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

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 beta-sheet content, with rings containing 6, 10, and 14 residues exhibiting beta-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 Gram-positive 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 Gram-negative 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.