| dc.description.abstract | As the world population battles drug-resistant tuberculosis (TB), there is an urgent need for novel anti-tubercular drugs. This dissertation documents the studies of glyoxylate shunt enzymes, isocitrate lyase (ICL) and malate synthase (GlcB), in Mycobacterium tuberculosis (Mtb) as drug targets for the therapeutic development of TB. Two different drug discovery approaches were used. A mechanism based approach was utilized for isocitrate lyase, while a fragment based approach was applied for malate synthase, and both approaches employed X-ray crystallography as a primary technique. Through the mechanism based approach, an ICL inhibitor complexed crystal structure was solved to 2.6 Å resolution after the treatment with itaconate. From the structure, the active site cysteine (Cys191) underwent covalent modification to form an S-methylsuccinyl adduct. The inhibitory mechanism was based on the direct nucleophilic attack on the itaconate vinyl group by Cys191 after activation via a nearby general base. Additional crystal structure of ICL following the inactivation by 2-vinyl isocitrate (2-VIC) at 1.8 Å resolution confirmed the formation of an S-homopyruvoyl adduct of Cys191. The structure was consistent with the proposed inhibitory mechanism where 2-VIC first bound in the active site in the same manner as the substrate isocitrate. A base catalyzed aldo cleavage of the C2-C3 bond of 2-VIC then produced 2-vinyl glyoxylate and the aci-succinate. Cys191 was deprotonated to generate succinate, as in the lyase mechanism, followed by the Michael addition of Cys191 thiolate to 2-vinyl glyoxylate to form the final S-homopyruoyl adduct.
A fragment based approach was used to advance drug discovery and further probe the active site of Mtb GlcB. Two libraries of 1580 fragments were screened against GlcB using differential scanning fluorimetry (DSF) to identify binding hits, and 18 complexed crystal structures were solved at 1.9-2.5 Å resolutions. The fragment bound GlcB crystal structures captured the conformations of the active site, which have not been reported for Mtb GlcB. The movements of two loops around the active site gave rise to a second portal to the surface and the narrowing of the active site tunnel. This series of conformational changes was hypothesized as a pathway for substrate-product exchange. The structures of the enzyme at various stages of product formation and dissociation, as well as an apo enzyme structure, were further elucidated to confirm the hypothesis. As a result, a detailed, mechanism driven substrate-product exchange in catalysis was formulated. One novel interaction from the fragments and the enzyme was further incorporated into the existing phenyl-diketo acid (PDKA) inhibitor, providing new drug designs. The resulting lead molecule was 100 times more potent compared to the parent PDKA, and was shown to make the same interaction and induce the same movement in the active site as the original fragment. The comprehensive knowledge from the structural studies of the two glyoxylate shunt enzymes provided new information that could lead to a greater understanding of Mtb’s physiology and guide the discovery of more effective treatments of TB. | en |
