Pancreatic cancer is reported to be dependent on NAD salvage pathway for its growth and survival. Nicotinamide phosphoribosyl transferase (NAMPT), an enzyme that catalyzes the rate limiting step of NAD biosynthesis is over expressed in a number of cancers. Inhibition of NAMPT with first generation inhibitors has been demonstrated to result in anti-tumor efficacy in preclinical models. Clinical development of first generation NAMPT inhibitors has been hindered because of their poor pharmacological profile, high cytochrome inhibition and possibly mechanism-based toxicities. Therefore, our objective was to develop NAMPT inhibitors with the “best-in-class” profile with strategies for overcoming mechanism-based toxicities. Utilizing structure-guided drug design including determination of co-crystal structures and SAR-based approaches, we have identified a novel chemical series of inhibitors of NAMPT. Optimization of the series for transient target inhibition as a result of reduced binding strength coupled with desirable pharmacokinetic profile to minimize mechanism based toxicity resulted in identification of AU-4869 as the lead compound. AU-4869 showed potent cross-species activity and reduced strength of binding in comparison with first generation NAMPT inhibitors. Anti-proliferative activity of AU-4869 correlated well with NAD depletion in a pancreatic cancer cell line. The anti-proliferative activities were rescued in NAPRT-proficient cell lines with the addition of nicotinic acid due to the NAMPT independent salvage pathway for biosynthesis of NAD, confirming the mechanism of action through NAD depletion. AU-4869 exhibited desirable drug-like properties including solubility, permeability, metabolic stability, lack of CYP & hERG inhibition and pharmacokinetic exposure upon oral dosing. At well-tolerated doses, AU-4869 exhibited superior efficacy at MTD doses in mice xenograft models as compared to first generation inhibitors. Addition of nicotinic acid improved the tolerability of AU-4869 and reversed the effects of mechanism based toxicity in Rodents. Anti-tumor activities of AU-4869 in the presence of nicotinic acid in NAPRT-deficient pancreatic cancer models are currently being evaluated in preclinical models. Citation Format: Dinesh Chikanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Narasimha Rao, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra, Shekar Chelur, Chetan Pandit, Murali Ramachandra. Novel NAMPT inhibitors for the treatment of Pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A72.

KRAS is the frequently mutated isoform in RAS driven cancers. The G12C mutation is more predominantly associated with various tumor types over other changes in K-Ras. Although direct targeting of RAS is very challenging, it is possible to selectively target G12C mutant K-Ras using a covalent approach. Mutant specific covalent inhibitors with high selectivity against wild type K-Ras and other GTPases are expected to lead to efficacy with a very high degree of tolerability. Here, we report identification of lead compounds from two distinct chemical series that selectively target K-Ras G12C. Molecular modeling based on the reported crystal structures aided in the identification of these compounds. Covalent binding of the lead compounds to K-Ras G12C was demonstrated by MALDI-TOF. Lead compounds were potent in selectively inhibiting proliferation of cell lines with K-Ras G12C mutation but not with wild type K-Ras. The anti-proliferative activity of the lead compounds correlated well with their potency in a cellular mechanistic assay. Lead compounds from both series exhibited excellent drug-like properties including solubility, metabolic stability, permeability lack of CYP inhibition and desired exposure in pharmacokinetic studies. In a xenograft model of NSCLC, the lead compound demonstrated dose-dependent tumor growth inhibition with excellent tolerability upon oral dosing. In summary, we have identified a novel, potent and selective K-Ras G12C inhibitor with optimized drug-like properties including oral bioavailability and efficacy in a NSCLC derived xenograft model. Toxicity evaluation is ongoing towards progressing the lead compound to the clinic. Citation Format: Leena Khare Satyam, Dinesh Chikkanna, Aswani K. G, Vinayak V. Khairnar, Sreekanth Reddy, Vakkapatla Durgaprasad, Kowju Radhakrishna, Sunil K. Panigrahi, Anuradha Ramanathan, Kumari Mahasweta, Anirudha Lakshminarasimhan, Narasimha R. K, Vinutha R, Sreevalsam Gopinath, Suryakant Kumar, Mubarak H. Shah, Raghuveer Ramachandra, Kiran A. B, Chetan Pandit, Murali Ramachandra. Identification of novel covalent inhibitors of K-Ras G12C that are efficacious in a xenograft model of NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 339.

