Colin T. Mant

Rational Design of α-Helical Antimicrobial Peptides with Enhanced Activities and Specificity/Therapeutic Index

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 °C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the d-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with l-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected d-/l-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.

Role of Peptide Hydrophobicity in the Mechanism of Action of α-Helical Antimicrobial Peptides

ABSTRACT In the present study, the 26-residue amphipathic α-helical antimicrobial peptide V13KL (Y. Chen et al., J. Biol. Chem. 2005, 280:12316-12329, 2005) was used as the framework to study the effects of peptide hydrophobicity on the mechanism of action of antimicrobial peptides. Hydrophobicity was systematically decreased or increased by replacing leucine residues with less hydrophobic alanine residues or replacing alanine residues with more hydrophobic leucine residues on the nonpolar face of the helix, respectively. Hydrophobicity of the nonpolar face of the amphipathic helix was demonstrated to correlate with peptide helicity (measured by circular dichroism spectroscopy) and self-associating ability (measured by reversed-phase high-performance liquid chromatography temperature profiling) in aqueous environments. Higher hydrophobicity was correlated with stronger hemolytic activity. In contrast, there was an optimum hydrophobicity window in which high antimicrobial activity could be obtained. Decreased or increased hydrophobicity beyond this window dramatically decreased antimicrobial activity. The decreased antimicrobial activity at high peptide hydrophobicity can be explained by the strong peptide self-association which prevents the peptide from passing through the cell wall in prokaryotic cells, whereas increased peptide self-association had no effect on peptide access to eukaryotic membranes.

Prediction of peptide retention times in reversed-phase high-performance liquid chromatography I. Determination of retention coefficients of amino acid residues of model synthetic peptides

We have examined the contribution of individual amino acid residues to peptide retention on reversed-phase (RP) columns by measuring their effect on retention of a model synthetic peptide: Ac-Gly-X-X-(Leu)3-(Lys)2-amide, where X is substituted by the 20 amino acids found in proteins. Consistently similar resolution of the 20 peptides on several RP columns enabled the determination of empirical sets of retention coefficients, describing the hydrophobicity of amino acid residues at pH 2.0 and pH 7.0. The much superior resolution and selectivity obtained with acetonitrile, compared to 2-propanol and methanol, confirmed its value as the best organic eluent for most practical purposes. The necessity of using peptides rather than alkylphenones as internal standards for peptide retention prediction is demonstrated and underlined by the predictive accuracy of our coefficients when applied to the resolution of a mixture of five commercially available synthetic peptide standards on several RP columns. Rules for retention time prediction for linear elution gradients, employing our hydrophobicity parameters, of peptides of known composition are presented and enable the researcher to correct for: (a) instrument variations, (b) varying length or diameter of RP columns, (c) varying n-alkyl chain length and ligand density of RP packings and (d) column aging.

ALPHA -HELICAL PROTEIN ASSEMBLY MOTIFS

This review will focus on a-helical protein assembly motifs where the a-helix is the major element of secondary structure involved in the folding and stability of the structure and may also be involved in function by binding to receptor molecules. Apart from the three types of a-helical motifs discussed, i.e. those motifs that form autonomously folded protein domains; those motifs that only form a stable folded domain when dimerized; and a motif that requires other structural elements to contribute to the hydrophobic core to stabilize a folded domain, we will present examples of more complex protein assemblies that have combined two different motifs to form a functional molecule.

Effects of ion-pairing reagents on the prediction of peptide retention in reversed-phase high-resolution liquid chromatography

We have examined the resolution, on reversed-phase columns, of a series of model synthetic peptides and commercially available synthetic peptide standards under gradient elution conditions, using a water-acetonitrile mobile phase containing hydrophilic (phosphoric acid) or hydrophobic (trifluoroacetic acid, heptafluorobutyric acid) ion-pairing reagents. Increasing hydrophobicity or concentration of the ion-pairing reagents increased peptide retention times. It was clearly shown that these reagents effected changes in peptide retention time solely through interaction with the basic residues in the peptide. In general, each positive charge, whether originating from a lysine, arginine or histidine side-chain, or from an N-terminal α-amino group, exerts an equal effect on peptide retention. Different counterions have different effects on the change in peptide retention time per positively charged residue due to their differences in hydrophobicity. However, increasing concentrations of a specific counterion have an essentially equal effect per positively charged residue. These effects are also column dependent (n-alkyl chain length and ligand density). These results, demonstrating a simple relationship between peptide retention in different ion-pairing systems, enabled the determination of rules for prediction of peptide retention times in one ion-pairing system from observed or predicted retention times in another system. The small average deviation of predicted and observed retention times for a series of basic peptides was good evidence for the value of this predictive method. This study provides a clear understanding of the effect of changing counterionn hydrophobicity or concentration on peptide retention, and thus can be extremely beneficial in the purification of peptides and for providing proof of peptide homogeneity.

Prediction of peptide retention times in reversed-phase high-performance liquid chromatography II. Correlation of observed and predicted peptide retention times factors and influencing the retention times of peptides

Abstract We have assessed the accuracy of a set of amino acid residue retention coefficients by applying them to the prediction of the retention times of 58 peptides under linear gradient elution conditions (solvent A = 0.1% trifluoroacetic acid in water, and solvent B = 0.1% trifluoroacetic acid in acetonitrile). These coefficients were determined by examining the retention times of synthetic model peptides in reversed-phase chromatography. The high degree of correlation (0.98) between predicted observed retention times not only indicated good predictive accuracy for our coefficients but was also further evidence that composition is generally the major factor affecting peptide retention time. For optimum accuracy in retention time prediction on any single column, it was essential to include an internal peptide standard in each run to correct for run-to-run deviations and column aging. The resolution of five commercially available synthetic peptide standards was found to improve with increasing flow-rate and decreasing gradient steepness. Increasing temperature resulted in a decrease in peptide retention times and slightly improved resolution. Rules for retention time prediction are presented which not only enable the experimenter to correct for instrument and column (length, diameter, n -alkyl chain length and ligand density) specifications, but also allow the prediction of peptide retention times at any gradient steepness, flow-rate and temperature.

