Selective Inhibition Mechanism of Tryptophan-tRNA Synthetase by Chuangxinmycin.

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