Impact of Charge Distribution on the Membrane Binding and Antimicrobial Activity of Cationic Heptapeptides.

Antimicrobial peptides (AMPs) hold immense potential in combating community-acquired and MDR infections. The therapeutic efficiency of AMPs depends on their various physicochemical properties. We investigated how the hydrophobic-hydrophilic landscape of tryptophan-containing cationic heptapeptides (CAMPs) influenced their ability to bind to microbial membranes and their antimicrobial effectiveness against several ESKAPE pathogens. We found that charge delocalization over the peptide backbone enhanced antimicrobial activity, while charge localization at the C- and N-termini reduced efficacy (1.5-2.7 folds) for both the single and double tryptophan peptides. The double-tryptophan peptide P32* had increased activity over the single tryptophan peptide P32 (∼2.3 fold), and alleviated the effect of charge segregation on activity. Using steered molecular dynamics umbrella sampling simulations, we evaluated the peptides based on their affinity (ΔG) for a membrane-mimetic bilayer model of Pseudomonas aeruginosa. Peptides with higher membrane-binding affinity exhibited greater antimicrobial potency. All peptides exhibited negligible hemolytic activity (HC10% ≤ 10 μM) and retained membranolytic antibacterial activity. The calculated binding free energies of the peptide(s) and the bilayer exhibited a strong linear correlation with the experimental minimum inhibitory concentration (MIC) values, with a negative slope. More favorable binding free energies (i.e., more negative values) corresponded to lower MICs, as stronger interactions with the membrane enhanced the membranolytic properties of these peptides. We demonstrated that integrating molecular simulations with experimental validation enables the use of membrane-binding free energy as a quantitative predictor of peptide activity. This approach provides a rational framework for the future design and optimization of membrane-active CAMPs.