Masood Jelokhani-Niaraki

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 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 β-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 β-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.

Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes.

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.

Effect of α, α-dialkyl amino acids on the protease resistance of peptides

A tryptic [EC 3.4.21.4] digestion assay of 2-aminoisobutyric acid (Aib)-containing peptides was carried out to investigate the effect of α,α-dialkyl amino acid residues on the protease resistance. The introduction of Aib residues to the P1′ positions exhibited a 19-fold higher protease resistance than the peptide with Aib residues introduced to the P2 position or the non-Aib peptide. The peptide having Aib residues introduced to the P1′ and P2 positions resulted in complete resistance.

Antifreeze protein from shorthorn sculpin: Identification of the ice‐binding surface

Shorthorn sculpins, Myoxocephalus scorpius, are protected from freezing in icy seawater by alanine‐rich, α‐helical antifreeze proteins (AFPs). The major serum isoform (SS‐8) has been reisolated and analyzed to establish its correct sequence. Over most of its length, this 42 amino acid protein is predicted to be an amphipathic α‐helix with one face entirely composed of Ala residues. The other side of the helix, which is more heterogeneous and hydrophilic, contains several Lys. Computer simulations had suggested previously that these Lys residues were involved in binding of the peptide to the {11–20} plane of ice in the <−1102> direction. To test this hypothesis, a series of SS‐8 variants were generated with single Ala to Lys substitutions at various points around the helix. All of the peptides retained significant α‐helicity and remained as monomers in solution. Substitutions on the hydrophilic helix face at position 16, 19, or 22 had no obvious effect, but those on the adjacent Ala‐rich surface at positions 17, 21, and 25 abolished antifreeze activity. These results, with support from our own modeling and docking studies, show that the helix interacts with the ice surface via the conserved alanine face, and lend support to the emerging idea that the interaction of fish AFPs with ice involves appreciable hydrophobic interactions. Furthermore, our modeling suggests a new N terminus cap structure, which helps to stabilize the helix, whereas the role of the lysines on the hydrophilic face may be to enhance solubility of the protein.

Modulation of specificity in cyclic antimicrobial peptides by amphipathicity

Amphipathicity of antimicrobial peptides is known to be a factor important for both antimicrobial as well as anti-eukaryotic activity. We have previously shown in cyclic antimicrobial peptides related to the head-to-tail cyclic decameric peptide gramicidin S, that changes in ring size can modulate amphipathicity through changes in secondary structure [1,2]. In a separate study we showed that the systematic incorporation of enantiomeric substitutions within the framework of a highly amphipathic cyclic tetradecameric peptide, GS14, resulted in disruption of structure and reduced amphipathicity relative to GS14 [3]. In both cases the reduction of peptide amphipathicity caused the dissociation of hemolytic activity from antimicrobial activity and resulted in peptides with a high specificity (therapeutic index).

Variation in Ring Size of Cyclic Antimicrobial Peptides Results in Diversity and Selectivity of Biological Activity

In continuing our efforts to understand the structure-function relationships in the cationic cyclic antimicrobial peptides [e.g.,1,2], the ring size was varied from 10 to 16 amino acids in a series of cyclic peptides based on the structure of GS 10 (a decameric analog of the antimicrobial peptide gramicidin S) to study the effect of changes in peptide size, structure, hydrophobicity and amphipathicity on biological activity. Similar to gramicidin S, GS 10 adopts a structure composed of an antiparallel β-sheet and two type II′ β-turns, is highly hemolytic and also quite active against a broad spectrum of bacteria and fungi [2]. The diversity in physicochemical and biological properties of the designed cyclic peptides of this study reveals the complex nature of their mechanism of biological activity.

Interaction of gramicidin S and its biologically active analogs with phospholipid bilayers

Gramicidin S (GS) is a cyclic decameric peptide, which is biologically active as a potent antibiotic against a wide range of bacteria and fungi [1]. GS is also very hemolytic against human erythrocytes. Interaction of GS with lipid bilayers of cell membranes is believed to play a major role in its biological activity. In order to develop insight into the mechanism of interaction of GS and GS-like biologically active peptides with lipid bilayers, we have utilized a series of GS analogs with distinct structural and functional features in a comparative study.

Effect of environment on the stability of helical Aib-model peptides

It is well known that peptides containing Aib residues form αand/or 3 10 helical structures leading to voltage-dependent ion channel in lipid membranes. We have previously reported the synthesis of Aib-containing gramicidin analogs [1,2] and model-peptides, Ac-(Aib–Lys–Aib–Ala)n -NH2 (n = 1–5, BKBA-N , N denotes the number of residues). Among the Aib-peptides, the longer peptide, BKBA-20 may aggregate and form alamethicin-like barrel-stave conformations in phospholipid bilayers [2]. In the present study, to characterize the structures and ion-selectivities of the Aib-peptides containing charged or aromatic residue, we have synthesized novel Aib-peptides containing Glu, Ser, Gly and Trp residues (Figure 1). The conformational stabilities of the synthesized peptides in aqueous and phospholipid membrane environments have been investigated by means of CD spectroscopy.