Effects of single d‐amino acid substitutions on disruption of β‐sheet structure and hydrophobicity in cyclic 14‐residue antimicrobial peptide analogs related to gramicidin S

Abstract

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure-activity studies of de novo-designed 14-residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single L-amino acid with its corresponding D-enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274, 13181]. The diastereomer with a D-Lys substituted at position 4 caused the greatest improvement in specificity vs. other L to D substitutions within the cyclic 14-residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted beta-sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275, 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a D- or L-amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the D- and L-substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic D-amino acids D-Lys and D-Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the L-diastereomers, which demonstrated strong hemolysis. All of the L-substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the D-substituted peptides tended to improve based on the hydrophilicity of the residue. D-Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic D-amino acid substitutions had superior antimicrobial activity vs. the L-enantiomers although substitution of a hydrophobic D-amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted beta-sheet structure on antimicrobial activity.

Structure-Activity Relationships of Diastereomeric Lysine Ring Size Analogs of the Antimicrobial Peptide Gramicidin S

“…7B). A similar pattern of behavior was reported in studies of other analogs of GS14 (6,8,18,19). The possible basis of these and other aspects of the behavior of these peptides are explored under "Discussion."…”

Strand Length-Dependent Antimicrobial Activity and Membrane-Active Mechanism of Arginine- and Valine-Rich β-Hairpin-Like Antimicrobial Peptides

“…The effects of the quenching reagent on peptide fluorescence intensities were assessed by the quenching constant (K SV ), which was estimated using the Stern-Vol- Outer membrane permeabilization assay. Outer membrane permeabilization activity was determined using the fluorescent dye N-phenyl-1-napthylamine (NPN) as described by Lee et al (19). E. coli UB1005 cells were obtained from the State Key Laboratory of Microbial Technology, Shandong University.…”

Cathelicidin peptide SMAP‐29: comprehensive review of its properties and potential as a novel class of antibiotics

“…For example, diastereomers of the potent but hemolytic antimicrobial peptide melittin were found to have lost hemolytic activity while retaining antibacterial activity and the ability to completely lyse both Gram-positive and Gram-negative bacteria [Oren and Shai, 1997]. Single D-amino acid substitutions of the peptide gramicidin S were also able to improve specificity for microbial cells [Lee et al, 2004].…”