Thiophene‐Engineered Lipid II Recognition Enables Potent Membranolytic Eradication of MRSA

Methicillin‐resistant Staphylococcus aureus (MRSA) remains a significant global health threat, necessitating the development of new antimicrobials. We report the rational design of a small molecule TPL(II)‐07, that combines multiple functional modules: (i) 2‐phenylthiophene for specific recognition and binding to lipid II, a key intermediate in peptidoglycan biosynthesis; (ii) an adamantane fragment with high interfacial permeability to destabilize lipid packing; (iii) a cationic lysine moiety to disperse biofilms; and (iv) a moderate aliphatic chain to disrupt membrane integrity. Among seven analogues, TPL(II)‐07 exhibited the highest potency, highlighting the synergistic contribution of each component. Mechanistic assays confirmed preferential binding of lipid II, bactericidal potency, biofilm dispersion, and the generation of reactive oxygen species, effective against both drug‐resistant (MIC 19 µM) and drug‐sensitive (MIC 9 µM) S. aureus strains. TPL(II)‐07 demonstrated excellent biocompatibility, with IC 50 > 50 mM in HEK‐293 and WI38 cells and negligible hemolytic activity. In addition, it suppressed key virulence traits, including hemolysin activity, biofilm formation, and staphyloxanthin production. Further, a borate‐functionalized guar gum hydrogel incorporating TPL(II)‐07 achieved effective infection control and enhanced wound repair in a murine excisional model, establishing thiophene‐based lipid II targeting as a versatile platform for next‐generation antimicrobials with dual antibacterial and wound‐healing potential.