Effect of aminoisobutyric acid (Aib) substitutions on the antimicrobial and cytolytic activities of the frog skin peptide, temporin-1DRa

Temporin-1DRa (HFLGTLVNLAKKIL.NH(2)), first isolated from the skin of the California red-legged frog Rana draytonii, shows broad-spectrum antimicrobial activity but its therapeutic potential is limited by its toxicity against mammalian cells. The cytolytic properties of cationic alpha-helical peptides are determined by a complex interaction between cationicity, hydrophobicity, conformation, and amphipathicity. This study has investigated the cytolytic properties of conformationally constrained analogs of temporin-1DRa containing alpha-aminoisobutyric acid (Aib) substitutions. Cytolytic activity was determined against the bacteria Escherichia coli and Staphylococcus aureus, the opportunistic yeast pathogen, Candida albicans, human erythrocytes, HepG2 hepatoma-derived cells, and L929 fibroblasts. Aib substitutions at Gly(4), Asn(8), and Ala(10) increased both % helicity, determined in methanol solution, and hydrophobicity resulting in increases in both antimicrobial potencies and toxicities against the mammalian cells. Substitution at Leu(6) resulted in an appreciable decrease in cytolytic activity against all cells whereas the substitutions at His(1), Phe(2), Leu(3), Thr(5), and Val(7) had only minor effects on activity. Substitutions at Leu(9), Ile(13), Leu(14) produced analogs with decreased helicity and hydrophobicity that retained activity against microorganisms but showed appreciably lower cytolytic activities against mammalian cells. In particular, the fourfold increase in therapeutic index [ratio of LC(50) against erythrocytes to minimum inhibitory concentration (MIC) against microorganisms] of [Aib(13)]temporin-1DRa identifies it as a compound with potential for development as a therapeutically valuable anti-infective agent.     

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.     

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.     

Antimicrobial properties of the frog skin peptide, ranatuerin-1 and its [Lys-8]-substituted analog

The predicted conformation of ranatuerin-1 (SMLSVLKNLG(10)KVGLGFVACK(20)INK QC), an antimicrobial peptide first isolated from the skin of the bullfrog Rana catesbeiana, comprises three structural domains: alpha-helix (residues 1-8), beta-sheet (residues 11-16) and beta-turn (residues 20-25). Circular dichroism studies confirm significant alpha-helical character in 50% trifluoroethanol. Replacement of Cys-19 and Cys-25 by serine resulted only in decreased antimicrobial potency but deletion of either the cyclic heptapeptide region [residues (19-25)] or the N-terminal domain [residues (1-8)] produced inactive analogs. Substitution of the glycine residues in the central domain of the [Ser-19, Ser-25] analog by lysine produced inactive peptides despite increased alpha-helical content and cationicity. The substitution Asn-8-->Lys gave a ranatuerin-1 analog with increased alpha-helicity and cationicity and increased potency against a range of Gram-positive and Gram-negative bacteria and against C. albicans but only a small increase (21%) in hemolytic activity. In contrast, increasing alpha-helicity and hydrophobicity by the substitution Asn-22-->Ala resulted in a 3.5-fold increase in hemolytic activity. Effects on antimicrobial potencies of substitutions of neutral amino acids at positions 4, 18, 22, and 24 by lysine were less marked. Strains of pathogenic E. coli from different groups showed varying degrees of sensitivity to ranatuerin-1 (MIC between 5 and 40 microM) but [Lys-8] ranatuerin-1 showed increased potency (between 2- and 8-fold; P < 0.01) against all strains. The data demonstrate that [Lys-8] ranatuerin-1 shows potential as a candidate for drug development.     

