Structural Superimposition

Abstact

Tuberculosis remains the leading cause of death from a single infectious agent, and rising multi-drug resistance in Mycobacterium tuberculosis (Mtb) underscores the urgent need for new antibiotics. Here, we characterize BVL3572S, a hydroxamic acid-containing compound that is bactericidal against Mtb. The primary targets of BVL3572S are the pyridoxal phosphate (PLP; active form of vitamin B6)-dependent aminotransferases HisC (Rv1600) and AlaA (Rv0337c; formerly AspC), simultaneously impacting L-histidine and L-alanine biosynthesis. X-ray crystallography revealed a covalent PLP-BVL3572S adduct within the HisC active site. The formation of the adduct followed by its release suggests a futile cycle that depletes PLP. Consistently, isotopic labeling revealed widespread perturbation of amino acid biosynthesis. CRISPRi and Tn-seq analyses additionally indicated disruptions in central metabolism, cell envelope integrity, and redox balance. BVL3572S displayed strong synergy with the antitubercular drug D-cycloserine. Collectively, our findings establish BVL3572S as a promising lead compound acting through a previously unexploited, multitarget mechanism.