Enhanced proteolytic resistance of cationic antimicrobial peptides through lysine side chain analogs and cyclization

While cationic antimicrobial peptides (CAPs) are compelling candidates for antimicrobial therapy, their clinical development is largely hampered by their rapid and non-specific enzymatic degradation in physiological fluids. We have earlier de novo designed and synthesized a novel category of CAPs typified by the sequence KKKKKK-AAFAAWAAFAA-NH₂ (termed "6K-F17") that have remarkable membrane-penetrating power, are highly selective for bacterial rather than host membranes, and are non-cytotoxic. Here we pursue the design and validation of the Lys chain-shortened 6K-F17 analogs 6Dap-F17 (Dap = diaminopropionic acid), 6Dab-F17 (Dab = diaminobutyric acid), and 6Orn-F17 (Orn = ornithine). Intriguingly, although initially designed to specifically resist trypsin vs. their original Lys sites, all three derivatives of 6K-F17 showed markedly improved stability not only against trypsin, but also against the major proteolytic enzymes elastase and proteinase K at a 1:100 enzyme-to-peptide (E:P) ratio. When the least stable analog, 6Dap-F17, was then cyclized ('stapled') - with reduced main chain hydrophobicity to avoid erythrocyte hemolysis - the peptide became robust towards all three enzymes up to 60 min at a 1:100 E:P ratio, and retained strong presence even at an enhanced 1:1 E:P ratio, as determined by HPLC and mass spectrometry. These results suggest that the application of Lys chain-shortening, either alone or in combination with macrocyclization, may enhance metabolic stability of CAPs, and thus their clinical potential.