Engineering of antimicrobial peptide Brevinin-1pl: arginine, lysine, and histidine substitutions enhance antimicrobial-anticancer efficacy with reduced cytotoxicity

Abstract

Introduction: Antimicrobial peptides (AMPs) are promising candidates for combating multidrug-resistant infections, but their clinical application is often limited by challenges such as poor selectivity and high cytotoxicity. This study aimed to optimize the therapeutic potential of brevinin-1pl, a frog-derived AMP with broad-spectrum antimicrobial and anticancer activities. Methods: Major experimental approaches encompassed antibacterial activity evaluation, hemolytic potential assessment, bactericidal rate determination via time-kill kinetics, SYTOX Green-based membrane integrity analysis, and MTT assays for anti-proliferative effects. Results: Substitutions with arginine (brevinin-1pl-2R and brevinin-1pl-5R) enhanced activity against Gram-positive bacteria but reduced efficacy against Gram-negative strains. Lysine substitution (brevinin-1pl-6K) decreased activity against Gram-positive bacteria due to reduced hydrophobicity. In contrast, histidine substitution (brevinin-1pl-3H) showed diminished activity against Gram-negative bacteria (e.g., MRSA MIC increased from 2 µM to 4 µM) but reduced hemolysis, indicating improved selectivity. Mechanistic studies using SYTOX green assays confirmed membrane disruption as a primary mode of action, while suggesting alternative mechanisms for Gram-positive Enterococcus faecium and Gram-negative Escherichia coli. The brevinin-1pl and its analogues demonstrated significant inhibitory efficacy against both MCF-7 breast cancer cells and H838 non-small cell lung cancer cells at a concentration of 10–4 M. Notably, brevinin-1pl-3H exhibited low cytotoxicity toward normal HaCaT cells despite its high hydrophobicity, suggesting potential for dermatological applications. Conclusion: These findings demonstrate that strategic amino acid substitutions can optimize the therapeutic potential of AMPs, offering a promising approach to develop peptides with enhanced efficacy and reduced clinical side effects.