Role of helicity of α-helical antimicrobial peptides to improve specificity

Abstract

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLKKTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Gram-positive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.

Figure 1

Representation of the parent peptide A12L/A20L as helical nets showing the polar/hydrophilic face (circled residues) and non-polar/ hydrophobic face (boxed residues) and helical wheel, the lysine residue at position 13 of the sequence is denoted by a triangle. In the helical nets, the D-amino acid substitution sites are shown in bold and italic, while in the helical wheel, three single substitution sites are shown with solidarrows on the non-polar face as a solidarc and hollowarrows on the polar face as an openarc, respectively, Ac denotes N^α-acetyl, and amide denotes C^α-amide. One-lettercodes are used for the amino acid residues

Figure 2

Effect of peptide L12_D/L20_Don the surface of negatively-stainedS. aureus(left) andP. aeruginosa(right) by scan electron microscopy. Untreated bacterial cells were shown in panels A and B. Treated bacterial cells with peptide L12_D/L20_D revealed disrupted cell membranes in panelsC and D

Figure 3

Fluorescence emission spectra (left) and Stern-Volmer plot (right) of peptides with various liposome models at 25°C. Stern-Volmer plots were obtained by the sequential addition of the fluorescence quencher KI. Results of three peptides were plotted as follows: parent peptide P in PanelsA and B, peptide K22_D in PanelsC and D, and peptide L12_D/L20_D in PanelsE and F, respectively. HEPES buffer, PC/cholesterol lipsomes and PC/PG lipsomes were presented by solidsquares, hollowcircles and solidtriangles, respectively

Figure 4

Relationships of peptide helicity, hydrophobicity with the numbers of D-amino acid substitutions. The experimental data from Table 2 and least squares fit analysis were used. The results showed correlations of helicity and the number of D-amino acid substitutions with R = 0.942 on the polar face (A) and R = 0.954 on the non-polar face (B); correlations of hydrophobicity and the number of D-amino acid substitutions with R = 0.967 on the polar face (C) and R = 0.924 on the non-polar face (D); correlations between hydrophobicity and helicity with R = 0.955 on the polar face (E) and R = 0.913 on the non-polar face (F)