Hosahalli Subramanya

Benzimidazole derivatives as potential dual inhibitors for PARP-1 and DHODH.

Poly (ADP-ribose) polymerases (PARPs) play diverse roles in various cellular processes that involve DNA repair and programmed cell death. Amongst these polymerases is PARP-1 which is the key DNA damage-sensing enzyme that acts as an initiator for the DNA repair mechanism. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the pyrimidine biosynthetic pathway which is an important target for anti-hyperproliferative and anti-inflammatory drug design. Since these enzymes share a common role in the DNA replication and repair mechanisms, it may be beneficial to target both PARP-1 and DHODH in attempts to design new anti-cancer agents. Benzimidazole derivatives have shown a wide variety of pharmacological activities including PARP and DHODH inhibition. We hereby report the design, synthesis and bioactivities of a series of benzimidazole derivatives as inhibitors of both the PARP-1 and DHODH enzymes.

Discovery of Pyridyl Bis(oxy)dibenzimidamide Derivatives as Selective Matriptase Inhibitors

Matriptase belongs to trypsin-like serine proteases involved in matrix remodeling/degradation, growth regulation, survival, motility, and cell morphogenesis. Herein, we report a structure-based approach, which led to the discovery of sulfonamide and amide derivatives of pyridyl bis(oxy)benzamidine as potent and selective matriptase inhibitors. Co-crystal structures of selected compounds in complex with matriptase supported compound designing. Additionally, WaterMap analyses indicated the possibility of occupying a distinct pocket within the catalytic domain, exploration of which resulted in >100-fold improvement in potency. Co-crystal structure of 10 with matriptase revealed critical interactions leading to potent target inhibition and selectivity against other serine proteases.

Discovery of azetidine based ene-amides as potent bacterial enoyl ACP reductase (FabI) inhibitors

A novel and potent series of ene-amides featuring azetidines has been developed as FabI inhibitors active against drug resistant Gram-positive pathogens particularly staphylococcal organisms. Most of the compounds from the series possessed excellent biochemical inhibition of Staphylococcus aureus FabI enzyme and whole cell activity against clinically relevant MRSA, MSSA and MRSE organisms which are responsible for significant morbidity and mortality in community as well as hospital settings. The binding mode of one of the leads, AEA16, in Escherichia coli FabI enzyme was determined unambiguously using X-ray crystallography. The lead compounds displayed good metabolic stability in mice liver microsomes and pharmacokinetic profile in mice. The in vivo efficacy of lead AEA16 has been demonstrated in a lethal murine systemic infection model.

Structure-guided discovery of 1,3,5 tri-substituted benzenes as potent and selective matriptase inhibitors exhibiting in vivo antitumor efficacy.

Matriptase is a serine protease implicated in cancer invasion and metastasis. Expression of matriptase is frequently dysregulated in human cancers and matriptase has been reported to activate latent growth factors such as hepatocyte growth factor/scatter factor, and proteases such as urokinase plasminogen activator suggesting that matriptase inhibitors could have therapeutic potential in treatment of cancer. Here we report a structure-based approach which led to the discovery of selective and potent matriptase inhibitors with benzene as central core having 1,3,5 tri-substitution pattern. X-ray crystallography of one of the potent analogs in complex with matriptase revealed strong hydrogen bonding and salt-bridge interactions in the S1 pocket, as well as strong CH-π contacts between the P2/P4 cyclohexyl and Trp215 side-chain. An additional interaction of the pendant amine at cyclohexyl with Gln175 side-chain results in substantial improvement in matriptase inhibition and selectivity against other related serine proteases. Compounds 15 and 26 showed tumor growth inhibition in a subcutaneous DU-145 prostate cancer mouse model. These compounds could be useful as tools to further explore the biology of matriptase as a drug target.

