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.