Dissociation of Antimicrobial and Hemolytic Activities in Cyclic Peptide Diastereomers by Systematic Alterations in Amphipathicity

Abstract

We have investigated the role of amphipathicity in a homologous series of head-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activity (i.e. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-YP), designed on the basis of gramicidin S (GS), exists in a preformed highly amphipathic ␤-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of GS14. The ␤-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure inducibility in hydrophobic environments. Because the diastereomers all have the same composition, sequence, and intrinsic hydrophobicity, the amphipathicity of the diastereomers could be ranked based upon retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low antimicrobial activity in the diastereomers. The latter may be the result of increased affinity of highly amphipathic peptides to outer membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The best diastereomer exhibited 130-fold less hemolytic activity compared with GS14, as well as greatly increased antimicrobial activities, resulting in improvement in therapeutic indices of between 1,000-and 10,000-fold for a number of microorganisms. The therapeutic indices of this peptide were between 16-and 32-fold greater than GS for Gram-negative microorganisms and represents a significant improvement in specificity over GS. Our findings show that a highly amphipathic nature is not desirable in the design of constrained cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.

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."…”

“…GS14dK4) usually exhibit antimicrobial activity comparable with that of GS itself but are considerably less hemolytic (8,21). Like GS, these materials are also targeted against lipid bilayer membranes, but they exhibit an enhanced capacity to discriminate between bacterial and animal cell membranes (8,21).…”

“…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

“…4) although it is generally assumed that the principal target of GS is the membrane lipid bilayer rather than the protein components. Recent studies have shown that structural analogs of GS can be designed with markedly reduced hemolytic activity and enhanced microbial activity, suggesting the possibility of GS derivatives as potent oral or injectable broad-spectrum antibiotics [9,[20][21][22]. Our finding would provide a new insight into the molecular design and development of novel GS-based antibiotics (see Fig.…”

“…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.…”