Selective Inhibition Mechanism of Mycobacterium tuberculosis Tryptophan-tRNA Synthetase by Chuangxinmycin

Tuberculosis (TB), caused by Mycobacterium tuberculosis (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 in vitro and in vivo, 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 trpS, rather than anthranilate synthase (TrpE). Overexpression of trpS in Mtb results in a 128-fold increase in the MIC of CM, indicating a fundamental cause of resistance, whereas overexpression of trpE leads to modest resistance, suggesting a secondary effect. Conversely, knockdown of trpS or trpE enhances the susceptibility of Mtb to CM. Meanwhile, promoters of trpS 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.