Susan W. Farmer

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.

Interactions of bacterial cationic peptide antibiotics with outer and cytoplasmic membranes of Pseudomonas aeruginosa.

ABSTRACT Polymyxins B and E1 and gramicidin S are bacterium-derived cationic antimicrobial peptides. The polymyxins were more potent than gramicidin S against Pseudomonas aeruginosa, with MICs of 0.125 to 0.25 and 8 μg/ml, respectively. These peptides differed in their affinities for binding to lipopolysaccharide, but all were able to permeabilize the outer membrane of wild-type P. aeruginosaPAO1 strain H103, suggesting differences in their mechanisms of self-promoted uptake. Gramicidin S caused rapid depolarization of the bacterial cytoplasmic membrane at concentrations at which no killing was observed within 30 min, whereas, conversely, the concentrations of the polymyxins that resulted in rapid killing resulted in minimal depolarization. These data indicate that the depolarization of the cytoplasmic membrane by these peptides did not correlate with bacterial cell lethality.

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.

Screening and Characterization of Surface-Tethered Cationic Peptides for Antimicrobial Activity

There is an urgent need to coat the surfaces of medical devices, including implants, with antimicrobial agents to reduce the risk of infection. A peptide array technology was modified to permit the screening of short peptides for antimicrobial activity while tethered to a surface. Cellulose-amino-hydroxypropyl ether (CAPE) linker chemistry was used to synthesize, on a cellulose support, peptides that remained covalently bound during biological assays. Among 122 tested sequences, the best surface-tethered 9-, 12-, and 13-mer peptides were found to be highly antimicrobial against bacteria and fungi, as confirmed using alternative surface materials and coupling strategies as well as coupling through the C and N termini of the peptides. Structure-activity modeling of the structural features determining the activity of tethered peptides indicated that the extent and positioning of positive charges and hydrophobic residues were influential in determining activity.

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.

Antimicrobial constituents of Rhus glabra

The antimicrobial activity of the methanol extract and isolated constituents of Rhus glabra (Anacardiaceae), a species used in folk medicine by North American native people, was evaluated against 11 microorganisms, including gram-positive and gram-negative bacteria. The extract was subsequently fractionated and monitored by bioassays leading to the isolation of three antibacterial compounds, the methyl ester of 3,4,5-trihydroxybenzoic acid (methyl gallate) (minimal inhibition concentration (MIC) 12.5 micrograms/ml), 4-methoxy-3,5-dihydroxybenzoic acid (MIC 25 micrograms/ml) and gallic acid (MIC > 1000 micrograms/ml). The first two compounds are reported here for the first time from Rhus glabra. Their structures were established using spectroscopic and chemical methods.

Interaction of Cationic Peptides with Bacterial Membranes

A common feature of cationic peptides is that their site of action is at the membrane due to channel formation, and that they tend to possess strong selectivity towards their target membrane. For example, although moth cecropin and bee melittin are members of the same family of peptides that adopt amphipathic a-helical structures, the cecropins are strongly antibacterial and demonstrate minimal eukaryotic selectivity (i.e., toxicity), whereas melittin is a weak antibacterial compound but a potent toxin. Whereas the basis for selectivity is not completely understood, it has been shown to be due to the size of the transmembrane electrical potential gradient (up to -140 mV in bacterial cytoplasmic membranes compared with about -20 mV or less in eukaryotic membranes) and the lipid composition (bacterial membranes contain a large number of anionic lipids such as phosphatidyl glycerol and cardiolipin and lack cholesterol in their membranes). Gram-negative bacteria have an additional, outer membrane, and our data suggests that a further level of selectivity is expressed there in that there are Gram-positive bacteria-selective peptides that interact poorly with the outer membrane but (presumably) well with cytoplasmic membranes, whereas we have identified peptides that interact with the outer membrane, but are not bactericidal and thus do not interact with cytoplasmic membranes. Although specific details may vary depending on the peptide, enough data exist to present a general model for the mechanism of action of cationic peptides against Grara-negative bacteria. This process is described below, and can be summarized as a sequence of events involving interaction with lipopolysac-

Antimicrobial Compounds from Alnus Rubra

AbstractThe methanol extract of the bark of Alnus rubra showed antimicrobial activity against Gram-positive and Gram-negative bacteria. Diarylheptanoid and its glycoside (oregonin) were identified as the two constituents responsible for this activity.

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).

Disruption of the β-Sheet Structure of Cyclic Peptides by Single Amino Acid Substitution: Influence on Prokaryotic and Eukaryotic Cell Viability

We have studied the effect of ring size on cyclic β-sheet peptides and showed that we were able to dissociate antimicrobial activity from hemolytic activity [1]. The 14-residue peptide, GS 14, displayed weak antimicrobial activity and strong hemolytic activity. In a subsequent study of GS 14 analogs, we reversed this profile to create peptides with strong antimicrobial activity and weak hemolytic activity. Single amino acids within the GS 14 sequence were substituted with their corresponding D- or L-enantiomers at all 14 ring positions. These analogs had reduced amphipathicity and disrupted β-sheet structure when compared to GS 14 (represented in Figure 1, left) [2,3]. Of these analogs, the D-Lys substitution at position 4 (Figure 1, right) yielded the largest increase in therapeutic index (lytic specificity for prokaryotic vs eukaryotic cells) [3]. Here, we substitute other amino acids (both D- and L-enantiomers) at position 4 and study their influence on hemolytic and antimicrobial activity.