Discovery and Optimization of Phenoxazinone Derivatives as Potent Antitubercular Agents Targeting FabD Protein.

Tuberculosis (TB) therapy demands novel agents with distinct structures, targets, and mechanisms. Herein, through phenotypic screening, we identified a phenoxazinone scaffold questiomycin A (QA) with potent activity against Mycobacterium tuberculosis (MTB) H37Rv strain (MIC: 0.41 μg/mL) and broad-spectrum activity against drug-resistant clinical isolates (MIC: 0.16-0.31 μg/mL). Structural optimization of QA yielded lead B10 with enhanced activity against drug-resistance MTB (MIC: 0.063-0.25 μg/mL) and good intracellular antimycobacterial activity. In addition, B10 showed improved in vitro and in vivo safety, optimized pharmacokinetic profiles (t1/2, 5.34 h; Cmax, 229.97 ng/mL; AUC0-24 h, 738.12 ng·h/mL). Preliminary mechanistic investigations revealed that B10 disrupts mycobacterial envelope integrity by inhibiting Malonyl CoA-acyl carrier protein transacylase (encoded by FabD gene), an unexploited target in the essential mycolic acid biosynthesis pathway. This work establishes QA derivatives as first-in-class fresh scaffolds against FabD as a potential target for MDR-TB therapy, offering an effectively strategy to discover novel anti-TB lead compounds.