Crystal structure of Mycobacterium tuberculosis mycothione reductase in complex with a specific inhibitor reveals competition with NADP (H) binding

Tuberculosis (TB) remains a major health threat, while the increasing occurrence of drug‐resistant strains underscores the need for new antitubercular drugs. A promising strategy to combat TB is based on disrupting the mycobacterial redox homeostasis by inhibiting an NADPH‐dependent oxidoreductase, mycothione reductase (Mtr). Using high‐throughput screening, we recently identified potent and selective Mtr inhibitors. Here we report high‐resolution X‐ray structures of Mtr from Mycobacterium tuberculosis and Mycobacterium xenopi, including the M. tuberculosis enzyme complexed with a novel inhibitor, Respiri‐1093. Our findings demonstrate that Respiri‐1093 competes with the NADP(H) binding rather than mycothione binding. Analysis of the binding site explains the observed selectivity of the inhibitor towards the M. tuberculosis enzyme. These results provide a structural basis for rational drug design.