Tachyplesin-1 (TP1; 1) is a cationic β-hairpin antimicrobial peptide with a membranolytic mechanism of action. While it possesses broad-spectrum, potent antimicrobial activity, 1 is highly hemolytic against mammalian erythrocytes, which precludes it from further development. In this study, we report a template-based approach to investigate the structure-function and structure-toxicity relationships of each amino acid of 1. We modulated charge and hydrophobicity by residue modification and truncation of the peptide. Antimicrobial activity was then assessed against six key bacterial pathogens and two fungi, with toxicity profiled against mammalian cells. The internal disulfide bridge Cys7-Cys12 of 1 was shown to play an important role in broad-spectrum antimicrobial activity against all pathogenic strains tested. Novel peptides based on the progenitor were then designed, including 5 (TP1[F4A]), 12 (TP1[I11A]), and 19 (TP1[C3A,C16A]). These had 26- to 64-fold improved activity/toxicity indices and show promise for further development. Structural studies of 5 (TP1[F4A]) and 12 (TP1[I11A]) identified a conserved β-hairpin secondary structure motif correlating with their very high stablility in mouse and human plasma. Membrane binding affinity determined by surface plasmon resonance confirmed their selectivity toward bacterial membranes, but the degree of membrane binding did not correlate with the degree of hemolysis, suggesting that other factors may drive toxicity.
Keywords: amphiphilicity; antibiotic; antimicrobial activity/toxicity index; antimicrobial peptide; membrane; resistance; tachyplesin-1; β-hairpin.