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.

The difficulty of accessing the mitochondrial matrix has limited the targeting of therapeutics to this organelle. Here, we report, to our knowledge, the first successful delivery of an active DNA alkylating agent--chlorambucil--to mitochondria, and describe unexpected features that result from rerouting this drug within the cell. Mitochondrial targeting of this agent dramatically potentiates its activity, and promotes apoptotic cell death in a variety of cancer cell lines and patient samples. This retention of activity is observed even in cells with resistance to chlorambucil or disabled apoptotic triggering.