The clinical translation of cationic alpha-helical antimicrobial peptides (AMPs) has been hindered by structural instability, proteolytic degradation, and in vivo toxicity from non-specific membrane lysis. Although analyses of hydrophobic content and charge distribution have informed the design of synthetic AMPs with increased potency and reduced in vitro hemolysis, non-specific membrane toxicity in vivo continues to impede AMP drug development. Here, we analyzed a 58-member library of stapled AMPs (StAMPs) based on Magainin-II, and applied the insights from structure-function-toxicity measurements to devise an algorithm for the design of stable, protease-resistant, potent, and nontoxic StAMP prototypes. We show that a lead double-stapled StAMP named Mag(i+4)1,15(A9K,B21A,N22K,S23K) can kill multidrug resistant Gram-negative pathogens, such as colistin-resistant Acinetobacter baumannii in a mouse peritonitis-sepsis model, without observed hemolysis or renal injury in murine toxicity studies. Inputting the amino acid sequences alone, we further generated membrane-selective StAMPs of pleurocidin, CAP18, and esculentin, highlighting the generalizability of our design platform.

A novel mechanism of monolayer poration leading to the rapid exfoliation and lysis of microbial membranes is reported.