Rational design of protease-resistant lipopeptides for combating antibiotic-resistant bacteria

Given the surge in antibiotic resistance, antimicrobial peptides have emerged as promising alternatives to conventional antibiotics; however, their therapeutic potential remains hindered by proteolytic instability and insufficient potency. Herein, we integrated an anti-proteolytic Arg-Pro scaffold with N-terminal fatty acid modification to improve peptide stability and membrane-binding affinity. A series of lipopeptides was synthesized and systematically evaluated for antimicrobial efficacy, stability, and cytotoxicity, yielding C12RP7 as the lead candidate. C12RP7 exhibited potent broad-spectrum activity against 20 tested strains, including multi-drug resistant pathogens, with a high therapeutic index (TI = 64.20) and low toxicity. It also retained functional stability in the presence of physiological salts, serum, and proteases. In murine models of peritonitis and enteritis, C12RP7 treatment significantly reduced bacterial burden, attenuated systemic inflammation, restored intestinal barrier integrity, and modulated the gut microbiota. These findings support C12RP7 as a protease-resistant, potent, and biocompatible lipopeptide with potential for treating bacterial infections.