Modulation of Structure and Antibacterial and Hemolytic Activity by Ring Size in Cyclic Gramicidin S Analogs

Abstract

We have evaluated the effect of ring size of gramicidin S analogs on secondary structure, lipid binding, lipid disruption, antibacterial and hemolytic activity. Cyclic analogs with ring sizes ranging from 4 to 14 residues were designed to maintain the amphipathic character as found in gramicidin S and synthesized by solid phase peptide synthesis. The secondary structure of these peptides showed a definite periodicity in ␤-sheet content, with rings containing 6, 10, and 14 residues exhibiting ␤-sheet structure, and rings containing 8 or 12 residues being largely disordered. Peptides containing 4 or 6 residues did not bind lipopolysaccharide, whereas longer peptides showed a trend of increasing binding affinity for lipopolysaccharide with increasing length. Destabilization of Escherichia coli outer membranes was only observed in peptides containing 10 or more residues. Peptides containing fewer than 10 residues were completely inactive and exhibited no hemolytic activity. The 10-residue peptide showed an activity profile similar to that of gramicidin S itself, with activity against Grampositive and Gram-negative microorganisms as well as yeast, but also showed high hemolytic activity. Differential activities were obtained by increasing the size of the ring to either 12 or 14 residues. The 14-residue peptide showed no antibiotic activity but exhibited increased hemolytic activity. The 12-residue peptide lost activity against Gram-positive bacteria, retained activity against Gram-negative microorganisms and yeast, but displayed decreased hemolytic activity. Biological activities in the 12-residue peptide were optimized by a series of substitutions in residues comprising both hydrophobic and basic sites resulting in a peptide that exhibited activities comparable with gramicidin S against Gramnegative microorganisms and yeast but with substantially lower hemolytic activity. Compared with gramicidin S, the best analog showed a 10-fold improvement in antibiotic specificity for Gram-negative microorganisms and a 7-fold improvement in specificity for yeast over human erythrocytes as determined by a therapeutic index. These results indicate that it is possible to modulate structure and activities of cyclic gramicidin S analogs by varying ring sizes and further show the potential for developing clinically useful antibiotics based on gramicidin S.

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

“…This result is incongruous with our results, which indicate that GS14 perturbs model lipid membranes very strongly and is a strong inhibitor of A. laidlawii B growth. However, it has been suggested that the weak antimicrobial activity of GS14 against Gram-negative bacteria may be attributable to its binding preferentially to the negatively charged lipopolysaccharides in the cell walls of those organisms (7,24). This suggestion is supported by studies showing that the affinity of GS14 for lipopolysaccharide is at least an order of magnitude greater than those of the other diastereomers examined (8).…”

“…Gramicidin S (GS) 1 is a cyclic decapeptide that exhibits strong antibiotic activity against a broad spectrum of Gramnegative and Gram-positive bacteria and against several pathogenic fungi (3)(4)(5)(6)(7)(8)(9). Numerous studies by us and others have shown that disruption of the barrier properties of cell membranes is the basis of the antimicrobial and hemolytic properties of GS (3,4,10).…”

Gramicidin S identified as a potent inhibitor for cytochrome bd‐type quinol oxidase

“…From the screening of hundreds of natural antibiotics in the Kitasato Institute for Life Sciences Chemical Library [19], we found the inhibitory activity of GS for Escherichia coli cytochrome bd. Recent studies have shown that GS analogs can be designed with the markedly improved therapeutic index, suggesting the possibility of GS derivatives as potent broad-spectrum antibiotics [9,[20][21][22]. Our finding would provide a new insight into molecular design and development of new GS analogs.…”

Isothermal Titration Calorimetry Studies of the Binding of a Rationally Designed Analogue of the Antimicrobial Peptide Gramicidin S to Phospholipid Bilayer Membranes

“…In fact, ITC measures the overall enthalpy (heat changes) which can include (1) heat changes accompanying conformational changes in the peptide molecules; (2) heat changes associated with formation of new noncovalent bonding such as electrostatic, van der Waals and the hydrogen bonding; (3) heat changes associated with desolvation energies such as the displacement or the release of the ordered water molecules from both peptide and membrane surfaces; and (4) heat changes associated with the perturbations of the lipid membrane structure as a result of the binding. The overall enthalpy can thus be conveniently written as (5) The probable contribution of changes in the conformation of GS14dK4 upon transfer from aqueous to membrane environment to the overall enthalpy change is difficult to estimate. Although CD, FTIR, and 1 H NMR spectroscopic studies have all shown that the transfer of GS14dK4 from aqueous to membrane or membrane mimetic environments is accompanied by an increase in β-sheet content (9,11,24), such studies have also shown that the conformational changes that accompany that process are relatively small.…”