Rational design of alpha-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 degrees 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.     

Structure-activity relationships of de novo designed cyclic antimicrobial peptides based on gramicidin S

The cyclic beta-sheet structure possessed by the 10-residue antibiotic peptide gramicidin S was taken as the structural framework for the de novo design of biologically active peptides with membrane-active properties. We have shown from previous studies that gramicidin S is a broad-spectrum antibiotic effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, but is toxic to human red blood cells. We tested the effect of ring size on antimicrobial activity and hemolytic activity on peptides varying from 4 to 16 residues. Interestingly, we were able to dissociate hemolytic activity and antimicrobial activity by increasing the ring size of the peptide to 14 residues (peptide GS14). Furthermore, we increased specificity for microbial membranes while decreasing toxicity to red blood cells by substituting enantiomers (D-amino acids for L-amino acids and vice versa) into the GS14 sequence. The enantiomeric substitutions all disrupted beta-sheet structure in benign medium and decreased peptide amphipathicity. The least amphipathic peptide, produced by substituting a D-Lys at position 4 of GS14 (peptide GS14K4), also had the highest therapeutic index, i.e., highest degree of specificity for microbial cells over human cells. Solution structures of GS14 analogs solved by NMR spectroscopy showed that the D-amino acid side chain was located on the nonpolar face of GS14K4. Another analog, a beta-sheet peptide with reduced amphipathicity (peptide GS14 K3L4), also had a lysine (lysine 3) on the nonpolar face as determined by the NMR structure. Both GS14K4 and GS14 K3L4 had reduced amphipathicity relative to GS14 and much higher therapeutic indices. Finally, the alteration of the nonpolar face hydrophobicity of GS14K4 analogs provided a range of activities and specificities, where the peptides with the intermediate hydrophobicities among the series had the highest therapeutic indices. The optimal peptide hydrophobicities varied depending on the microorganism being tested, with higher hydrophobicity requirements against Gram-positive bacteria and yeast compared with Gram-negative microorganisms. The net result of these studies suggests that it is possible to rationally design a cyclic membrane-active antimicrobial peptide with high specificity towards prokaryotic (bacterial and fungal) membranes and minimal toxicity to eukaryotic (e.g., mammalian) membranes.     

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 beta-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 beta-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.     

Development of the structural basis for antimicrobial and hemolytic activities of peptides based on gramicidin S and design of novel analogs using NMR spectroscopy

The structures of 14-residue head-to-tail cyclic gramicidin S peptides have been investigated to develop the structural rationale for their antimicrobial and hemolytic profiles. The basis for these studies is GS14 (cyclo(VKLKVdYPLKVKLdYP)), designed as an extension of the naturally occurring antimicrobial peptide. The structure of GS14 has been determined using NMR methods and was found to exist in a highly amphipathic antiparallel beta-sheet conformation. Systematic enantiomeric substitutions within the framework of the GS14 peptide were found to decrease the amphipathicity of this molecule. These results indicated that there was a direct correlation between the high amphipathic character and potent hemolytic activity in the diastereomers, whereas an inverse correlation existed between amphipathicity and antimicrobial function. To define the structural consequences of changing the amphipathic nature of GS14 analogs to maximize antimicrobial activity and to minimize hemolysis, NMR structures were determined in water and the membrane-mimetic solvent trifluoroethanol. The structures show that these attributes are the result of induction of the beta-sheet character in a membrane environment and the positioning of charged side chains on the hydrophobic face of the cyclic framework, thus decreasing the amphipathicity and directed hydrophobicity of these molecules. Implications for the design of more effective antimicrobials are discussed.     

Effects of residue 5-point mutation and N-terminus hydrophobic residues on temporin-SHc physicochemical and biological properties

