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 notable antibacterial and antibiofilm activities of MLS0315771, a competitive phosphomannose isomerase (MPI) inhibitor, against a broad range of Gram-positive bacteria. MLS0315771 exhibited notable inhibitory and bactericidal effects against Staphylococcus aureus and Enterococcus faecium. The MIC₅₀/MIC₉₀ values were 25/25 µM for MSSA, 12.5/12.5 µM for MRSA, and 6.25/6.25 µM for linezolid-resistant E. faecium. This compound also significantly delayed bacterial proliferation at subinhibitory concentrations and reduced 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 moderate, cell line-dependent cytotoxicity toward mammalian cells, suggesting a limited but measurable therapeutic window. Collectively, these findings demonstrate that MLS0315771 acts as a novel membrane-targeting antibacterial agent with notable antibacterial activity against MDR and biofilm-forming Gram-positive pathogens, highlighting its potential as a candidate lead compound for future antimicrobial development.