Refining Selectivity of Branched CAMPs through Modulation of Hydrophobic–Hydrophilic Balance by Incorporation of N-Terminal ω- Amino Acid Residues

Abstract

High selectivity for pathogenic strains over mammalian host cells is a fundamental prerequisite for an antimicrobial therapeutic. Hydrophobic–hydrophilic balance has been a cornerstone for modulating AMP performance. Here, we have refined the selectivity of 2BWWR-7dL by modulating the hydrophilic–hydrophobic balance through a novel concept of the introduction of N-terminal ω-amino acid residues. C3–7dL, the lead analog, exhibited improved broad-spectrum antimicrobial potency (1.5×) against several ESKAPE pathogens and antibiotic-resistant Gram-positive strains, MRSA and VRSA, with reduced hemolytic activity and cytotoxicity toward mammalian cell lines, and an improved therapeutic index over the template peptide. Additionally, C3–7dL had potent antibiofilm activity against the model strain P. aeruginosa (>95% inhibition/eradication), salt tolerance, fast killing kinetics (∼5 min), protease and serum stability, absence of resistance development for over 45 days of continuous exposure, and a membranolytic mechanism of action, making it a very prospective therapeutic. Our in-depth mechanistic investigations through various biophysical assays on the model Gram-negative bacterium P. aeruginosa established that while a high charge-density (high hydrophilicity) promoted the outer-membrane binding (through LPS interaction) and depolarization of membranes, high hydrophobicity facilitated inner-membrane permeabilization and the intracellular leakage. We also established that the hydrophobic–hydrophilic balance was critical for attaining selectivity toward microbial membranes over mammalian membranes through ITC and fluorescence experiments. C3–7dL possessed the optimum trade-off between its hydrophilicity and hydrophobicity, which accounted for its best activity and biosafety, among the designed analogs of 2BWWR-7dL. Such intricate mechanistic investigations will enable the design of effective and biosafe antimicrobial therapeutics for the future.