhibition or no response. The effects in xenografts correlated with TC50 values for MPC-9528 for these cell lines in culture, which ranged from 260 pM to 24 nM. The TC50 values also correlated well with basal cellular NAD levels, IC50 values for MPC-9528-induced NAD depletion, and Nampt protein expression, but not with expression of three other enzymes involved in NAD metabolism – Naprt, Qprt or Parp1. The mechanism of cell death induced by MPC-9528 was cell type dependent and did not correlate with MPC-9528 potency in culture. Conclusions: NAD levels in cancer cell lines are primarily dependent upon the Nampt pathway. The differential sensitivity of tumor cells to the Nampt inhibitor MPC-9528 is likely due to the magnitude of NAD production, which is a function of Nampt expression. MPC-9528 has the greatest effect on tumor cell lines with lower Nampt expression; therefore, a companion diagnostic based upon Nampt expression in primary tumor specimens could be used to select patients most likely to respond to MPC-9528 monotherapy in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 577. doi:10.1158/1538-7445.AM2011-577

Abstract LB-393: The cancer metabolism inhibitor MPC-9528 induces tumor regression in xenograft models with multiple dosing schedules by causing rapid and sustained reduction in tumor NAD

Introduction : MPC-9528 is a potent, selective, orally bioavailable inhibitor of nicotinamide phosphoribosyltransferase (Nampt), which catalyzes the rate-limiting step for NAD biosynthesis from nicotinamide. Inhibition of Nampt by MPC-9528 results in cell death as a consequence of NAD depletion and inhibition of ATP synthesis. MPC-9528 is a potent tumoricidal agent of cancer cell lines of diverse origin and shows anti-tumor activity in multiple xenograft models. Here we explore the determinants of efficacy in xenograft models by comparing the effects of MPC-9528 oral dosing schedules in terms of both tumor NAD depletion and survival. Methods : HT1080 human fibrosarcoma cells were implanted subcutaneously into athymic mice (nu/nu) for tumor studies. Dosing was initiated when median tumor volume was >100 mm 3 . MPC-9528 was dosed orally, either once weekly, once daily, or twice daily, for a total of two or three weeks. For pharmacodynamic (PD) studies, animals were dosed with MPC-9528 and tumors were collected at various times post-dosing for NAD determination by LC-MS/MS. Results : MPC-9528 (75 mg/kg) dosed once weekly for three weeks in a HT1080 xenograft model resulted in 75% tumor regression on study Day 23, 8 days after the last dose. The ED 50 for anti-tumor activity with this schedule was 44 mg/kg and doses at or below 35 mg/kg showed no anti-tumor activity. All doses were equally well-tolerated with Conclusions : MPC-9528 administration results in regression of tumors when plasma concentrations of compound are maintained above a threshold. MPC-9528 shows comparable efficacy when given intermittently, on a weekly schedule, or continuously on a once, or twice daily schedule. This unique attribute of MPC-9528 reflects its mechanism of action as a cell metabolism inhibitor that functions by inhibiting NAD synthesis in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-393. doi:10.1158/1538-7445.AM2011-LB-393

Abstract LB-288: MPI-0486348: A novel inhibitor of nicotinamide phosphoribosyltransferase (Nampt) induces tumor regression in a preclinical model

