Comparison of biophysical and biologic properties of alpha-helical enantiomeric antimicrobial peptides

Abstract

In our previous study (Chen et al. J Biol Chem 2005, 280:12316-12329), we utilized an alpha-helical antimicrobial peptide V(681) as the framework to study the effects of peptide hydrophobicity, amphipathicity, and helicity on biologic activities where we obtained several V(681) analogs with dramatic improvement in peptide therapeutic indices against gram-negative and gram-positive bacteria. In the present study, the D-enantiomers of three peptides--V(681), V13A(D) and V13K(L) were synthesized to compare biophysical and biologic properties with their enantiomeric isomers. Each D-enantiomer was shown by circular dichroism spectroscopy to be a mirror image of the corresponding L-isomer in benign conditions and in the presence of 50% trifluoroethanol. L- and D-enantiomers exhibited equivalent antimicrobial activities against a diverse group of Pseudomonas aeruginosa clinical isolates, various gram-negative and gram-positive bacteria and a fungus. In addition, L- and D-enantiomeric peptides were equally active in their ability to lyse human red blood cells. The similar activity of L- and D-enantiomeric peptides on prokaryotic or eukaryotic cell membranes suggests that there are no chiral receptors and the cell membrane is the sole target for these peptides. Peptide D-V13K(D) showed significant improvements in the therapeutic indices compared with the parent peptide V(681) by 53-fold against P. aeruginosa strains, 80-fold against gram-negative bacteria, 69-fold against gram-positive bacteria, and 33-fold against Candida albicans. The excellent stability of D-enantiomers to trypsin digestion (no proteolysis by trypsin) compared with the rapid breakdown of the L-enantiomers highlights the advantage of the D-enantiomers and their potential as clinical therapeutics.

Figure 1. Space-filling model of peptides V₆₈₁ and V13K_L

Hydrophobic amino acids on the non-polar face of the helix are green; hydrophilic amino acids on the polar face of the helix are gray; peptide backbone is colored white. The Lys substitution at position 13 (V13K_L) on the non-polar face of the helix is blue. The models were created by the pymol v0.98. The peptide sequences are shown in Table 1.

Figure 2. Circular dichroism (CD) spectra of peptides V₆₈₁ and d-V₆₈₁ (panel A), V13K_L and d-V13K_D (panel B) and peptides V13A_D and d-V13A_L (panel C) at pH 7.4 and 5 °C, in 50 mm aq. potassium phosphate buffer (KP buffer) containing 100 mm KCl

In panels (A–C), solid symbols represent the CD spectra of peptide analogs in KP buffer without trifluoroethanol (TFE), whilst open symbols represent CD spectra obtained in the presence of 50% TFE; the symbols used are: circles for l-peptides V₆₈₁, V13K_L, and V13A_D and diamonds for d-peptides d-V₆₈₁, d-V13K_D, and d-V13A_L.

Figure 3. Peptide retention behavior during reversed-phase high-performance liquid chromatography (RP-HPLC) with increasing temperature

Column and conditions: RP-HPLC, SB-C₈ column (150 × 2.1 mm I.D.; 5 µm particle size, 300 Å pore size), linear A–B gradient (0.5% acetonitrile/min) at a flow rate of 0.35 mL/min, where eluent A is 0.2% aqueous trifluoroacetic acid (TFA) and eluent B is 0.2% TFA in acetonitrile. The retention times of the peptides during the change of temperature are shown in panel (A). In panel (B), the retention time of peptides are normalized to 5 °C through the expression $(t_{\mathrm{R}}^t-t_{\mathrm{R}}^5)$, where $t_{\mathrm{R}}^t$ is the retention time at a specific temperature of an antimicrobial peptide or the random coil peptide, and $t_{\mathrm{R}}^5$ is the retention time at 5 °C. In panel (C), the retention behavior of the peptides was normalized to that of the random coil peptide C through the expression ($t_{\mathrm{R}}^t-t_{\mathrm{R}}^5$ for peptides) minus ($t_{\mathrm{R}}^t-t_{\mathrm{R}}^5$ for C). In panels (A–C), the symbols used are: circles for V₆₈₁ and d-V₆₈₁, diamonds for V13A_D and d-V13A_L, squares for V13K_L, d-V13K_D and triangles for random coil peptide C.

Figure 4. Time study of peptide hemolysis of human red blood cells

Closed symbols were used for l-peptides and open symbols were used for d-peptides. Different symbols were used to represent the different concentrations of peptides during the hemolysis study.

Figure 5. Peptide stability to proteolysis by trypsin

Closed symbols were used for l-peptides and open symbols were used for d-peptides. Circles denote V₆₈₁ and d-V₆₈₁, squares denote V13K_L and d-V13K_D, and diamonds denote V13A_D and d-V13A_L.