Dysregulated fatty acid metabolism is thought to be a hallmark of cancer, wherein fatty acids function both as an energy source and as signals for enzymatic and transcriptional networks contributing to malignancy. Fatty acid-binding protein 5 (FABP5) is an intracellular protein that facilitates transport of fatty acids and plays a role in regulating the expression of genes associated with cancer progression such as cell growth, survival, and metastasis. Overexpression of FABP5 has been reported to contribute to an aggressive phenotype and a poor survival correlation in several cancers. Therefore, inhibition of FABP5 is considered as a therapeutic approach for cancers. Phenotypic screening of a library of covalent compounds for selective sensitivity of cancer cells followed by medicinal chemistry optimization resulted in the identification of AUR104 with desirable properties. Chemoproteomic-based target deconvolution revealed FABP5 as the cellular target of AUR104. Covalent adduct formation with Cys43 of FABP5 by AUR104 was confirmed by mass spectrometry. Target occupancy studies using a biotin-tagged AUR104 demonstrated potent covalent binding to FABP5 in both cell-free and cellular conditions. Ligand displacement assay with a fluorescent fatty acid probe confirmed the competitive binding mode of AUR104 with fatty acids. Binding at the fatty acid site and covalent bond formation with Cys43 were also demonstrated by crystallography. Furthermore, AUR104 showed a high degree of selectivity against a broad safety pharmacology panel of enzymes and receptors. AUR104 exhibited potent anti-proliferative activity in a large panel of cell lines derived from both hematological and solid cancers with a high degree of selectivity over normal cells. Anti-proliferative activity in lymphoma cell lines correlated with inhibition of MALT1 pathway activity, cleavage of RelB/Bcl10 and secretion of cytokines, IL-10 and IL-6. AUR104 displayed desirable drug-like properties and dose-dependent oral exposure in pharmacokinetic studies. Oral dosing with AUR104 resulted in dose-dependent anti-tumor activity in DLBCL (OCI-LY10) and NSCLC (NCI-H1975) xenograft models. In a repeated dose MTD studies in rodents and non-rodents, AUR104 showed good tolerability with an exposure multiple of >500 over cellular EC50 for up to 8 hours. In summary, we have identified a novel covalent FABP5 inhibitor with optimized properties that showed anti-tumor activity in in vitro and in vivo models with acceptable safety profile. The data presented here strongly support clinical development of AUR104. Citation Format: Dinesh Chikkanna, Leena Khare Satyam, Sunil Kumar Pnaigrahi, Vinayak Khairnar, Manoj Pothuganti, Lakshmi Narayan Kaza, Narasimha Raju Kalidindi, Vijaya Shankar Nataraj, Aditya Kiran Gatta, Narasimha Rao Krishnamurthy, Sandeep Patil, DS Samiulla, Kiran Aithal, Vijay Kamal Ahuja, Nirbhay Kumar Tiwari, KB Charamannna, Pravin Pise, Thomas Anthony, Kavitha Nellore, Sanjeev Giri, Shekar Chelur, Susanta Samajdar, Murali Ramachandra. Discovery and preclinical evaluation of a novel covalent inhibitor of FABP5 for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1266.

