Structure-activity relationships of diastereomeric lysine ring size analogs of the antimicrobial peptide gramicidin S: mechanism of action and discrimination between bacterial and animal cell membranes

Abstract

Structure-activity relationships were examined in seven gramicidin S analogs in which the ring-expanded analog GS14 [cyclo-(VKLKVdYPLKVKLdYP)] is modified by enantiomeric inversions of its lysine residues. The conformation, amphiphilicity, and self-association propensity of these peptides were investigated by circular dichroism spectroscopy and reversed phase high performance liquid chromatography. (31)P nuclear magnetic resonance spectroscopic and dye leakage experiments were performed to evaluate the capacity of these peptides to induce inverse nonlamellar phases in, and to permeabilize phospholipid bilayers; their growth inhibitory activity against the cell wall-less mollicute Acholeplasma laidlawii B was also examined. The amount and stability of beta-sheet structure, effective hydrophobicity, propensity for self-association in water, ability to disrupt the organization of phospholipid bilayers, and ability to inhibit A. laidlawii B growth are strongly correlated with the facial amphiphilicity of these GS14 analogs. Also, the magnitude of the parameters segregate these peptides into three groups, consisting of GS14, the four single inversion analogs, and the two multiple inversion analogs. The capacity of these peptides to differentiate between bacterial and animal cell membranes exhibits a biphasic relationship with peptide amphiphilicity, suggesting that there may only be a narrow range of peptide amphiphilicity within which it is possible to achieve the dual therapeutic requirements of high antibiotic effectiveness and low hemolytic activity. These results were rationalized by considering how the physiochemical properties of these GS14 analogs are likely to be reflected in their partitioning into lipid bilayer membranes.

Fig. 1. CD spectra of GS14 and its diastereomeric derivatives dissolved in aqueous solution (A), 50% TFE (B), and in SDS-containing aqueous media (C)

The spectra shown are representative of GS14 (■), GS14dK₂(△), GS14d K₄ (○), GS14d K₉ (◆),GS14d K₁₁ (☆), GS14d K₂K₄ (*), and GS14d. K₂K₄K₉K₁₁ (▲)

Fig. 2. Effect of temperature on the retention times of GS14 and its di-astereomeric analogs on a RP-HPLC column

The temperature profiling curves of the four diastereomers obtained by single enantiomeric inversions of the Lys residues of GS14 are shown on an expanded scale in the right panel. Data are presented for GS14 (■), GS14d K₂(△), GS14d K₄ (○), GS14dK₉ (◆), GS14d K₁₁ (☆) GS14dK₂K₄ (*), and GS14dK₂K₄K₉K₁₁ (▲)

Fig. 3. Proton-decoupled ³¹P NMR powder patterns exhibited by mixtures of GS14 and some of its dia-stereomeric Lys derivatives with DPEPE

The spectra shown were acquired at the temperatures indicated using mixtures with an overall lipid: peptide ratio of 25:1.

Fig. 4. Temperature-dependent changes in the relative areas of the isotropic peak (~2 ppm downfield) in the ³¹P NMR spectra exhibited by DPEPE mixtures with GS14 and the various diaste-reomeric Lys derivatives studied

Data are presented for GS14 (■), GS14d K₂(△), GS14d K₄ (○), GS14d K₉ (◆), GS14d K₁₁ (☆), GS14d K₂K₄ (*), and GS14d K₂K₄K₉K₁₁ (▲)

Fig. 5. Peptide induced dye release from large unilamellar POPC vesicles

Data are presented as a function of peptide concentration for the following peptides: GS14 ((■), GS14d K₂ (△), GS14dK₄(○), GS14d K₉ (◆), GS14d K₁₁ (☆), GS14d K₂K₄ (*), and GS14d. K₂K₄K₉K₁₁(▲)

Fig. 6. Effect of GS14 and its diastereomeric analogs on the growth of A. laidlawii B

Data are presented as a function of peptide concentration for the following peptides: GS14 (■), GS14dK₂ (△), GS14dK₄ (○), GS14dK₉ (◆), GS14dK₁₁ (☆), GS14dK₂K₄ (*), and GS14dK₂K₄K₉K₁₁ (▲).

Fig. 7. Plots of the therapeutic indices (A) and the antibiotic (B, upper line) and hemolytic activities (B, lower line) of GS14 and its lysine diastereomers as a function of the retention times of the peptides on a RP-HPLC column

Antibiotic activities were measured against A. laidlawii B and are expressed as the negative logarithm of the apparent LD₅₀ values determined from the data presented in Fig. 6. The hemolytic activity data were obtained from Ref. . The therapeutic indices are the ratios between the apparent LD₅₀ values for inhibition of A. laidlawii B growth and the hemolytic activity and are thus a gauge of the capacity of these peptides to discriminate between A. laidlawii B cells and erythrocytes. The symbols represent data points obtained with the following peptides: GS14 (■), GS14dK₂ (△), GS14dK₄ (○), GS14dK₉ (◆), GS14dK₁₁ (☆), GS14dK₂K₄ (*), and GS14dK₂K₄K₉K₁₁ (▲).

Fig. 8. Molecular models of the antibiotic peptides GS14 (A) and GS14-dK₄ (B and C)

The left images show side views of the peptide backbone and the orientations of the Lys (K) and hydrophobic (unlabeled) side chains relative to the ring plane. The right images show top views of the peptide backbone with the * symbols marking the positions of the inward oriented Lys C_α protons of GS14 and the dashed lines representing connectivities where the distance and orientation of the amide carbonyls and amide protons are favorable for cross-ring hydrogen bonding. The GS14 model shown was obtained from minimized structures published by Gibbs et al. (27), and the GS14-dK₄ models were obtained from the NMR solution structures in water (B) and 30% TFE (C) determined by McInnes et al. (21).