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first step in the recycling of nicotinamide into NAD. Cancer cells develop dependence on Nampt due to increased energy requirements and the elevated activity of NAD consuming enzymes such as sirtuins and mono and poly(ADP-ribose) polymerases (PARPs). Identification of Nampt as the target of an orphan cytotoxic compound using chemical proteomics spawned a Nampt drug discovery program at Myriad. We have developed a potent and selective inhibitor of Nampt that inhibits the growth of cancer cells in culture and induces tumor regression in a mouse xenograft model. Materials and Methods: In vitro Nampt activity was measured in a coupled biochemical assay based on the production of fluorescent resorufin. Viability was determined by measuring cellular ATP levels at 72 hrs following addition of compound. Nampt cellular activity was assayed by measuring levels of NAD and NAD-dependent poly (ADP)-ribosylation (PAR). Pharmacokinetic studies were conducted in female CD-1 mice. Anti-tumor activity was evaluated in an HCT116 human colon carcinoma xenograft in nu/nu mice. Results: MPI-0486348 inhibited Nampt activity in vitro with an average IC50 value of 40 pM. Depending upon the time of compound exposure, MPI-0486348 showed TC50 values of 180 pM to 20 nM in HCT116 cells. MPI-0486348 treatment also reduced cellular NAD levels and nuclear PAR levels, with IC50 values of 170 pM and 120 pM, respectively. These cellular effects of MPI-0486348 were prevented by coadministration of nicotinic acid, demonstrating on target mechanism of action. In mouse pharmacokinetic studies, absolute oral bioavailability of 47% was determined using 40% Captisol as the vehicle at an oral dose of 10 mg/kg. The same dose in rats gave an absolute oral bioavailability of 46%. Finally, in HCT116 tumor-bearing mice, a single oral dose of MPI-0486348 (50 or 75 mg/kg) resulted in 31% and 67% regression, respectively. Conclusions : MPI-0486348 is an orally bioavailable small molecule inhibitor of Nampt and shows promising preclinical results in a human xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-288.

Abstract 3673: Identifying the target of an orphan compound: CB30865 is a Nampt inhibitor

Background: Drug discovery based on cellular phenotypes is impeded by the challenge of identifying the molecular target. To alleviate this problem, Myriad Pharmaceuticals has developed a chemical proteomics process to identify cellular proteins that bind to small molecules. CB30865 is a potent and selective cytotoxic compound of previously unknown mechanism of action (1). Using our technology, we have unequivocally demonstrated that CB30865 cytotoxicity is due to inhibition of nicotinamide phosphoribosyltransferase (Nampt). Nampt catalyzes the first step in the synthesis of NAD from nicotinamide. Cancer cells develop dependence on Nampt due to increased energy requirements and the elevated activity of NAD consuming enzymes such as sirtuins and mono and poly(ADP-ribose) polymerases. Materials and Methods: Direct target affinity purification (DTAP) was performed using analogs of CB30865 (MPI-0479883, MPI-0482594) coupled to beads. Purified proteins were identified by nano-flow reversed-phase liquid chromatography and tandem mass spectrometry using an LTQ-Orbitrap. In vitro Nampt activity was measured in a coupled biochemical assay based on the production of fluorescent resorufin. Nampt cellular activity was assayed by measuring levels of NAD and NAD-dependent polyADP-ribosylation. Results: As reported (1), 3-pyridyl, but not 2-pyridyl, analogs of CB30865 were potently cytotoxic. A 55 kD protein was purified efficiently using DTAP with MPI-0479883 (a 3-pyridyl CB30856 analog) but not MPI-0482594 (a 2-pyridyl analog). Preincubation of cellular lysates with uncoupled 3-pyridyl but not 2-pyridyl analogs potently blocked binding of the 55 kD protein. Based on LC-MS/MS analyses, the 55 kD protein was identified as Nampt. Immunoblotting of DTAPs using a Nampt specific antibody confirmed the MS identification. Furthermore, biochemical and cellular assays of Nampt activity demonstrated pyridyl-isomer specific enzyme inhibition. Finally, cellular Nampt inhibition and cytotoxicity were completely rescued by treating cells with 10 µM nicotinic acid as an alternate NAD source. Conclusions: The chemical proteomics DTAP approach is a powerful tool to identify molecular targets of orphan compounds. We demonstrate this here through identification of Nampt as the molecular target of CB30865 and further implicate NAD metabolism as a functional target for cancer. (1) Skelton LA, Ormerod MG, Titley J, Kimbell R, Brunton LA, Jackman AL. A novel class of lipophilic quinazoline-based folic acid analogues: cytotoxic agents with a folate-independent locus. Br J Cancer. 1999 Apr;79(11-12):1692-701. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3673.