Questiomycin A demonstrates antibacterial activity against Mycobacterium tuberculosis by directly targeting FabD

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health threat, particularly due to the rise of multidrug-resistant TB (MDR-TB). Current treatment limitations highlight the urgent need for new therapeutics to improve efficacy and shorten therapy duration. Through high-throughput screening, we identified Questiomycin A (QM), a phenoxazinone compound with promising anti-tuberculosis activity against drug-sensitive and drug-resistant Mtb strains. Transcriptomic profiling indicated disruption of cell wall-associated pathways, while thermal proteome profiling showed that QM binding substantially increased the thermal stability of FabD (malonyl CoA-acyl carrier protein transacylase, MCAT), a key enzyme in mycolic acid biosynthesis. Surface plasmon resonance, enzymatic assays, and genetic manipulation confirmed QM directly binds to and inhibits FabD. This inhibition damages cell wall integrity, increases permeability, disturbs intracellular pH homeostasis, and collapses the proton motive force, ultimately leading to bacterial death. Pharmacokinetic evaluation in six-week-old male Balb/c mice following a single oral dose demonstrates rapid absorption but limited drug exposure. Together, these findings reveal that QM targets FabD to compromise cell wall homeostasis in Mtb, providing a foundation for developing novel anti-tubercular agents targeting FabD.