Sivapriya Kirubakaran

Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics

Background: Several new ATP-competitive mTOR inhibitors have been described, but their kinome-wide selectivity profiles have not been disclosed. Results: Four different profiling technologies revealed a different spectrum of targets for four recently described mTOR inhibitors. Conclusion: Diverse heterocyclic mTOR inhibitors have unique pharmacology. Significance: Profiling data guide choices of mTOR inhibitors for particular applications and provide new potential targets for medicinal chemistry efforts. An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC50 = 2 nm) relative to other inhibitors. In vitro biochemical profiling at 10 μm revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μm but did show that PP242 efficiently inhibited the RET receptor (EC50, 42 nm) and JAK1/2/3 kinases (EC50, 780 nm). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μm and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

Design, Synthesis, and Docking Studies of New Torin2 Analogs as Potential ATR/mTOR Kinase Inhibitors

Targeting DNA damage and response (DDR) pathway has become an attractive approach in cancer therapy. The key mediators involved in this pathway are ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia-mutated, Rad3-related kinase (ATR). These kinases induce cell cycle arrest in response to chemo- and radio-therapy and facilitate DNA repair via their major downstream targets. Targeting ATP-binding site of these kinases is currently under study. Torin2 is a second generation ATP competitive mTOR kinase inhibitor (EC50 = 250 pmol/L) with better pharmacokinetic profile. Torin2 also exhibits potent biochemical and cellular activity against ATM (EC50 = 28 nmol/L) and ATR (EC50 = 35 nmol/L) kinases. In this study, eight new Torin2 analogs were designed and synthesized through multistep synthesis. All the synthesized compounds were characterized by NMR and mass analysis. The newly synthesized analogs were evaluated for their anti-cancer activity via CellTiter-Glo® assay. Additionally, compounds 13 and 14 also showed significant inhibition for ATR and mTOR substrates, i.e., p-Chk1 Ser 317 and p70 S6K Thr 389, respectively. Compounds 13 and 14 displayed promising anti-cancer activity with HCT-116 cell lines in the preliminary study. Further, a comparative model of ATR kinase was generated using the SWISS-MODEL server and validated using PROCHECK, ProSA analysis. Synthesized compounds were docked into the ATP-binding site to understand the binding modes and for the rational design of new inhibitors.

Effect of co-crystallization on physico-chemical properties of Gefitinib

Well-controlled crystallization of the API’s (Active Pharmaceutical Ingredients) is often an important factor in pharmaceutical industries. The development of new crystallization methods to design the products with specific physico-chemical properties is a complex and challenging issue. This existing challenge impetus us to explore the possible ways to control the crystallization of gefitinib. Gefitinib, [N-(3-chloro-4-fluro-phenyl)-7-methoxy-6-(3-morpholin-4-yl propoxy) quinazolin-4amine] is an anticancer drug used in the treatment of non-small cell lung cancer. Gefitinib exists in five polymorphic forms (i) anhydrous (ii) solvate of MeOH (iii) solvate of DMSO, (iv) monohydrate and (v) trihydrate forms. Each polymorph has different physical properties and leads to large variation in the biopharmaceutical performance. The bioavailability of pure gefitinib is low and there is a need to enhance the solubility. With this regard, our aim is to isolate the more specific crystalline polymorph of gefitinib and to enhance the solubility of the preferred form. Here we present, the effect of selective amino acids used for co-crystallization with gefitinib and the results will be discussed in detail. [1] Pao ,W. et al. Proc. Natl. Acad. Sci. U.S.A.101 (36): 13306–11, 2004. [2] Sordella, R. et al. Science. 305 (5687): 1163–7, 2004. [3].Thorat, S.H. et al. CrystEngComm, 16, 8638-8641, 2014.