Selective Inhibition Mechanism of <i>Mycobacterium tuberculosis</i> Tryptophan-tRNA Synthetase by Chuangxinmycin

Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (Mtb), represents a global health challenge, necessitating new treatments with distinct mechanisms of action (MOA) to combat drug resistance. Chuangxinmycin (CM), characterized by its indole-dihydrothiopyran heterocyclic skeleton, exhibits potent antibacterial activity both <i>in vitro</i> and <i>in vivo</i>, with a minimum inhibitory concentration (MIC) of 0.25 μg/mL against Mtb. However, the MOA of CM against Mtb has remained obscure. Through comprehensive genetic, chemical rescue, and protein-drug interaction studies, coupled with biochemical analyses, we reveal that CM selectively binds and inhibits tryptophanyl-tRNA synthetase (TrpRS) encoded by <i>trpS</i>, rather than anthranilate synthase (TrpE). Overexpression of <i>trpS</i> in Mtb results in a 128-fold increase in the MIC of CM, indicating a fundamental cause of resistance, whereas overexpression of <i>trpE</i> leads to modest resistance, suggesting a secondary effect. Conversely, knockdown of <i>trpS</i> or <i>trpE</i> enhances the susceptibility of Mtb to CM. Meanwhile, promoters of <i>trpS</i> in CM-resistant Mtb mutants exhibit increased activity compared to the wild type. Furthermore, drug-protein interaction and biochemical assays have confirmed that while CM effectively inhibits TrpRS, mutants of TrpE show decreased affinity for tryptophan. These results establish that CM exerts its anti-Mtb effects by interfering with the tryptophan-tRNA linkage essential for protein synthesis.