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  • In conclusion we have conducted a


    In conclusion, we have conducted a structure-based virtual screen of a novel non-active site pocket of TS-DHFR. Our screen identified Fmoc-Val-OPfp , which inhibits DHFR by 52%, displays mixed noncompetitive inhibition with respect to dihydrofolate, and is not competitive with respect to NADPH. Furthermore, we conducted an SAR study utilizing derivatives of compound which led to the development of covalent compounds designed to target Cys44. Compound demonstrated time-dependent inhibition through covalent interaction via a disulfide bond and selectively inhibits DHFR, while mutation of Cys44 interferes with binding of with the non-active site pocket. Work is currently underway to obtain co-crystal structures in order to validate our modeling and obtain a better understanding of the interactions between compound and the proposed TS-DHFR non-active site pocket. The discovery of offers an excellent starting point for further lead optimization of covalent inhibitors to the DHFR allosteric site. Acknowledgments This work is supported by NIH Grant (AI083146) to K.S.A., and Training Grant (5T32AI007404-23) to D.J.C.
    Introduction According to WHO 2017 report, tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, ranking above HIV/AIDS. In 2016, there were an estimated 1.3 million TB deaths among HIV-negative people and an additional 374,000 deaths among HIV-positive people. Mycobacterium tuberculosis (Mtb), the causative agent of TB in humans, is a slow-growing acid-fast bacterium with a highly impermeable cell wall. Mtb is an opportunistic pathogen that is able to survive within macrophages in a latent form for decades and reactivates in immune compromised individuals such as those with a concurrent HIV infection.1, 2 The spread of multidrug-resistant (MDR) TB and the emergence of extensively drug-resistant (XDR) TB lead to revitalization of antitubercular drug discovery efforts.3, 4, 5 The discovery of bedaquiline (inhibitor of mitochondrial ATP synthase) and delamanid (inhibitor of mycolic acid biosynthesis) after a gap of more than 40 years offered some relief in treatment of MDR-TB but the two agents have certain pronounced issues like hERG toxicity and ADME issues.3, 6, 7 Also, very few chemical entities (TBA- 354 (nitroimidazole), PBTZ169 (benzothiazinone), and Q203 (imidazopyridine) are in phase I clinical studies. Therefore, there is a significant unmet medical need for safer, more effective TB drugs with different mechanisms. The folate pathway plays an essential role in cell survival by generating 5, 10-methylene tetrahydrofolate as a one-carbon donor for the synthesis of deoxythymidine monophosphate (dTMP), purines, methionine and histidine. Disruption of this pathway leads to the critical deficiency of these key molecules, impaired DNA replication and ultimately cell death. Although DHFR is a validated drug target for bacterial and protozal infections, it is not currently invoked for TB therapy. Therefore, designing antifolate compounds that inhibit Mtb-DHFR activity and also the growth of Mtb may be a promising strategy for TB drug discovery and development.10, 11, 12 Methotrexate (MTX), pyrimethamine, and trimetrexate, clinically approved antifolates, are potent inhibitors of the Mtb-DHFR but they fail to inhibit the growth of Mtb, most likely due to an inability to permeate the lipid-rich cell wall. Compounds that have been reported as Mtb-DHFR inhibitors majorly belong to the triazine or pyrimidine class and are shown in Fig 114, 15, 16, 17, 18, 19.