| dc.description.abstract | Isocitrate lyase 1 & 2 are essential enzymes for Mycobacterium tuberculosis pathogenicity and antibiotic resistance, and thereby, are appealing targets for the treatment of latent tuberculosis. Since high-throughput screening campaigns were unsuccessful in identifying suitable inhibitors, these enzymes present an opportunistic venture for a rational approach. Understanding the catalytic mechanism would assist in new strategies for inhibiting the isocitrate lyases (ICLs). The retro-aldol catalytic cleavage and the nucleophilic active-site cysteine inspired an adaptation of isocitrate analogs as ICL mechanism-based inactivators. Finally, a mechanistic comprehension of ICL inhibition by known inactivators and substrate analogs imparts essential structural-activity features of potential inhibitors. This disseration assembled kinetic data and mutagenesis studies to explain the catalytic mechanism of ICL in depth. Besides the recapitulation on discussion of M. tuberculosis ICL1 rate limiting step(s), Lys189 was identified as a potential catalytic base involving the first deprotonation step during isocitrate cleavage. Hence, Lys189, along with Cys191, comprises another susceptible nucleophilic active-site residue for development of novel inactivators. On the other hand, characterization of 2-vinyl-(2R,3S)-isocitrate revealed the first mechanism-based inactivator of ICLs. Catalysis of 2-vinyl-(2R,3S)-isocitrate unmasks 2- vinyl-glyoxylate which then forms a covalent Michael adduct with the conserved cysteine. However, the maximal inactivation rate is slow (kvinact = 0.08 min^-1) and 2-vinyl-glyoxylate is susceptible to reaction with thiols. Hence, substrate analogs were evaluated to find a more suitable scaffold for the next generation of ICL inactivators. Cis-di-carboxylate compounds were found to be better replacements for succinate than their trans-isomers, prompting the investigation of cis-epoxy-succinate as a potential ICL inactivator. Cis-epoxy-succinate, a succinate analog, rapidly inactivates both ICLs via formation of thioether adducts with active-site conserved cysteines. In comparison with 2-vinyl- (2R,3S)-isocitrate, ICL1 inactivation efficiency (vkinact/vKinact) is improved by 750-fold. Exogenous thiols did not interfere with the inactivation. Neither did cis-epoxy-succinate show any inhibition effects on all eight of the tricarboxylic cycle enzymes tested. Cis-epoxysuccinate also demonstrated anti-mycobacteria effects (ICv50 = 100 µM) under conditions in which ICL activity is indispensable. Differential gel electrophoresis of E.coli lysate, pretreated with and without cis-epoxy-succinate, asserted that cis-epoxy-succinate has a notable selectivity for ICL. | |