Temporin-SHc (FLSHIAGFLSNLF_amide) first isolated from skin extraction of the Tunisian frog Pelophylax saharica, which shows potent antimicrobial activity against Gram-positive bacteria and is highly active against yeasts and fungi without hemolytic activity at antimicrobial concentrations. The peptide adopts well-defined α-helical conformation when bound to SDS micelles. In this study, we explored the effects of residue at position 5 and the N-terminus hydrophobic character on the hydrophilic/polar face of temp-SHc, on its biological activities (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity and helicity). Antibacterial and hemolytic properties of temporin-SHc derivatives depend strongly on physicochemical properties. Therefore, slight decreasing amphipathicity together with hydrophobicity and helicity by the substitution Ile⁵ → Leu decreased antimicrobial potency approximately twofold without changing of hemolytic activity. It is noteworthy that a conservative amino acid substitution decreases the antimicrobial activity, underlining the differences between Leu/Ile side chains insertion into the lipid bilayer. While the modification of N-terminal hydrophobic character by four residue inversion decreased amphipathicity (twofold) of (4-1)L₅temp-SHc and resulted in an increase in antibacterial activity against E. coli, E. faecalis and C. parapsilosis of at least fourfold, its therapeutic potential is limited by its drastic increase of hemolysis (LC₅₀ = 2 μM). We found that the percentage of helicity of temp-SHc analog is directly correlated to its hemolytic activity. Last, the hydrophobic N-terminal character is an important determinant of antimicrobial activity.     

Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides

In our previous study, we utilized a 26-residue amphipathic alpha-helical antimicrobial peptide L-V13K (Chen et al., Antimicrob Agents Chemother 2007, 51, 1398-1406) as the framework to study the effects of peptide hydrophobicity on the mechanism of its antimicrobial action. In this study, we explored the effects of net charge and the number of positively charged residues on the hydrophilic/polar face of L-V13K on its biological activity (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity, helicity, and peptide self-association). The net charge of V13K analogs at pH 7 varied between -5 and +10 and the number of positively charged residues varied from 1 to 10. The minimal inhibitory concentrations (MIC) against six strains of Pseudomonas aeruginosa as well as other gram-negative and gram-positive bacteria were determined along with the maximal peptide concentration that produces no hemolysis of human red blood cells (MHC). Our results show that the number of positively charged residues on the polar face and net charge are both important for both antimicrobial activity and hemolytic activity. The most dramatic observation is the sharp transition of hemolytic activity on increasing one positive charge on the polar face of V13K i.e., the change from +8 to +9 resulted in greater than 32-fold increase in hemolytic activity (250 microg/ml to <7.8 microg/ml, respectively).     

Effects of single D-amino acid substitutions on disruption of beta-sheet structure and hydrophobicity in cyclic 14-residue antimicrobial peptide analogs related to gramicidin S.

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure-activity studies of de novo-designed 14-residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single L-amino acid with its corresponding D-enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274, 13181]. The diastereomer with a D-Lys substituted at position 4 caused the greatest improvement in specificity vs. other L to D substitutions within the cyclic 14-residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted beta-sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275, 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a D- or L-amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the D- and L-substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic D-amino acids D-Lys and D-Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the L-diastereomers, which demonstrated strong hemolysis. All of the L-substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the D-substituted peptides tended to improve based on the hydrophilicity of the residue. D-Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic D-amino acid substitutions had superior antimicrobial activity vs. the L-enantiomers although substitution of a hydrophobic D-amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted beta-sheet structure on antimicrobial activity.     

Antimicrobial and cytolytic properties of the frog skin peptide, kassinatuerin-1 and its L- and D-lysine-substituted derivatives

Kassinatuerin-1, a 21-amino-acid C-terminally alpha-amidated peptide first isolated from the skin of the African frog Kassina senegalensis, adopts an amphipathic alpha-helical conformation in a membrane-mimetic solvent (50% trifluoroethanol) and shows broad-spectrum antimicrobial activity. However, its therapeutic potential is limited by its relatively high cytolytic activity against mammalian cells. The antimicrobial and cytolytic properties of a peptide are determined by an interaction between cationicity, hydrophobicity, alpha-helicity and amphipathicity. Replacement of the C-terminal alpha-amide group in kassinatuerin-1 by carboxylic acid decreased both cationicity and alpha-helicity, resulting in an analog with decreased potency against Escherichia coli (4-fold) and Staphylococcus aureus (16-fold). Low cytolytic activities against human erythrocytes (LD50>400 microM) and L929 fibroblasts (LD50=105 microM) were also observed. Increasing cationicity, while maintaining amphipathic alpha-helical character, by progressively substituting Gly7, Ser18, and Asp19 on the hydrophilic face of the alpha-helix with L-lysine, increased antimicrobial potency against S. aureus and Candida albicans (up to 4-fold) but also increased hemolytic and cytolytic activities. In contrast, analogs with d-lysine at positions 7, 18 and 19 retained activity against Gram-negative bacteria but displayed reduced hemolytic and cytolytic activities. For example, the carboxylic acid derivative of [D-Lys7, D-Lys18, D-Lys19]kassinatuerin-1 was active (minimum inhibitory concentration (MIC)=6-12.5 microM) against a range of strongly antibiotic-resistant strains of E. coli but showed no detectable hemolytic activity at 400 microM and was 4-fold less cytolyic than kassinatuerin-1. However, the reduction in alpha-helicity produced by the D-amino acid substitutions resulted in analogs with reduced potencies against Gram-positive bacteria and against C. albicans.     

