Rationally Designed Highly Amphipathic Antimicrobial Peptides Demonstrating Superior Bacterial Selectivity Relative to the Corresponding α-Helix Peptide

De novo design of antimicrobial peptides is a pivotal strategy for developing new antibacterial agents, leveraging its rapid and efficient nature. (XXYY)_n, where X represents cationic residues, Y denotes hydrophobic residues, and n varies from 2 to 4, is a classical α-helix template. Based on which, numerous antimicrobial peptides have been synthesized. Herein, we hypothesize that the amphipathy of this type of α-helix template can be further enhanced based on the principles of α-helical protein folding, characterized by a rotation occurring every 3.6 amino acid residues, and propose the highly amphipathic template XXYYXXYXXYYX (where X represents cationic residues and Y denotes hydrophobic residues). Accordingly, the amino acid composition and arrangement of the α-helix peptide (RRWF)₃ are adjusted, yielding the highly amphipathic counterpart H-R (RRWFRRWRRWFR). The structure-activity relationship of which is further explored through the substitution of residues at positions 8 and 12. Notably, the highly amphipathic peptides exhibit enhanced antimicrobial activity and reduced hemolytic toxicity compared to (RRWF)₃, resulting in superior bacterial selectivity. The most highly amphipathic peptide, H-R, demonstrates potent activity against biofilms and multidrug-resistant bacteria, low propensity for resistance, and high safety and effectiveness in vivo. The antibacterial mechanisms of H-R are also preliminarily investigated in this study. As noted, H-R represents promising antimicrobial candidates for addressing infections associated with drug-resistant bacteria.