Incyclinide exhibits potent antibacterial and anti-biofilm activity against Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) and biofilm-associated infections remain major therapeutic challenges. Here, we investigated the antibacterial activity and mechanism of Incyclinide against S. aureus . Incyclinide showed potent activity against 40 clinical isolates, with a minimum inhibitory concentration (MIC) of 2 µM for 81.25% of MSSA isolates and 91.66% of MRSA isolates. In representative MSSA and MRSA strains, 1× MIC completely inhibited planktonic growth, while time-kill assays demonstrated concentration-dependent bactericidal activity; at 8× MIC, bacterial counts decreased by 3.2 log 10 CFU/mL in NCTC 8325 and 4.3 log 10 CFU/mL in USA300 after 24 h. Incyclinide also markedly reduced the biomass of mature, pre-established biofilms. Proteomic profiling of Incyclinide-treated USA300 identified 59 differentially expressed proteins enriched in membrane-related functions. Transmission electron microscopy and fluorescence assays revealed severe cell envelope damage, increased membrane permeability, and membrane depolarization. Exogenous phospholipids antagonized antibacterial activity, with phosphatidylethanolamine and cardiolipin showing stronger effects than phosphatidylglycerol. In addition, resistance induction and whole-genome sequencing identified recurrent clpX mutations; clpX deletion increased the MIC from 2 to 8 µM, whereas complementation restored susceptibility. Molecular docking suggested favorable binding between Incyclinide and ClpX, with a predicted binding energy of −9.4 kcal/mol. Incyclinide also exhibited low hemolytic activity, negligible cytotoxicity toward BEAS-2B and human umbilical vein endothelial cell up to 128 µM, and significant protection in a Galleria mellonella infection model. Collectively, these findings identify Incyclinide as a promising anti- S . aureus agent with potent activity against MRSA and mature biofilms, acting primarily through membrane disruption, with ClpX potentially contributing to susceptibility. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) and biofilm-associated infections pose significant clinical challenges due to limited therapeutic options. Here, we identify Incyclinide, a tetracycline derivative, as a potent anti- S . aureus agent. It exhibits potent bactericidal activity against clinical MRSA isolates and effectively eradicates mature biofilms. Mechanistically, Incyclinide disrupts membrane integrity, causing depolarization and leakage. Notably, we identify the essential chaperone ClpX as a key molecular determinant of susceptibility through genetic and docking studies. Incyclinide demonstrates a favorable safety profile and superior efficacy compared with vancomycin in a Galleria mellonella infection model. Our findings highlight Incyclinide as a promising candidate featuring a dual membrane-targeting and intracellular mechanism, offering a strategic advantage against drug-resistant and biofilm-embedded pathogens.