Discovery of O-(3-carbamimidoylphenyl)-l-serine amides as matriptase inhibitors using a fragment-linking approach

Matriptase is a cell-surface trypsin-like serine protease of epithelial origin, which cleaves and activates proteins including hepatocyte growth factor/scatter factor and proteases such as uPA, which are involved in the progression of various cancers. Here we report a fragment-linking approach, which led to the discovery of O-(3-carbamimidoylphenyl)-l-serine amides as potent matriptase inhibitors. The co-crystal structure of one of the potent inhibitors, 6 in complex with matriptase catalytic domain validated the working hypothesis guiding the development of this congeneric series and revealed the structural basis for matriptase inhibition. Replacement of a naphthyl group in 6 with 2,4,6-tri-isopropyl phenyl resulted in 10 with improved matriptase inhibition, which exhibited significant primary tumor growth inhibition in a mouse model of prostate cancer. Compounds such as 10, identified using a fragment-linking approach, can be explored further to understand the role of matriptase as a drug target in cancer and inflammation.

AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei--Crystal structure, mode of action, and biological activity.

Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram‐negative bacterium. Current therapeutic options are largely limited to trimethoprim‐sulfamethoxazole and β‐lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl‐ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl‐ACP reductase present in B. pseudomallei. In this study, we evaluated AFN‐1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN‐1252 stabilized BpmFabI and inhibited the enzyme activity with an IC50 of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X‐ray structure of BpmFabI with AFN‐1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN‐1252 formed a symmetrical tetrameric structure with one molecule of AFN‐1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN‐1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors.

Dihydroorotate dehydrogenase Inhibitors Target c-Myc and Arrest Melanoma, Myeloma and Lymphoma cells at S-phase

Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. Inhibition of this enzyme impedes cancer cell proliferation but the exact mechanisms of action of these inhibitors in cancer cells are poorly understood. In this study, we showed that cancer cells, namely melanoma, myeloma and lymphoma overexpressed DHODH protein and treatment with A771726 and Brequinar sodium resulted in cell cycle arrest at S-phase. Transfection with DHODH shRNA depleted DHODH protein expression and impeded the proliferation of melanoma cells. shRNA knockdown of DHODH in combination with DHODH inhibitors further reduced the cancer cell proliferation, suggesting that knockdown of DHODH had sensitized the cells to DHODH inhibitors. Cell cycle regulatory proteins, c-Myc and its transcriptional target, p21 were found down- and up-regulated, respectively, following treatment with DHODH inhibitors in melanoma, myeloma and lymphoma cells. Interestingly, knockdown of DHODH by shRNA had also similarly affected the expression of c-Myc and p21 proteins. Our findings suggest that DHODH inhibitors induce cell cycle arrest in cancer cells via additional DHODH-independent pathway that is associated with p21 up-regulation and c-Myc down-regulation. Hence, DHODH inhibitors can be explored as potential therapeutic agents in cancer therapy.

Abstract B100: ASN003, a unique B-RAF inhibitor with additional selective activity against PI3K and mTOR kinases, shows strong antitumor activity in multiple xenograft models