Strategies for transformation of naturally-occurring amphibian antimicrobial peptides into therapeutically valuable anti-infective agents

The emergence of strains of pathogenic microorganisms with resistance to commonly used antibiotics has necessitated a search for novel types of antimicrobial agents. Many frog species produce amphipathic alpha-helical peptides with broad spectrum antimicrobial activity in the skin but their therapeutic potential is limited by varying degrees of cytolytic activity towards eukaryotic cells. Methods for development of such peptides into anti-infective drugs are illustrated by the example of temporin-1DRa (HFLGTLVNLAK KIL.NH(2)). Studies with model alpha-helical peptides have shown that increase in cationicity promotes antimicrobial activity whereas increases in hydrophobicity, helicity and amphipathicity promote hemolytic activity and loss of selectivity for microorganisms. Analogs of temporin-1DRa in which each amino acid is replaced by L-lysine and D-lysine were synthesized and their cytolytic activities tested against a range of microorganisms and human erythrocytes. Small changes in structure produced marked changes in conformation, as determined by retention time on reversed-phase HPLC, and in biological activity. However, peptides containing the substitutions (Val(7) -->L-Lys), (Thr(5)-->D-Lys) and (Asn(8)-->D-Lys) retained the high solubility and potent, broad spectrum antimicrobial activity of the naturally occurring peptide but were appreciably (up to 10-fold) less hemolytic. In contrast, analogs in which Leu(9) and Ile(13) were replaced by the more hydrophobic cyclohexylglycine residue showed slightly increased antimicrobial potencies (up to 2-fold) but a 4-fold increase in hemolytic activity. The data suggest a strategy of selective increases in cationicity concomitant with decreases in helicity and hydrophobicity in the transformation of naturally-occurring antimicrobial peptides into non-toxic therapeutic agents.     

New potent antimicrobial peptides from the venom of Polistinae wasps and their analogs

Four new peptides of the mastoparan family, characterized recently in the venom of three neotropical social wasps collected in the Dominican Republic, Polistes major major, Polistes dorsalis dorsalis and Mischocyttarus phthisicus were synthesized and tested for antimicrobial potency against Bacillus subtilis, Staphylococcus aureus, Escherichia coli (E.c.) and Pseudomonas aeruginosa, and for hemolytic and mast cells degranulation activities. As these peptides posses strong antimicrobial activity (minimal inhibitory concentration (MIC) values against Bacillus subtillis and E.c. in the range of 5–40 μM), we prepared 40 of their analogs to correlate biological activities, especially antimicrobial, with the net positive charge, hydrophobicity, amphipathicity, peptide length, amino acid substitutions at different positions of the peptide chain, N-terminal acylation and C-terminal deamidation. Circular dichroism spectra of the peptides measured in the presence of trifluoroethanol or SDS showed that the peptides might adopt -helical conformation in such anisotropic environments.     

Conformation and other biophysical properties of cyclic antimicrobial peptides in aqueous solutions.

