Repurposing PFM01 as a novel antibacterial agent against Staphylococcus aureus

Staphylococcus aureus infections pose a significant threat to global human health, particularly with the widespread transmission of methicillin-resistant S. aureus (MRSA) strains, which has created an urgent need for the development of new antimicrobial agents. PFM01 is an N-alkylated derivative of Mirin, an MRE11 endonuclease inhibitor. It was initially discovered to block the repair of double-strand DNA breaks and demonstrated preliminary anti-tumor potential; however, its antimicrobial activity had not yet been explored. Here, the anti- S . aureus activity of PFM01 was determined by broth microdilution minimum inhibitory concentration (MIC) testing and time-kill kinetics. The in vitro cytotoxicity was assessed by CCK-8 assay and hemolysis test. The potential mechanisms were systematically investigated by whole-genome sequencing, drug affinity responsive target stability (DARTS), and molecular docking analyses. PFM01 exhibited potent antibacterial activity against Gram-positive bacteria (including S. aureus , Enterococcus faecalis , Enterococcus faecium , and Streptococcus lactis ), with MIC ₅₀ values ranging from 6.25 to 12.5 μM (1.83–3.67 μg/mL). Time-kill curve analysis confirmed its bactericidal capacity against free-floating S. aureus (including bacteria in the logarithmic growth phase and stationary phase-retained cells). Furthermore, PFM01 inhibited S. aureus biofilm formation in a concentration-dependent manner and possesses the ability to remove mature biofilms. In vitro cellular safety tests of PFM01 indicated low cytotoxicity and no significant hemolytic activity. Moreover, PFM01 altered the membrane permeability and potential of S. aureus , and its antibacterial activity was counteracted by the exogenous phospholipids. Whole-genome sequencing identified genetic mutations in the rapZ , mupR , mprF, and clpX genes of PFM01-resistant S. aureus strains; however, the antibacterial activity of PFM01 against mprF and clpX knockout S. aureus strains showed no significant change compared with wild-type strains. Proteomic analysis further revealed that PFM01 primarily might interfere with purine metabolism, amino acid biosynthesis, and the phosphotransferase system. DARTS experiments suggested that glycerol-3-phosphate cytidylyltransferase (TarD) might be a potential target, and molecular docking results further confirmed a stable interaction between PFM01 and TarD (binding energy: −7.4 kcal/mol). In summary, PFM01 may exert its antibacterial effect by disrupting the bacterial cell membrane structure and targeting TarD, and holds promise as a therapeutic agent for S. aureus infections.