Drug repurposing: Antimicrobial and antibiofilm effects of MLS0315771 against Gram-positive bacteria

The escalating prevalence of multidrug-resistant (MDR) and biofilm-forming Staphylococcus aureus has created an urgent demand for alternative therapeutic strategies beyond conventional antibiotics. In this study, we report for the first time the potent antibacterial and antibiofilm activities of MLS0315771, a competitive phosphomannose isomerase (MPI) inhibitor, against a broad range of Gram-positive bacteria. MLS0315771 exhibited strong inhibitory and bactericidal effects against both MSSA and MRSA, as well as Enterococcus faecium strains resistant to linezolid, with MIC₅₀/₉₀ values of 6.25/6.25 μM. This compound also significantly delayed bacterial proliferation at subinhibitory concentrations and eradicated mature biofilms in vitro. Time-kill assays revealed that MLS0315771 displayed time-dependent bactericidal activity comparable or superior to vancomycin. Proteomic profiling of S. aureus exposed to MLS0315771 and functional assays confirmed that MLS0315771 disrupts bacterial membrane integrity and induces depolarization, thereby impairing essential membrane functions. Checkerboard analyses further indicated that unsaturated long-chain fatty acids antagonized its antibacterial activity, supporting a membrane-targeting antibacterial mechanism. Moreover, MLS0315771 displayed low hemolytic activity and minimal cytotoxicity toward mammalian cells, suggesting a favorable therapeutic index. Collectively, these findings demonstrate that MLS0315771 acts as a novel membrane-targeting antibacterial agent with potent activity against MDR and biofilm-forming Gram-positive pathogens, highlighting its potential as a promising lead compound for future antimicrobial development.