Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

Significance The tricarboxylic acid (TCA) cycle plays a central role in the metabolism of many pathogens, but few inhibitors of this pathway currently exist. Our study describes the first small molecule inhibitor, to our knowledge, of the fumarate hydratase enzyme in the TCA cycle of Mycobacterium tuberculosis. We also report the discovery of an allosteric regulatory site that confers selectivity of the inhibitor’s activity over the homologous human enzyme. Our findings illustrate the potential to target vulnerable metabolic enzymes effectively and selectively, despite their high degree of evolutionary conservation. Enzymes in essential metabolic pathways are attractive targets for the treatment of bacterial diseases, but in many cases, the presence of homologous human enzymes makes them impractical candidates for drug development. Fumarate hydratase, an essential enzyme in the tricarboxylic acid (TCA) cycle, has been identified as one such potential therapeutic target in tuberculosis. We report the discovery of the first small molecule inhibitor, to our knowledge, of the Mycobacterium tuberculosis fumarate hydratase. A crystal structure at 2.0-Å resolution of the compound in complex with the protein establishes the existence of a previously unidentified allosteric regulatory site. This allosteric site allows for selective inhibition with respect to the homologous human enzyme. We observe a unique binding mode in which two inhibitor molecules interact within the allosteric site, driving significant conformational changes that preclude simultaneous substrate and inhibitor binding. Our results demonstrate the selective inhibition of a highly conserved metabolic enzyme that contains identical active site residues in both the host and the pathogen.