Nicotinamide phosphoribosyl transferase (NAMPT) is the enzyme that catalyzes the rate limiting step in the salvage pathway of Nicotinamide Adenine Dinucleotide (NAD) biosynthesis. NAMPT is reported to be overexpressed in a number of cancer and inflammatory indications. Because of the requirement of NAD for a number of key biochemical pathways, inhibition of NAMPT has been shown to result in antitumor efficacy in preclinical models. NAMPT inhibitors have also been reported to enhance sensitivity to a number of targeted agents and overcome resistance to available therapies such as bortezomib resistance in multiple myeloma. In view of the sub-optimal profile of the reported first generation NAMPT inhibitors with respect to pharmacokinetics and drug-drug interaction, we sought to develop NAMPT inhibitors with the "best-in-class" profile for overcoming mechanism-based toxicities and/or resistance to current therapies. Utilizing structure-guided drug design and SAR-based approaches, we have optimized two chemical series of inhibitors of NAMPT. Determination of co-crystal structures with several de novo designed hits greatly aided in the identification of lead compounds that exhibited potent inhibition of NAMPT. Lead compounds were highly active in inhibiting proliferation that correlated well with cellular NAD depletion of cell lines derived from multiple myeloma, prostate and breast cancers. The anti-proliferative activities were rescued in NAPRT- proficient cell lines with the addition of NA due to the NAMPT independent salvage pathway for biosynthesis of NAD, confirming the mechanism of action through NAD depletion. Lead compounds exhibited desirable drug-like properties including solubility, permeability, metabolic stability, lack of CYP inhibition and pharmacokinetic exposure. In a xenograft model of pancreatic cancer, treatment with lead compounds resulted in regression of tumors with no signs of toxicity. Recent reports demonstrating the overexpression of NAMPT in bortezomib-resistant cells and a synergistic efficacy with a combination of NAMPT inhibitor and bortezomib against the resistant cells prompted us to evaluate the combination with our optimized leads. The combination of lead NAMPT inhibitors with bortezomib showed synergistic killing of cultured multiple myeloma cell lines. Evaluation of efficacy of the lead compounds as a single agent or in combination with bortezomib in xenograft models of multiple myeloma is currently underway. Modulation of NAD levels at lower doses of NAMPT inhibitors in combination with bortezomib could overcome the limitations such as mechanism-based toxicity and/or resistance of both these therapies and provide an effective treatment option for multiple myeloma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B210. Citation Format: Dinesh Chikkanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Aparna Satyanandan, Narasimha Rao, S Karthikeyan, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra, Hosahalli Subramanya, Chetan Pandit, Murali Ramachandra. Novel inhibitors of nicotinamide phosphoribosyl transferase and their evaluation in combination with bortezomib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B210.

Nicotinamide phosphoribosyl transferase (NAMPT) is the enzyme that catalyzes the rate limiting step in the salvage pathway of Nicotinamide Adenine Dinucleotide (NAD) biosynthesis. NAMPT is reported to be overexpressed in a number of cancer and inflammatory indications. Because of the requirement of NAD as a co-factor or substrate for a number of key biochemical pathways including those catalyzed by PARP1, Sirtuins and ADP-ribosyl cyclase, inhibition of NAMPT has been shown to result in anti-tumor efficacy in preclinical models. Two NAMPT Inhibitors FK866/APO866 and GMX1778 are currently in clinical trials for oncology indications. In the presence of these clinical agents, cultured cell lines show development of resistance due to mutations underscoring the potential need for inhibitors from distinct chemical series. Here, we report a structure-guided drug design based approach for identification of lead compounds from two chemical series selectively targeting NAMPT. Determination of co-crystal structures with several de novo designed hits greatly aided in the identification of lead compounds that exhibited potent inhibition of NAMPT against both wild type and resistance mutants (G217R and H191R) Lead compounds were highly active in inhibiting proliferation that correlated well with cellular NAD depletion in several cancer cell lines. Normal cells and selected cancer cell lines have an NAMPT independent salvage pathway for biosynthesis of NAD, which is dependent on nicotinic acid phosphoribosyltransferase (NAPRT) and Nicotinic acid (NA). The anti-proliferative activities were fully rescued in NAPRT- proficient cell lines with the addition of NA, confirming the mechanism of action through specific NAD depletion. Lead compounds from both series exhibited excellent drug-like properties including solubility, metabolic stability and permeability, and desired exposure in pharmacokinetic studies. Anti-tumor activities of these compounds including NA rescue in NAPRT-proficient tumor models are currently being evaluated in preclinical models. Citation Format: Murali Ramachandra, Chetan Pandit, Hosahalli Subramanya, Dinesh Chikkanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Aparna Satyanandan, Narasimha Rao, Arnab Bera, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra. Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5389. doi:10.1158/1538-7445.AM2013-5389