Mode of Action Study of Para-aminosalicylic Acid and Structure, Function and Inhibitor Study of the Isocitrate Dehydrogenase-2 in Mycobacterium tuberculosis

Tuberculosis (TB) killed 1.5 million people and rivaled AIDS, becoming the leading cause of death from infectious disease in 2014. The prevalence of multidrug resistant TB has intensified the current therapeutic procedure, making it urgent to find novel anti-tubercular agents and to come up with solutions to retard the emergence of the drug resistance. This dissertation focuses on the identification of drug targets, the exploration of drug resistance mechanisms, and the identification of novel inhibitors. In the first part, the mechanism of action of the classic anti-tubercular drug, para-aminosalicylic acid (PAS), was explored through genetic, cell viability and molecular modeling studies. Dihydrofolate reductase (DHFR) was identified to be the putative intracellular target of PAS. In addition, the molecular mechanism of PAS resistance was intensively investigated for the clinically relevant Rv2671 up-regulation mutant. Biochemical assays showed that Rv2671 exhibited a low DHFR activity with a high Km for the substrate, 7, 8-dihydrofolate. X-ray crystal structure of the Rv2671 in complex with NADP+ and tetrahydrofolate (THF) further confirmed the structural similarity between Rv2671 and DHFR. These studies together suggested that PAS resistance of this mutant is derived from the ability to complement the DHFR activity with the high level of Rv2671. The second part of this dissertation details the characteristics of Mycobacterium tuberculosis isocitrate dehydrogenase-2 (Mtb IDH2). The kinetic study of Mtb IDH2 suggested that it catalyzes an ordered sequential reaction by binding NADP+ first. X-ray crystal structure revealed the fairly conserved active site and dissimilar overall structure compared to human IDHs (HIDHs), suggesting a potential for drug selectivity. A screening of known inhibitors of mutant HIDHs and a high-throughput screening of Mtb whole cell active compounds were further implemented to identify inhibitors for Mtb IDH2. Two compounds from the screenings exhibited IC50s below 10 μM. The enzyme structure and the modest potency inhibitors of Mtb IDH2 can serve as viable starting points for the follow-up inhibitor development of Mtb IDH2