As a step towards understanding the mechanism of the biological activity of cyclic antimicrobial peptides, the biophysical properties and conformations of four membrane-active cyclic peptide antibiotics, based on gramicidin S (GS), were examined in aqueous environments. These cyclic peptides, GS10 [cyclo(VKLdYP)2], GS12 [cyclo(VKLKdYPKVKLdYP)], GS14 [cyclo(VKLKVdYPLKVKLdYP)] and [d-Lys]4GS14 [cyclo(VKLdKVdYPLKVKLdYP)] (d-amino acid residues are denoted by d and are underlined) had different ring sizes of 10, 12 and 14 residues, were different in structure and amphipathicity, and covered a broad spectrum of hemolytic and antimicrobial activities. GS10, GS12 and [d-Lys]4GS14 were shown to be monomeric in buffer systems with ionic strength biological environments. GS14 was also monomeric at low concentrations, but aggregated at concentrations > 50 microm. The affinity of peptides for self-assembly and interaction with hydrophobic surfaces was related to their free energy of intermolecular interaction. The effects of variations in salt and organic solvent (trifluoroethanol) concentration and temperature on peptide conformation were also examined. Similar to GS, GS10 proved to have a stable and rather rigid conformation in different environments and over a broad range of temperatures, whereas GS12, GS14 and [d-Lys]4GS14 had more flexible conformations. Despite its conformational similarity to GS10, GS14 had unique physicochemical properties due to its tendency to aggregate at relatively low concentrations. The biophysical data explain the direct relation between structure, amphipathicity and hydrophobicity of the cyclic peptides and their hemolytic activity. However, this relation with the antimicrobial activity of the peptides is of a more complex nature due to the diversity in membrane structures of microorganisms.     

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH₂ (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH₂ (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.     

Structure‐activity relationship of indolicidin,a Trp‐rich antibacterial peptide

A series of Trp and Arg analogs of antibacterial indolicidin (Ind) was synthesized and the antimicrobial and hemolytic activities were investigated. [L⁹]Ind, [L¹¹]Ind, [K⁸,L⁹]Ind and [K^{6, 8},L⁹]Ind showed desirable characteristics, exhibiting negligible hemolytic activity while keeping strong antibacterial activity. The results indicated that the Trp residue at position 11 essentially contributes to both activities and one can not be exchanged for the other, whereas the Trp residues at positions 4 and 9 play important roles in antimicrobial and hemolytic activities, respectively. The Trp residues at positions 6 and 8 play no important roles in biological activities. We then found that the retro analog of Ind showed higher antibacterial activity than Ind against both Gram‐positive and Gram‐negative bacteria but remarkably lower hemolytic activity than that of Ind. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae)

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH₂ (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8–Cys23 and Cys11–Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.     

Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy.

Mastoparan B (MP-B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic alpha-helical peptide. MP-B possesses a variety of biological activities, such as mast cells degradation histamine release, erythrocyte lysis and inhibition of the growth of gram-positive and gram-negative bacteria. In order to study the relationship between the structure and the biological activity of MP-B, we used four analogs by replacing amino acids with alanine. Tertiary structures of MP-B and its analogs in 2,2,2-trifluoroethanol (TFE)-containing aqueous solution have been determined by NMR spectroscopy and molecular modeling. The results indicate that [Ala4]MP-B and [Ala12]MP-B with higher hydrophobicity adopt a higher content of amphiphilic helical structures, and have better antimicrobial and hemolytic activities than MP-B. However, [Ala3]MP-B and [Ala9]MP-B with lower hydrophobicity have disordered structures. [Ala3]MP-B and [Ala9]MP-B have low antimicrobial activity and much less hemolytic activity relative to MP-B. It is likely that tryptophan residue in MP-B and appropriate hydrophobicity of MP-B to induce alpha-helical structure is essential for the antibacterial and hemolytic activity of MP-B. This study can aid understanding of the structure-activity relationship of MP-B and to design peptides to possess lytic activity.     

Deletion of all cysteines in tachyplesin I abolishes hemolytic activity and retains antimicrobial activity and lipopolysaccharide selective binding

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.     

Structure-activity studies of normal and retro pig cecropin-melittin hybrids.

