Biochemical property and membrane-peptide interactions of de novo antimicrobial peptides designed by helix-forming units.

Typical peptides composed of Phe, Ile, and Arg residues have not been reported, and the effect of the helix-forming unit (HFU) composed of the tripeptide core on biological activity remains unclear. In this study, multimers of the 3-residue HFU were designed to investigate the structure-function relationships. The in vitro biological activities of the peptides were determined. We used synthetic lipid vesicles and intact bacteria to assess the interactions of the peptides with cell membranes. The well-studied peptide melittin was chosen as a control peptide. The results showed that the antimicrobial and hemolytic activities of the peptides increased with the number of HFUs. HFU3 had optimal cell selectivity as determined by the therapeutic index. HFU3 and HFU4 exhibited strong resistance to salts, pH, and heat. CD spectra revealed that the peptides except HFU2 displayed α-helix-rich secondary structures in the presence of SDS or trifluoroethanol (TFE). The peptides interacted weakly with zwitterionic phospholipids (mimicking mammalian membranes) but strongly with negatively charged phospholipids (mimicking bacterial membranes), which corresponds well with the data for the biological activities. There was a correlation between the cell selectivity of the peptides and their high binding affinity with negatively charged phospholipids. Cell membrane permeability experiments suggest that the peptides targeted the cell membrane, and HFU3 showed higher permeabilization of the inner membrane but lower permeabilization of the outer membrane than melittin. These findings provide the new insights to design antimicrobial peptides with antimicrobial potency by trimers.

Novel design of short antimicrobial peptides derived from the bactericidal domain of avian ß-defensin-4.

Short antimicrobial peptides were designed and synthesized by C-terminal truncation and residue substitution of avian ß-defensin-4. The biological activity of these peptides was examined to elucidate the quantitative structure-activity relationships and find a lead peptide for the development of a novel antimicrobial peptide. The results showed that the truncation of the avian ß-defensin-4 eliminated the hemolysis of the peptide. The GLI13 derivative, developed by substituting the Cys of the truncated peptide with Ile, led to increased antimicrobial activity. These results suggest that the peptides with antimicrobial activity can be derived by truncating the avian ß-defensin-4. We further developed the GLI13 derivative with an increased net charge by residue substitution. The results showed that the GLI13-5 with 5 net charges had the highest cell selectivty. An increase in the net charge from 6 to 8 did not result in the improvement of antimicrobial potency. Membrane-simulating experiments showed that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. A membrane depolarization assay showed that GLI13-5 killed bacteria by targeting the cytoplasmic membrane. These results suggest that the short peptide developed by truncation of linear ß-defensin may be a promising candidate for future antibacterial agents.

The effects of Leu or Val residues on cell selectivity of α-helical peptides.

In this study, the peptides were designed to compare the effect of multiple Leu or Val residues as the hydrophobic side of an α-helical model on their structure, function, and interaction with model membranes. The Leu-rich peptides displayed 4- to 16-fold stronger antimicrobial activity than Val-rich peptides, while Val-containing peptides showed no haemolysis and weak cytotoxicity. The peptides LR and VR showed an α-helical-rich structure under a membranemimicking environment. Different cell selectivity for Leu- or Val-containing peptides correlated with the targeted cell membranes. The Leu-rich peptide LR(W) and Val-rich peptide VR(W) interacted preferentially with negatively charged phospholipids over zwitterionic phospholipids. VR(W) displayed no interaction with zwitterionic phospholipids, which was consistent with its lack of haemolytic activity. The ability of LR to depolarize bacterial cells was much greater than that of VR. Val- and Leu-rich peptides appeared to kill bacteria in a membrane-targeted fashion, with different modes of action. Leu-rich peptides appeared to be active via a membrane-disrupting mode, while Val-rich peptides were active via the formation of small channels.