Comparison of Biophysical and Biologic Properties of α‐Helical Enantiomeric Antimicrobial Peptides

In our previous study (Chen et al. J Biol Chem 2005, 280:12316–12329), we utilized an α‐helical antimicrobial peptide V681 as the framework to study the effects of peptide hydrophobicity, amphipathicity, and helicity on biologic activities where we obtained several V681 analogs with dramatic improvement in peptide therapeutic indices against gram‐negative and gram‐positive bacteria. In the present study, the d‐enantiomers of three peptides – V681, V13AD and V13KL were synthesized to compare biophysical and biologic properties with their enantiomeric isomers. Each d‐enantiomer was shown by circular dichroism spectroscopy to be a mirror image of the corresponding l‐isomer in benign conditions and in the presence of 50% trifluoroethanol. l‐ and d‐enantiomers exhibited equivalent antimicrobial activities against a diverse group of Pseudomonas aeruginosa clinical isolates, various gram‐negative and gram‐positive bacteria and a fungus. In addition, l‐ and d‐enantiomeric peptides were equally active in their ability to lyse human red blood cells. The similar activity of l‐ and d‐enantiomeric peptides on prokaryotic or eukaryotic cell membranes suggests that there are no chiral receptors and the cell membrane is the sole target for these peptides. Peptide d‐V13KD showed significant improvements in the therapeutic indices compared with the parent peptide V681 by 53‐fold against P. aeruginosa strains, 80‐fold against gram‐negative bacteria, 69‐fold against gram‐positive bacteria, and 33‐fold against Candida albicans. The excellent stability of d‐enantiomers to trypsin digestion (no proteolysis by trypsin) compared with the rapid breakdown of the l‐enantiomers highlights the advantage of the d‐enantiomers and their potential as clinical therapeutics.

Hydrophilic-interaction chromatography of peptides on hydrophilic and strong cation-exchange columns

Hydrophilic-interaction chromatography (HILIC) was recently introduced as a potentially useful separation mode for the purification of peptides and other polar compounds. The elution order of peptides in HILIC, which separates solutes based on hydrophilic interactions, should be opposite to that obtained in reversed-phase chromatography, which separates solutes based on hydrophobic interactions. Three series of peptides, two of which consisted of positively charged peptides (independent of pH at pH less than 7) and one of which consisted of uncharged or negatively charged peptides (dependent on pH), and which varied in overall hydrophilicity/hydrophobicity, were utilized to examine the separation mechanism and efficiency of HILIC on hydrophilic and strong cation-exchange columns.

Effect of peptide chain length on peptide retention behaviour in reversed-phase chromatogrphy

The use of amino acid retention or hydrophobicity coefficients for the prediction of peptide retention time and/or the elution order on hydrophobic stationary phases is based on the premise that amino acid composition is the major factor affecting peptide retention in reversed-phase chromatography. Although this assumption generally agrees well for small peptides (up to ca. 15 residues), the retention times of increasingly larger peptides are less than expected from a simple summation of retention coefficients. In the present study, we report the synthesis of four series of peptide polymers which vary significantly in overall hydrophobicity and polypeptide chain length (5-50 amino acid residues, Ac = acetyl): Ac-(G-L-G-A-K-G-A-G-V-G)n-amide (n = 1-5), Ac-(G-K-G-L-G)n-amide (n = 1, 2, 4, 6, 8, 10), Ac-(L-G-L-K-A)n-amide (n = 1, 2, 4, 6, 8, 10) and Ac-(L-G-L-K-L)n-amide (n = 1, 2, 4). From the retention behaviour of these peptide polymers on C4, C8 and C18 stationary phases under gradient elution conditions, we have clearly established the effect of polypeptide chain length and hydrophobicity on peptide retention. This, in turn, has enabled us to extend the utility of retention time prediction for peptides containing up to 50 residues by introducing a peptide chain-length correction.

Mixed-mode hydrophilic and ionic interaction chromatography rivals reversed-phase liquid chromatography for the separation of peptides

Abstract Peptide separations based upon mixed-mode hydrophilic and ionic interactions with a strong cation-exchange column have been investigated. The peptide separations were generally achieved by utilizing a linear increasing salt (sodium perchlorate) gradient in the presence of acetonitrile (29–90%, v/v) at pH 7. The presence of acetonitrile in the mobile phase promotes hydrophilic interactions with the hydrophilic stationary phase, these hydrophilic interactions becoming increasingly important to the separation process as the acetonitrile concentration is increased. At acetonitrile concentrations of 20–50% (v/v) in the mobile phase, the peptides utilized in this study were eluted in order of increasing net positive charge, indicating that ionic interactions were dominating the separation process. Peptides with the same net positive charge were also well resolved by an hydrophilic interaction mechanism, being eluted in order of increasing hydrophilicity (decreasing hydrophobicity). At higher acetonitrile concentrations (70–90%, v/v), column selectivity was changed dramatically, with hydrophilic interactions now dominating the separation process. Under these conditions, specific peptides may be eluted earlier or later than less highly charged peptides, depending upon their hydrophilic/hydrophobic character. This mixed-mode methodology was compared to reversed-phase liquid chromatography of the peptides at pH 2 and pH 7. The results of this comparison suggested that mixed-mode hydrophilic-ion-exchange chromatography on a strong cation-exchange column rivals reversed-phase liquid chromatography for peptide separations.