Leslie H. Kondejewski

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.

New ice‐binding face for type I antifreeze protein

Type I antifreeze protein (AFP) from winter flounder is an alanine‐rich, 37 amino acid, single α‐helix that contains three 11 amino acid repeats (Thr‐X2‐Asx‐X7), where X is generally Ala. The regularly spaced Thr, Asx and Leu residues lie on one face of the helix and have traditionally been thought to form hydrogen bonds and van der Waals interactions with the ice surface. Recently, substitution experiments have called into question the importance of Leu and Asn for ice‐binding. Sequence alignments of five type I AFP isoforms show that Leu and Asn are not well conserved, whereas Ala residues adjacent to the Thr, at right angles to the Leu/Asn‐rich face, are completely conserved. To investigate the role of these Ala residues, a series of Ala to Leu steric mutations was made at various points around the helix. All the substituted peptides were fully α‐helical and remained as monomers in solution. Wild‐type activity was retained in A19L and A20L. A17L, where the substitution lies adjacent to the Thr‐rich face, had no detectable antifreeze activity. The nearby A21L substitution had 10% wild‐type activity and demonstrated weak interactions with the ice surface. We propose a new ice‐binding face for type I AFP that encompasses the conserved Ala‐rich surface and adjacent Thr.

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 β-sheet content, with rings containing 6, 10, and 14 residues exhibiting β-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.

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.

Hydrophilic interaction/cation-exchange chromatography for separation of cyclic peptides

Abstract Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is a novel high-performance technique which provides unique selectivities compared to reversed-phase chromatography (RPLC) for peptide separations. Separations by HILIC/CEX are effected by linear increasing salt gradients in the presence of acetonitrile (up to 90%), which promotes hydrophilic interactions overlayed on ionic interactions with the ion-exchange matrix. In the present study, the utility of HILIC/CEX has been extended to the separation of cyclic peptides in the form of synthetic model analogues of gramicidin S: Series 1 comprised six 10-residue cyclic peptide analogues which exhibited amphipathic, rigid β-pleated sheet conformation; Series 2 comprised 14-residue cyclic diastereomeric analogues of gramicidin S, where only the enantiomeric configuration of a single amino acid side-chain is varied from peptide to peptide. Observation of the retention behaviour of these two series of cyclic peptides during HILIC/CEX and RPLC confirmed not only the excellent complementarity of these two chromatographic modes but also highlighted the dramatic separations achievable by the mixed-mode approach.

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.

Analysis of synthetic peptides by high-performance liquid chromatography.

Publisher Summary High-performance liquid chromatography (HPLC) has proved extremely versatile for the isolation/purification of peptides varying widely in their sources, quantity, and complexity. High-performance chromatographic techniques are suited to the purification of a single peptide from the kind of complex peptide mixtures encountered following solid-phase peptide synthesis, where impurities are closely related to the peptide of interest (deletion, terminated, or chemically modified peptides), perhaps missing only one amino acid residue, and, hence, may be difficult to separate. This chapter focuses on HPLC applications of particular interest to researchers utilizing solid-phase synthesis methods: (1) problem-solving approaches to difficult peptide separations, (2) HPLC monitoring of formation of desired product, (3) and HPLC as a diagnostic tool to detect unwanted side-chain modification. The use of reversed-phase chromatography (RP-HPLC), the most important mode of HPLC for synthetic peptidepurification, is focussed; in addition, the exciting potential of a novel mixed-mode HPLC technique, hydrophilic interaction/cation-exchange chromatography (HILIC/CEC), is demonstrated.

Effect of ring size on conformation and biological activity of cyclic cationic antimicrobial peptides.

In a series of cyclic peptides based on GS10, an analogue of gramicidin S (GS), the ring size was varied from 10 to 16 amino acids. Alternative addition of basic and hydrophobic amino acids to the original GS10 construct generated a variety of even-numbered rings, i.e., GS10 [cyclo-(VKLdYPVKLdYP)], GS12 [cyclo-(VKLKdYPKVKLdYP)], GS14 [cyclo-(VKLKVdYPLKVKLdYP), and GS16 [cyclo-(VKLKVKdYPKLKVKLdYP)] (d stands for d-enantiomers). The odd-numbered analogues (11-, 13-, and 15-mers) were derived from these four peptides by either addition or deletion of single basic (Lys) or hydrophobic (Leu or Val) amino acids. The resulting peptides, divided into three groups on the basis of peptide ring size (10- to 12-meric, 13- and 14-meric, and 15- and 16-meric), illustrated a diverse spectrum of biological activity correlated to their ring size, degree of beta-structure disruption, charge, hydrophobicity, amphipathicity, and affinity for lipid membranes. Two of these peptides with potent antimicrobial activities and high therapeutic indexes (4.5- to 10-fold compared with GS) are promising candidates for development of broad-spectrum antibiotics.

A method for the facile solid-phase synthesis of gramicidin S and its analogs

A simple method is described for the facile synthesis of gramicidin S and six other analogs, using standard solidphase synthetic technology and a single solution-phase cyclization step. The peptides were purified to homogeneity and characterized by plasma desorption time-of-flight mass spectrometry and NMR spectroscopy. Complete 1H NMR assignments for all seven peptides (in aqueous solution) are presented. Unlike previous approaches, the presented method is simple, automatable, rapid (less than three days), high-yielding, requires no side-chain protection during cyclization, and appears to be generally applicable to the preparation of a variety of related head-to-tail cyclic peptides.

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).