Various genes in RAS-RAF and PI3K pathways are frequently mutated in a wide variety of solid tumors. Concurrent double mutations are also observed quite often in a broad range of tumor types. Combined inhibition of both pathways has been shown to impart greater efficacy in multiple tumor models in animals. In order to simultaneously block these pathways, we have discovered and characterized several molecules showing dual inhibition of the RAS-RAF and PI3K pathways. ASN003 has been identified as a lead compound with potent and highly selective inhibitory activity against B-RAF, PI3K and mTOR kinases (low nM IC50). Within the PI3K family, ASN003 has high selectivity for inhibition of PI3Kα and PI3Kδ over PI3Kβ. In cell-based mechanistic studies, ASN003 did not increase phospho-ERK levels in the KRAS mutant cell line HCT116 and thus may not cause paradoxical activation of RAS/MAPK pathway in tumors. Consistent with its dual inhibition profile, ASN003 showed strong antiproliferative activity in cell lines with B-RAF and PI3K pathway mutations as well as vemurafenib (B-RAF inhibitor)-resistant cell lines. ASN003 suppressed the phosphorylation of downstream targets of B-RAF, PI3K and mTOR in pharmacodynamic studies in multiple tumor models, indicating appropriate target engagement. In in vivo efficacy studies, ASN003 showed regression in a B-RAFV600E mutant A375 xenograft model and also caused significant tumor growth inhibition in RKO and A2058 tumor models, which harbor mutations in PIK3CA or PTEN genes, respectively, in addition to the B-RAF V600E mutation. Dual targeting of the B-RAF and PI3K pathways with ASN003 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. Due to the lack of paradoxical activation, treatment with ASN003 may not cause skin toxicities seen with pure B-RAF inhibitors. ASN003 is currently in preclinical development and is expected to enter Phase I/II clinical trials by early 2016. Citation Format: Scott K. Thompson, Mahaboobi Jaleel, Vijay Kumar Nyavanandi, Murali Ramachandra, Hosahalli Subramanya, Aravind Basavaraju, Vaibhav Sihorkar, Roger A. Smith, Niranjan Rao, Sandeep Gupta, Sanjeeva P. Reddy. ASN003, a unique B-RAF inhibitor with additional selective activity against PI3K and mTOR kinases, shows strong antitumor activity in multiple xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B100.

Abstract B274: Discovery and characterization of a highly selective inhibitor of B-RAF, PI3K, and mTOR kinases with antitumor activity in B-RAF and B-RAF/PI3K pathway double mutant xenograft models.

The RAS-RAF-MAPK and PI3K pathways are two major signaling pathways involved in the initiation and progression of a broad range of tumors. These two pathways are frequently activated in the majority of solid tumors through mutations in various components of the pathways. Inhibition of one pathway often leads to the activation of the other pathway. Preclinical studies have shown that simultaneous inhibition of these two pathways leads to greater efficacy in a broader range of tumor types. For example, despite the presence of the B-RAFV600E mutation, colorectal cancer tumors do not respond to B-RAF selective inhibitors. In contrast, the combination of B-RAF or MEK inhibitors with PI3K pathway inhibitors shows significant tumor growth inhibition in colorectal cancer xenograft models in mice. Currently, there are several clinical trials ongoing using combinations of inhibitors targeting both pathways. We employed a rational drug design approach to discover and develop a single compound with dual inhibition of both RAS-RAF-MAPK and PI3K pathways. EN3352 was identified as a lead compound with potent inhibitory activity against B-RAF, PI3K and mTOR kinases, with low nanomolar IC50 values. Profiling of EN3352 in a panel of 292 kinases showed that it is highly selective for these three kinases. Within the PI3K family, EN3352 is selective for inhibition of PI3Kα versus PI3Kβ. EN3352 showed broader anti-proliferative activity in tumor cell lines compared to the B-Raf selective inhibitors, vemurafenib and dabrafenib. EN3352 has good oral bioavailability in preclinical species and showed inhibition of phosphorylation of the downstream targets of B-RAF, PI3K and mTOR in various tumor models in mice. EN3352 showed regression in a B-RAFV600E mutant A375 xenograft model and also showed significant tumor growth inhibition in a RKO model, which harbors mutations in both B-RAF and PIK3CA genes. Dual targeting of the B-RAF and PI3K pathways with EN3352 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. (Disclosure: Funding for this research was provided by Endo Pharmaceuticals Inc.) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B274. Citation Format: Sanjeeva P. Reddy, Mahaboobi Jaleel, Vijay K. Nyavanandi, Murali Ramachandra, Hosahalli Subramanya, Aravind Basavaraju, Vaibhav Sihorkar, Roger A. Smith, Sandeep Gupta, Scott K. Thompson. Discovery and characterization of a highly selective inhibitor of B-RAF, PI3K, and mTOR kinases with antitumor activity in B-RAF and B-RAF/PI3K pathway double mutant xenograft models. [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 B274.

Abstract 5389: Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT).

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