Amphipathicity-driven optimization of substance P-derived antimicrobial peptides for infected wound healing

Infected wounds require therapeutics that simultaneously control bacterial infection and promote tissue repair. Here, we modified the neuropeptide substance P (SP) scaffold to generate two short antimicrobial peptides, SP-1 and SP-2. SP-2 was derived from SP-1 through a single L/K positional exchange to enhance amphipathic organization without changing amino acid composition. Peptide structure, antibacterial activity, and wound-healing activity were evaluated in vitro and in vivo. SP-1 and SP-2 showed antibacterial activity against the tested strains, with MIC values ranging from 4 to 16 μM, whereas native SP showed no detectable activity. Both analogs could rapidly kill bacteria accompanied by membrane damage. Compared with SP-1, SP-2 showed faster bactericidal kinetics, stronger antibiofilm activities, stronger suppression of LPS-induced inflammatory responses, better proteolytic stability, and smaller self-assembled particles. SP-1 and SP-2 also retained SP-associated wound-healing activity. In an S. aureus-infected mouse full-thickness wound model, SP-2 reduced recoverable bacterial burden more effectively than either SP-1 or vancomycin and improved wound repair. However, SP-2 exhibited concentration-dependent hemolysis and potential systemic toxicity at 60 mg/kg, indicating the need to further improve its therapeutic window. Together, this study provides a strategy for developing multifunctional short antimicrobial peptides from neuropeptide scaffolds for infected wound repair.