Advances in antimicrobial peptide immunobiology

Antimicrobial peptides are ancient components of the innate immune system and have been isolated from organisms spanning the phylogenetic spectrum. Over an evolutionary time span, these peptides have retained potency, in the face of highly mutable target microorganisms. This fact suggests important coevolutionary influences in the host-pathogen relationship. Despite their diverse origins, the majority of antimicrobial peptides have common biophysical parameters that are likely essential for activity, including small size, cationicity, and amphipathicity. Although more than 900 different antimicrobial peptides have been characterized, most can be grouped as belonging to one of three structural classes: (1) linear, often of alpha-helical propensity; (2) cysteine stabilized, most commonly conforming to beta-sheet structure; and (3) those with one or more predominant amino acid residues, but variable in structure. Interestingly, these biophysical and structural features are retained in ribosomally as well as nonribosomally synthesized peptides. Therefore, it appears that a relatively limited set of physicochemical features is required for antimicrobial peptide efficacy against a broad spectrum of microbial pathogens.During the past several years, a number of themes have emerged within the field of antimicrobial peptide immunobiology. One developing area expands upon known microbicidal mechanisms of antimicrobial peptides to include targets beyond the plasma membrane. Examples include antimicrobial peptide activity involving structures such as extracellular polysaccharide and cell wall components, as well as the identification of an increasing number of intracellular targets. Additional areas of interest include an expanding recognition of antimicrobial peptide multifunctionality, and the identification of large antimicrobial proteins, and antimicrobial peptide or protein fragments derived thereof. The following discussion highlights such recent developments in antimicrobial peptide immunobiology, with an emphasis on the biophysical aspects of host-defense polypeptide action and mechanisms of microbial resistance.     

Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae).

Seven peptides (XT-1-XT-7) with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of the diploid clawed frog, Xenopus tropicalis. Structural characterization of the peptides demonstrated that amino acid sequence similarity to antimicrobial peptides previously isolated from Xenopus laevis was low, suggesting that the species are not closely related phylogenetically. Peptides XT-5 and XT-3 are probably the orthologs of X. laevis peptide glycine-leucine amide (PGL(a)) and the N-terminal spacer region of prolevitide, respectively. XT-1, XT-6 and XT-7 show limited structural similarity to the spacer region of X. laevis procaeruleins and the paralogs XT-2 and XT-4 are similar to corresponding regions of proxenopsin. Orthologs of the magainins were not identified. The C-terminally alpha-amidated peptide XT-7 (GLLGPLLKIAAKVGSNLL.NH2) showed the lowest minimum inhibitory concentrations against reference microorganisms (Staphylococcus aureus 5 microM, Escherichia coli 5 microM, and Candida albicans 40 microM) and was also active against clinical isolates of methicillin-resistant S. aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus group C, Shigella sonnei, Pseudomonas aeruginosa and Enterobacter cloacae. The peptide was, however, hemolytic against human erythrocytes (50% lysis at 70 microM). Circular dichroism studies showed that XT-7 has a random structure in aqueous solution, pH 7.0 but adopts an alpha-helical conformation in the presence of 50% trifluoroethanol. Decreasing the cationicity of XT-7 either by replacement of the C-terminal CONH2 group by COOH or by deletion of the Lys(8) residue produced analogs with greatly (>10-fold) decreased antimicrobial potencies.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris.

Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.     

Promotion of peptide antimicrobial activity by fatty acid conjugation

Three peptides, YGAA[KKAAKAA](2) (AKK), KLFKRHLKWKII (SC4), and YG[AKAKAAKA](2) (KAK), were conjugated with lauric acid and tested for the effect on their structure, antibacterial activity, and eukaryotic cell toxicity. The conjugated AKK and SC4 peptides showed increased antimicrobial activity relative to unconjugated peptides, but the conjugated KAK peptide did not. The circular dichroism spectrum of AKK showed a significantly larger increase in its alpha-helical content in the conjugated form than peptide KAK in a solution containing phosphatidylethanolamine/phosphotidylglycerol vesicles, which mimics bacterial membranes. The KAK and AKK peptides and their corresponding fatty acid conjugates showed little change in their structure in the presence of phosphatidylcholine vesicles, which mimic the cell membrane of eukaryotic cells. The hemolytic activity of the KAK and AKK peptides and conjugates was low. However, the SC4 fatty acid conjugate showed a large increase in hemolytic activity and a corresponding increase in helical content in the presence of phosphatidylcholine vesicles. These results support the model of antimicrobial peptide hemolytic and antimicrobial activity being linked to changes in secondary structure as the peptides interact with lipid membranes. Fatty acid conjugation may improve the usefulness of peptides as antimicrobial agents by enhancing their ability to form secondary structures upon interacting with the bacterial membranes.     

