Protective mechanism of action of the antifungal drug naftifine against <i>Mycobacterium abscessus</i> infection

<i>Mycobacterium abscessus,</i> a rapidly growing nontuberculous mycobacterium, causes chronic pulmonary infections that are difficult to treat due to extensive intrinsic drug resistance. Through high-content screening of 786 FDA-approved drugs against intracellular <i>M. abscessus</i> in human THP-1 macrophages, we identified naftifine, an antifungal allylamine, as a novel antimycobacterial agent with dual-acting therapeutic mechanisms. Naftifine demonstrated potent activity against reference strains and multidrug-resistant clinical isolates. It showed enhanced efficacy in intracellular environments compared to axenic culture, indicating significant host-directed effects. Mechanistic investigations revealed that naftifine operates through a unique dual mechanism. It directly targets bacteria by inhibiting MmpL3 (MAB_4508), the essential mycolic acid transporter, and modulates host immunity through autophagy activation via the mTOR pathway suppression. Whole-genome sequencing of spontaneous naftifine-resistant mutants identified point mutations (S302T and V299G) in MmpL3. Complementation studies confirmed MmpL3 as the primary molecular target. Cross-resistance analysis with other MmpL3 inhibitors (BM212 and AU1235) validated this target identification. Notably, naftifine represents the first MmpL3 inhibitor demonstrated to induce autophagy, distinguishing it from other MmpL3-targeting compounds. Naftifine-induced autophagy enhanced macrophage-mediated bacterial clearance and reduced infection-associated necrosis, improving host cell survival. <i>In vivo</i> studies demonstrated a significant reduction of pulmonary and splenic bacterial burden with reduced lung inflammation. Furthermore, naftifine exhibited synergistic activity with β-lactam antibiotics without antagonizing other clinically used antibiotics. This is the first report demonstrating the unique combination of MmpL3 inhibition and autophagy induction by a single compound against <i>M. abscessus</i>, establishing naftifine as a promising dual-action therapeutic candidate for treating multidrug-resistant infections.