Dual-targeting amphiphilic lipopeptides combat multidrug-resistant Gram-positive pathogens by inhibiting type I signal peptidase and disrupting bacterial membrane.

Antimicrobial resistance poses a significant threat to global health, particularly with multidrug-resistant (MDR) Gram-positive pathogens evading last-line treatments. We herein designed and synthesized novel amphiphilic lipopeptide derivatives to exert dual antibacterial mechanisms: SPase I inhibition and bacterial membrane disruption. Among these, compound A09 demonstrated potent in vitro activity against a series of MDR Gram-positive pathogens. Biochemical characterization revealed potent inhibition of Escherichia coli SPase I (LepB) with an IC50 of 4.475 μM and a Kd of 16.3 ± 11.4 μM. Mechanistic studies confirmed A09's membrane disruption and significant biofilm eradication capability. Critically, A09 exhibited substantial efficacy in vivo in a murine model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection. Furthermore, A09 displayed low hemolytic and cytotoxic effects, indicating a favorable safety profile. These results established A09 as a promising dual-mechanism anti-infective agent for treating drug-resistant Gram-positive infections.