Alchemical Simulation-Aided De Novo Design of Membrane-Active Antimicrobial Hepta-Peptides

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics. This study uses alchemical free energy simulations to design ultrashort, cationic, broad-spectrum membrane-active peptides. Previously, we identified broad-spectrum peptide P4 (LKWLKKL-NH₂, charge +4) with moderate activity (10-50 μM) but it was ineffective against Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). Here, we designed eight analogues of P4 by substituting side chains at the third and fourth positions while retaining the overall charge. Alchemical simulations ranked the peptides by assessing the changes in the POPE/POPG bilayer affinity (ΔΔ<i>G</i>) resulting from P4 mutation(s). Testing against <i>Acinetobacter baumannii</i>, <i>MRSA</i>, and <i>Candida albicans</i> showed a strong correlation between <i>in silico</i> ranking and experimental potency. Key findings include the following: (1) substituting L4 with W4 (P4^L4W) improved activity by 2- to 5-fold against all the strains; (2) W3 was crucial; (3) peptides were membranolytic; and (4) alanine substitutions compromised activity. This comprehensive approach links energetics, molecular interactions, and activity, accelerating the identification of therapeutic membrane-active peptides.