Van der Walt, MandelieOosthuizen, Carel B.Serian, MirunaLorenz, Christian D.Mason, A. JamesBester, Megan JeanGaspar, Anabella Regina Marques2025-09-102025-09-102025-05Van der Walt, M., Oosthuizen, C.B., Serian, M. et al. 2025, 'Enhanced gram-negative membrane disruption and in vivo efficacy via lysine-arginine enrichment of Opis16a', ACS Medicinal Chemistry Letters, vol. 16, no. 6, pp. 998-1007, doi : 10.1021/acsmedchemlett.5c00038.1948-5875 (online)10.1021/acsmedchemlett.5c00038http://hdl.handle.net/2263/104276SUPPORTING INFORMATION : Materials and methods; (Table S1) antimicrobial susceptibility profile; (Table S2) antibacterial activity against Gram-negative bacteria; (Figure S1) hydrogen bonding results; (Figure S2) peptide secondary structure analysis; (Figures S3 and S4) membrane permeabilization; (Figure S5) peptide starting structures.Infections complicate burn wound care, especially with the rise of antimicrobial resistance. Antimicrobial peptides (AMPs) offer the potential for advancing wound care by combating persistent infections. Opis16a, a scorpion venom-derived AMP, exhibits potent antibacterial activity by targeting Gram-negative membranes, causing rapid membrane disruption and bacterial cell death. Here, four novel Opis16a analogues were developed with improved membrane targeting and antibacterial efficacy. One analogue shows particular promise for topical application in Gram-negative burn wound infections. Enhanced peptide–lipid hydrogen bonding increases conformational stability, membrane insertion, and permeabilization rates. Substituting lysine residues in the C-terminal with arginine leads to the most consistent improvement in activity, selectivity for pathogen over HaCat cells, and stability in serum. In an in vivo Galleria mellonella burn wound model, a 5 mg/kg topical dose provides better protection than Opis16a against Enterobacter cloacae NICD 16103. These findings highlight the potential of optimized bactericidal AMPs to improve burn wound care.en© 2025 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0.Antimicrobial peptides (AMPs)Wound infectionsAntimicrobial resistance (AMR)Gram-negative bacteriaOpis16aOpis16aCterKRArginine substitutionMembrane permeabilizationMolecular dynamicsEnterobacter cloacae (E. cloacae)Article