Structural basis for inhibition of Mycobacterium tuberculosis α-methylacyl-CoA racemase by 2-arylthiopropanoyl-CoA inhibitor analogs

α-Methylacyl-CoA racemase (AMACR; P504S) is a pivotal enzyme involved in the β-oxidation of branched-chain fatty acids and bile acid intermediates, catalyzing the conversion between (2R)- and (2S)-2-methylacyl-CoA thioester epimers. The AMACR reaction enables downstream catabolism of these thioesters via stereospecific enzymes within the β-oxidation pathway. The AMACR homolog in Mycobacterium tuberculosis (MCR) has emerged as a tractable model for dissecting the mechanistic underpinnings of the racemization reaction and presents a promising therapeutic target given the pathogen's dependence on lipid metabolism for persistence and virulence. Previously, we reported the detailed molecular structure of wild-type MCR and in complex with a diverse set of acyl-CoA substrates. They revealed conserved active site residues that mediate substrate anchoring and epimerization and highlighted distinct molecular interactions that confer selectivity toward 2-methyl-branched substrates. Complementing these results, in this report, we present high-resolution structures for 2-arylthiopropanoyl-CoA inhibitors in complex with MCR and a comprehensive set of enzyme inhibition assays to delineate structure-activity relationships and probe competitive binding modes. Our findings underscore the importance of inhibitor side-chain branching and CoA anchoring in modulating enzymatic turnover and inhibition. Together, these data enhance our understanding of the racemization mechanism of MCR and establish a structural foundation for the rational design of selective inhibitors. Targeting MCR could represent a novel future therapeutic strategy for M. tuberculosis based on impairing cholesterol utilization.