Covalent Inactivation of Isocitrate Lyase by -2,3-Epoxy-Succinic Acid.

The isocitrate lyases (ICL1/2) are essential enzymes of (), the causative agent of tuberculosis. At present, no ICL1/2 inhibitors have progressed to clinical evaluation, despite extensive drug discovery efforts. Herein, we surveyed succinate analogs against ICL1 and found that dicarboxylic acids constrained in their conformations, such as maleic acid, comprise uncompetitive inhibitors of ICL1 and inhibit more potently than their -isomers. From this, we identified -2,3 epoxysuccinic acid (-EpS) as a selective, irreversible covalent inactivator of ICL1 (/= (5.0 ± 1.4) × 10 M s; = 200 ± 50 nM), the most potent inactivator of ICL1 yet characterized. Crystallographic and mass spectrometric analysis demonstrated that Cys of ICL1 was S-malylated by -EpS, and a crystallographic "snapshot" of inactivation lent insight into the chemical mechanism of this inactivation. Proteomic analysis of lysates showed that -EpS selectively labeled plasmid-expressed ICL1. Consistently, -EpS, but not its -isomer, inhibited the growth of under conditions in which ICL function is essential. These findings encourage the development of analogs of -2,3-epoxysuccinate as antituberculosis agents.