High therapeutic index α-helical antimicrobial peptides with repetitive subunit structures against multidrug-resistant bacteria.

De novo design of antimicrobial peptides (AMPs) offers a promising strategy to overcome the limitations of natural AMPs through rational design, providing potential solutions to address the growing risk of traditional antibiotic resistance. In this study, a series of new AMPs are generated using the α-helical template (XXFY)n and its β-sheet counterpart (KFKY)n (X = Lys, Dab, Orn, or Arg; F = Phe; Y = Leu, Ile, Phe, or Trp; K = Lys; n = 2, 3, 4, or 5), enabling a systematic investigation of their structure-activity relationships (SAR). The optimal peptide 27, designated as (OOFI)4 (O = Orn, I = Ile), demonstrates potent broad-spectrum antimicrobial activity against both standard and multidrug-resistant bacterial strains, along with low hemolytic toxicity. Furthermore, 27 exhibits a low propensity for inducing resistance, rapid bactericidal effects, robust membrane-disrupting activity, and immunomodulatory activity. Notably, peptide 27 also shows remarkable efficacy in treating multidrug-resistant P. aeruginosa 124-induced lung infections and MRSA-induced skin infections in murine models. In conclusion, the SAR analysis in this study offers novel insights into template-based AMP design, and the newly developed peptide 27 emerges as a promising candidate for treating clinically relevant drug-resistant bacterial infections.