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

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.