Multi-drug resistant tuberculosis (MDR-TB) is an emerging infectious disease threat classified as a category C priority pathogen by NIH. We are studying two enzymes from the MTB FAS-II pathway: InhA, the enoyl reductase and KasA, the ketoacyl synthase. Currently, a series of compounds have been designed and synthesized that are nanomolar inhibitors of the M. tuberculosis (MTB) enoyl reductase enzyme. These lead compounds inhibit the growth of both sensitive and drug resistant MTB strains with similar antibacterial potency (1-2 mg/mL), consistent with the hypothesis that compounds that do not require activation by the mycobacterial KatG enzyme will be active against isoniazid-resistant clinical strains. A current goal of this project is to optimize the existing lead compounds to the point at which they can be used in preclinical trials for the treatment of patients infected with drug resistant TB. The proposed research includes the following specific aims. In related studies, we are also investigating protein-protein interactions between components of the FAS-II pathway using two-hybrid methods, analytical ultracentrifugation and surface plasmon resonance (BiaCore). We hypothesize that interactions between components of the pathway modulate the inhibition of enzymes such as InhA by the front-line anti-TB drug isoniazid. Finally, we are also studying the interaction of InhA with the endogenous AcpM substrate. KasA is also a target for drug discovery and in this project we are using inter-ligand NOE NMR spectroscopy to identify lead compounds for enzyme inhibition. This project requires expression of KasA in M. smegmatis as well as the synthesis and purification of acyl-AcpM substrates for enzyme kinetic studies. |
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