Chimeric analogs of cecropin P1 and melittin with normal and retro sequences were synthesized to explore the effect of sequence, amide bond direction (helical dipole), charge, amphipathicity and hydrophobicity on their antibacterial activity and channel-forming ability. When viewed from the opposite end by rotation in the plane 180 degrees retro analogs have the same sequence as the parent with reversed amide bond and helical dipole directions. The expected activities were related to the important structural features and a series of assumptions were made. Retro analogs are expected to be inactive if both sequence and amide bond direction make critical contributions to the activity. CP1(1-10)M(2-9) amide, (SWLSKTAKKLIGAVLKVL), showed a broad antibacterial spectrum with high activity against the two Gram-negative and three Gram-positive bacteria tested. Retro-CP1(1-10)M(2-9) was less active compared to its normal peptide. CP1(1-9)M(1-8) and CP1(1-9)M(2-8) amides were found to be active against Gram-negative Escherichia coli and also Gram-positive Streptococcus pyogenes, but inactive against the other test organisms. The corresponding retro analogs were inactive against all the five bacteria tested. These results suggest that both sequence and amide bond direction (helix dipole) are important structural requirements for the activity of CP1-M hybrids. Acetylation of the N-terminal amine in both normal and retro analogs lowered their activity, indicating the contribution of free amine to the activity. These analogs form ion-conducting channels in lipid bilayers. The action of the peptides may be explained by self-aggregation and formation of ion-conducting pores across bacterial membranes. Conformational analysis obtained from CD measurements showed that all analogs form amphipathic alpha-helices in presence of 12-20% hexafluoro isopropanol. The retro CP1(1-10)M(2-9) amide showed higher helicity and is more potent compared to other retro analogs synthesized. These studies show the effect of small sequence modifications on the biological activity of the peptides and on their alpha-helical conformation in HFIP, the structure-inducing organic solvent.     

Mechanism of action and relationship between structure and biological activity of Ctx-Ha: a new ceratotoxin-like peptide from Hypsiboas albopunctatus

The increase in bacterial resistance to current antibiotics has led to the development of new active molecules. We have isolated the antimicrobial peptide Ctx-Ha from the skin secretion of the frog Hypsiboas albopunctatus. The aim of the present work was to elucidate the mechanism of action of this new antimicrobial peptide. The sequence similarity with Ceratotoxin, the pore size, and the pore-like release of carboxyfluorescein from vesicles indicated that Ctx(Ile21)-Ha has a mechanism of action based on the barrel- stave model. In a second part of this work, we synthesized three analogues to provide information about the relationship between the peptide's structure and its biological activity. Ctx(Ile21)-Ha-VD 16, Ctx(Ile21)- Ha-VD 5,16 and Ctx(Ile21)-Ha-I9K were designed to disrupt the peptide's helical structure and change the hydrophobicity/ hydrophilicity and amphipathicity of the apolar face in order to uncouple the antimicrobial activity of Ctx(Ile21)-Ha from its hemolytic activity. To evaluate the effects of the amino acid substitutions on peptide conformation, secondary structure was accessed using CD measurements. The peptides presented a high amount of α-helical structure in the presence of TFE and LPC. The CD data showed that destruction of the amphipathic α-helix by the replacing isoleucine by lysine is less harmful to the structure than D-amino acid substitutions. Biological tests demonstrated that all peptides have activity. Nevertheless, the peptide Ctx(Ile21)-Ha-I9K showed the highest value of therapeutic index. Our findings suggest that these peptides are potential templates for the development of new antimicrobial drugs. These studies highlight the importance of single amino acid modification as a tool to modulate the biological activity of antimicrobial peptides.     

Synthesis of analogs of peptides from Buthus martensii scorpion venom with potential antibiotic activity

Five analogs of a natural peptide (BmKn1) found in the venom of scorpion Buthus martensii Karsh have been synthesized and tested to compare their antimicrobial and hemolytic activity with the wild type. Circular dichroism spectra show that these peptides form an alpha helix structure and its amino acid positions predict an amphipathic nature. Results show that increasing hydrophobicity by substituting successively positions 5 and 9 of the sequence (on the hydrophobic side of the helix) with alanine, valine and leucine enhances antimicrobial activity and hemolysis. When changes are done on positions 7 and 10 (on the hydrophilic side) by introducing more positive charges with addition of lysine, both activities also increase. However, when negative charges are introduced instead (with glutamic acids), antimicrobial activity is observed but hemolysis is reduced to zero under the concentrations studied. Although strong inhibitory activity begins at low concentrations (10 μg/mL), some peptides level off inhibition and no change is observed as concentrations are increased.     

De novo designed cyclic cationic peptides as inhibitors of plant pathogenic bacteria

Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of l-Phe with d-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 microM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t(R)), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a d-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t(R). The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.