A family of brevinin-2 peptides with potent activity against Pseudomonas aeruginosa from the skin of the Hokkaido frog, Rana pirica

Nine peptides displaying varying degrees of antimicrobial activity were extracted from the skin of the Hokkaido frog, Rana pirica. Five structurally related peptides were identified as members of the brevinin-2 family. These peptides were active against reference strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) and Gram-positive (Staphlococcus aureus) bacteria but displayed relatively low hemolytic activity. The most abundant peptide, brevinin-2PRa (680 nmol/g weight of dry skin) showed high potency [minimal inhibitory concentration (MIC) values between 6 and 12 microM] against a range of clinical isolates of P. aeruginosa. In addition, activity was unaffected by NaCl concentrations up to 200 mM. Cladistic analysis based on the primary structures of brevinin-2 peptides supports a close phylogenetic relationship between R. pirica and Japanese mountain brown frog Rana ornativentris. One peptide of the ranatuerin-2 family and one strongly hemolytic peptide of the brevinin-1 family were also isolated from the extract along with two members of the temporin family, temporin-1PRa (ILPILGNLLNGLL.NH(2)) and temporin-1PRb (ILPILGNLLNSLL.NH(2)) that atypically lacked basic amino acid residues and showed only very weak antimicrobial and hemolytic activity.     

Purification,structure-function analysis,and molecular characterization of novel linear peptides from scorpion Opisthacanthus madagascariensis

In the previous report [Biochem. Biophys. Res. Commun. 286 (2001) 820], we described a novel short linear peptide, IsCT, with cytolytic activity isolated from the venom of scorpion Opisthacanthus madagascariensis. From the same scorpion venom, we further purified and characterized three short linear peptides named IsCT2, IsCTf, and IsCT2f that shared high homology with IsCT, while with different C-terminal areas between IsCT/IsCT2 and IsCTf/IsCT2f. Structure-activity relationship was analyzed by performing vivo and vitro assays and circular dichroism spectroscopy. Like IsCT, IsCT2 showed broad activity spectra against microbes (Gram positive and negative bacteria as well as fungi) and relatively weak hemolytic activity against sheep red blood cells. It adopts an amphipathic alpha-helical structure in aqueous TFE and is able to disrupt the artificial membrane. However, the other two peptides IsCTf and IsCT2f showed no activity in antimicrobial or hemolytic assay. Furthermore, IsCTf and IsCT2f cannot form amphipathic alpha-helix while demonstrating random coil structure in aqueous TFE, which might result in their lost cytolytic activity. IsCTf and IsCT2f both exist in the crude venom and are proved to be enzymatic products from IsCT and IsCT2. Whether they have some other biological activity is still unclear. In addition, we got the cDNAs encoding the precursors of IsCT and IsCT2. Besides the signal peptide, they still contain an unusual acidic pro-peptide at the C-terminal that was quite different from other known precursors of scorpion venom peptides. The novel structure and biological activity of these peptides proposed them to be a new class in scorpion venom.     

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.     

Antimicrobial peptides derived from heme-containing proteins: Hemocidins

Deprived of heme and partially unfolded hemoglobin, myoglobin and cytochrome c display microbicidal activity against a broad spectrum of microorganisms with half maximal lethal dose estimated at micromolar concentrations. The intact proteins were ineffective. Antibacterial activity of these apohemoproteins was also sustained after digestion to approximately 50 amino acids long peptides but further fragmentation abolished microbicidal properties. The most active fragment of apomyoglobin (corresponding to 56–131 region) showed a pronounced effect on the E. coli membrane permeabilization and its action was sensitive to salt as well as to divalent cations concentrations. The membrane-directed effect was specific toward bacteria but no lipopolysaccharide binding properties were observed. No hemolytic properties, even at high peptide concentrations were found; however, a slight but dose-independent cytotoxic effect was observed on fibroblasts and hepatoma cells. The presented data suggest a `carpet-like' mechanism of the membrane-directed activity and may result from exceptional abilities of hemoprotein-derived peptides to form alpha-helical structures. We postulate that the antimicrobial peptides obtained from the heme-containing proteins should be named hemocidins, in contrast to, e.g., hemorphins displaying opioid-like activity.     

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.     

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.     

Role of amino acid residues within the disulfide loop of thanatin,a potent antibiotic peptide

Thanatin, a 21-residue peptide, is an inducible insect peptide with a broad range of activity against bacteria and fungi. It has a C-terminal disulfide loop, like the frog skin secretion antimicrobial peptides of the brevinin family. In this study, we tried to find the effect of a number of amino acids between the disulfide bond. Thanatin showed stronger antibacterial activity to Gram negative bacteria than other mutants, except Th1; whereas, the mutant peptides with deletion had higher activity to Gram positive bacteria than thanatin. An increase in the number of amino acid(s) using the alanine residue decreased the antibacterial activity in all of the bacteria. Th1 with deletion of threonine at position 15 (Thr(1)(2)) showed similar antibacterial activity against Gram-negative bacteria, but had higher activity against the Gram positive bacteria. In order to study the structure-function relationship, we measured liposome disruption by the peptides and CD spectra of the peptides. Th1 also showed the highest liposome leaking activity and alpha-helical propensity in the sodium dodecyl sulfate solution, compared with other peptides. Liposome disruption activity was closely correlated with the anti-Gram positive bacterial activity. All of the peptides showed no hemolytic activity. Th1 was considered to be useful as an antimicrobial peptide with broad spectrum without toxicity     

