Peptides that form transmembrane barrel-stave pores are potential alternative therapeutics for bacterial infections and cancer. However, their optimization for clinical translation is hampered by a lack of sequence-function understanding. Recently, we have de novo designed the first synthetic barrel-stave pore-forming antimicrobial peptide with an identified function of all residues. Here, we systematically mutate the peptide to improve pore-forming ability in anticipation of enhanced activity. Using computer simulations, supported by liposome leakage and atomic force microscopy experiments, we find that pore-forming ability, while critical, is not the limiting factor for improving activity in the submicromolar range. Affinity for bacterial and cancer cell membranes needs to be optimized simultaneously. Optimized peptides more effectively killed antibiotic-resistant ESKAPEE bacteria at submicromolar concentrations, showing low cytotoxicity to human cells and skin model. Peptides showed systemic anti-infective activity in a preclinical mouse model of Acinetobacter baumannii infection. We also demonstrate peptide optimization for pH-dependent antimicrobial and anticancer activity.

CEITEC Central European Institute of Technology Masaryk University Brno 625 00 Czech Republic

Czech Advanced Technology and Research Institute Palacký University Olomouc 779 00 Czech Republic

Department of Biochemical Sciences Laboratory Affiliated to Istituto Pasteur Italia Fondazione Cenci Bolognetti Sapienza University of Rome Rome 00185 Italy

Department of Chemistry School of Arts and Sciences University of Pennsylvania Philadelphia Pennsylvania 19104 United States

Department of Condensed Matter Physics Faculty of Science Masaryk University Brno 611 37 Czech Republic

Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno 625 00 Czech Republic

Departments of Bioengineering and Chemical and Biomolecular Engineering School of Engineering and Applied Science University of Pennsylvania Philadelphia Pennsylvania 19104 United States

Institute of Microbiology Czech Academy of Sciences BIOCEV Vestec 252 50 Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Olomouc 779 00 Czech Republic

Machine Biology Group Departments of Psychiatry and Microbiology Institute for Biomedical Informatics Institute for Translational Medicine and Therapeutics Perelman School of Medicine University of Pennsylvania Philadelphia Pennsylvania 19104 United States

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno 625 00 Czech Republic

National Reference Centre for Antibiotic Resistance Besançon 25030 France

Penn Institute for Computational Science University of Pennsylvania Philadelphia Pennsylvania 19104 United States

University of Franche Comté CNRS Chrono environment Besançon 25030 France

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