Design of Protegrin-1 Analogs with Improved Antibacterial Selectivity

Protegrin-1 (PG-1) is a cationic fi-hairpin pore-forming antimicrobial peptide having a membranolytic mechanism of action. It possesses in vitro a potent antimicrobial activity against a panel of clinically relevant MDR ESKAPE pathogens. However, its extremely high hemolytic activity and cytotoxicity toward mammalian cells prevent the further development of the protegrin-based antibiotic for systemic administration. In this study, we rationally modulated the PG-1 charge and hydrophobicity by substituting selected residues in the central beta-sheet region of PG-1 to design its analogs, which retain a high antimicrobial activity but have a reduced toxicity toward mammalian cells. In this work, eight PG-1 analogs with single amino acid substitutions and five analogs with double substitutions were obtained. These analogs were produced as thioredoxin fusions in Escherichia coli. It was shown that a significant reduction in hemolytic activity without any loss of antimicrobial activity could be achieved by a single amino acid substitution, V16R in the C-terminal beta-strand, which is responsible for the PG-1 oligomerization. As the result, a selective analog with a >= 30-fold improved therapeutic index was obtained. FTIR spectroscopy analysis of analog, [V16R], revealed that the peptide is unable to form oligomeric structures in a membrane-mimicking environment, in contrast to wild-type PG-1. Analog [V16R] showed a reasonable efficacy in septicemia infection mice model as a systemic antibiotic and could be considered as a promising lead for further drug design.

Automated summary

In this study, PG-1 analogs with reduced toxicity towards mammalian cells were designed by modifying the charge and hydrophobicity of PG-1. These analogs showed high antimicrobial activity against a range of clinically relevant pathogens and a significant decrease in hemolytic activity. One particularly promising analog, [V16R], demonstrated improved therapeutic efficacy in a mouse model of septicemia and could be a potential candidate for further drug development.