Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity

We have investigated the role of amphipathicity in a homologous series of h ead-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activit y (Le. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-Y P), designed on the basis of gramicidin S (GS), exists in a preformed highl y amphipathic beta-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each containe d a different single enantiomeric substitution within the framework of GS14 , The beta-sheet structure of all GS14 diastereomers was disrupted as deter mined by CD and NMR spectroscopy under aqueous conditions; however, all dia stereomers exhibited differential structure inducibility in hydrophobic env ironments. Because the diastereomers all have the same composition, sequenc e, and intrinsic hydrophobicity, the amphipathicity of the diastereomers co uld be ranked based upon retention time from reversed-phase high performanc e liquid chromatography, There was a clear correlation showing that high am phipathicity resulted in high hemolytic activity and low antimicrobial acti vity in the diastereomers, The latter may be the result of increased affini ty of highly amphipathic peptides to outer membrane components of Gram-nega tive microorganisms. The diastereomers possessing the most favorable therap eutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The be st diastereomer exhibited 130-fold less hemolytic activity compared with GS 14, as well as greatly increased antimicrobial activities, resulting in imp rovement in therapeutic indices of between 1,000- and 10,000 fold for a num ber of microorganisms. The therapeutic indices of this peptide were between 16- and 32-fold greater than GS for Gram-negative microorganisms and repre sents a significant improvement in specificity over GS, Our findings show t hat a highly amphipathic nature is not desirable in the design of constrain ed cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.