Comparison of the membrane interaction and permeabilization by the designed peptide Ac-MB21-NH2 and truncated dermaseptin S3

Ac-MB21-NH(2) (Ac-FASLLGKALKALAKQ-NH(2)) and dermaseptin S3(1-16)-NH(2) (ALWKNMLKGIGKLAGK-NH(2)) are cationic amphipathic peptides with antimicrobial activity against a broad spectrum of microorganisms including various fungi. The interaction of the peptides with liposomes was studied by exploiting the tryptophan fluorescence of F1W-Ac-MB21-NH(2) and dermaseptin S3(1-16)-NH(2). Spectral analysis and the use of quenchers indicate that the tryptophans of both peptides insert more deeply in anionic than in zwitterionic liposomes. Membrane insertion correlates with the formation of an alpha-helical peptide structure. Both peptides permeabilize liposomes composed of anionic, cylindric phospholipids more efficiently than liposomes formed of zwitterionic, conic (phospho)lipids.     

Alpha-helical antimicrobial peptides--using a sequence template to guide structure-activity relationship studies

An important class of cytolytic antimicrobial peptides (AMPs) assumes an amphipathic, alpha-helical conformation that permits efficient interaction with biological membranes. Host defence peptides of this type are widespread in nature, and numerous synthetic model AMPs have been derived from these or designed de novo based on their characteristics. In this review we provide an overview of the 'sequence template' approach which we have used to design potent artificial helical AMPs, to guide structure-activity relationship studies aimed at their optimization, and to help identify novel natural AMP sequences. Combining this approach with the rational use of natural and non-proteinogenic amino acid building blocks has allowed us to probe the individual effects on the peptides' activity of structural and physico-chemical parameters such as the size, propensity for helical structuring, amphipathic hydrophobicity, cationicity, and hydrophobic or polar sector characteristics. These studies furthermore provided useful insights into alternative modes of action for natural membrane-active helical peptides.     

Conformation-dependent antibiotic activity of tritrpticin,a cathelicidin-derived antimicrobial peptide

Tritrpticin, a Trp-rich cationic antimicrobial peptide with a unique amino acid sequence (VRRFPWWWPFLRR), is found in porcine cathelicidin cDNA. Tritrpticin has a broad spectrum of antibacterial and antifungal activities and hemolytic activity comparable to that of indolicidin. To investigate the mechanism of the bacterial killing action of tritrpticin and to identify structural features important for bacterial cell selectivity, we designed several tritrpticin analogs with amino acid substitutions of the Pro and Trp residues. Circular dichroism studies revealed that the substitution of Pro-->Ala (TPA) or Trp-->Phe (TWF) leads to significant conformational changes in SDS micelles, converting the beta-turn to alpha-helix or to poly-L-proline II helix, respectively. Compared to tritrpticin, TPA retained most of its antimicrobial activity, but showed enhanced hemolytic and membrane-disrupting activities. In contrast, TWF showed a 2-4-fold increase in antimicrobial activity against Gram-negative bacteria, but a marked decrease in both hemolytic and membrane-disrupting activities. Taken together, our findings suggest that compared with the beta-turn and alpha-helical structures, the poly-L-proline II helix is crucial for effective bacterial cell selectivity in tritrpticin and its analogs.     

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.     

Identification and optimization of an antimicrobial peptide from the ant venom toxin pilosulin

A template based on positional residue frequencies in the N-terminal stretch of natural alpha-helical antimicrobial peptides was used to prepare sequence patterns and to scan the Swiss-Prot Database, using the ScanProsite tool. This search identified a segment in pilosulin 1, a cytotoxic peptide from the venom of the jumper ant Myrmecia pilosula, as a potential novel antimicrobial peptide sequence. This segment, corresponding to the 20 N-terminal residues, was synthesized and its structural properties and biological activities were investigated. It showed a potent and broad spectrum antimicrobial activity including standard and multi-drug resistant gram-positive and gram-negative bacteria and Candida albicans, confirming the validity of the search method. A rational redesign approach resulting in four amino acid substitutions yielded a variant with improved antibacterial and significantly reduced hemolytic activity.