Biological activities of retro and diastereo analogs of a 13‐residue peptide with antimicrobial and hemolytic activities

Abstract: The biological activities of synthetic retro and diastereo analogs of PKLLKTFLSKWIG (SPFK), a 13‐residue peptide with antimicrobial and hemolytic activities, have been investigated. Retro peptides with C‐terminal acid and amide exhibited antibacterial activities comparable with those of SPFK. Their hemolytic activities were, however, only marginally lower. The diastereo analog with C‐terminal acid was not antibacterial and was weakly hemolytic. Amidation of this analog could restore antibacterial activity. Both retro analogs were unordered in aqueous medium but had a propensity for a helical structure in trifluoroethanol. However, diastereo analogs were unordered in both aqueous medium and trifluoroethanol. Thus, reversing the sequence in a short amphiphilic peptide may not always result in the selective loss of biological activity such as hemolytic activity. Also, introduction of enantiomeric amino acids in a short peptide to generate a diastereomer may result in loss of structure as well as antimicrobial and hemolytic activities, unless compensated by an increase in positive charges.     

Identification and characterization of novel host defense peptides from the skin secretion of the fungoid frog,Hydrophylax bahuvistara (Anura: Ranidae)

Two novel peptides (brevinin1 HYba1 and brevinin1 HYba2) were identified from the skin secretion of the frog Hydrophylax bahuvistara, endemic to Western Ghats, India, and their amino acid sequences were confirmed using cDNA cloning and LC/MS/MS. Antibacterial, hemolytic, and cytotoxic activities of brevinin1 peptides and their synthetic analogs (amidated C‐terminus) were investigated and compared. All the peptides except the acidic forms showed antibacterial activity against all tested Gram‐positive and Gram‐negative bacteria. They exhibited low hemolysis on human erythrocytes and showed potent cytotoxic activity against Hep 3B cancer cell line. Upon amidation, the peptides showed increased activity against the tested microbes without altering their hemolytic and cytotoxic properties. The study also emphasizes the need for screening endemic amphibian fauna of Western Ghats, as a potential source of host defense peptides with possible therapeutic applications in the future.     

Consequences of introducing a disulfide bond into an antibacterial and hemolytic peptide

Abstract: The effect of introducing a disulfide bridge between the N- and C-terminal ends on the structure and biological activities of the 13-residue linear peptide PKLLKTFLSKWIG(SPFK), which has both antibacterial and hemolytic activity, have been investigated. The terminal amino acids P and G in SPFK were replaced by cysteines to form a disulfide bridge. The linear peptides C(Acm)KLLKTFLSKWIC(Acm) and C(Acm) KLLKTFLSKWIC(Acm)-amide, where Acm is acetamidomethyl group, showed antibacterial activity but did not possess hemolytic activity unlike SPFK. Introduction of an S–S bridge resulted in enhanced hemolytic activity compared with SPFK. The hemolytic activity was particularly pronounced in the cyclic peptide CKLLKTFLSKWIC-amide. Circular dichroism studies indicate that the cyclic peptides tend to adopt distorted helical structures. The cyclic peptides also have a greater affinity for lipid vesicles, which could be the reason for the effective perturbation of the erythrocyte membrane.     

Effects of increasing hydrophobicity by N‐terminal myristoylation on the antibacterial and hemolytic activities of the C‐terminal cationic segments of human‐β‐defensins 1–3

Analogs of the cationic C‐terminal segments of human‐β‐defensins HBD1‐3, Phd1‐3 with a single disulfide bond, exhibited comparable antimicrobial activity that was salt sensitive. They did not show hemolytic activity. In this study, N‐terminal myristoylation was carried out on Phd1‐3 to examine whether increasing hydrophobicity would result in improved antibacterial activity. The antibacterial activity of the oxidized myristoylated peptides MPhd1‐3 and their reduced forms MPhd1r‐3r was determined. These peptides showed enhanced antibacterial activity as compared to Phd1‐3, on mid‐log phase and stationary phase of Staphylococcus aureus and Escherichia coli, except MPhd1r‐3r that were inactive on stationary‐phase E. coli. In the presence of 150 mm NaCl, MPhd1‐3 showed activity against S. aureus. MPhd1and two exhibited activity against E. coli but MPhd3 was inactive. Zeta potential measurements indicated that MPhd1‐3 were more effective in surface charge neutralization of bacteria as compared to Phd1‐3. MPhd1‐3 exhibited hemolytic activity to varying extents with MPhd1 being most hemolytic. The data indicate that myristoylation enhances antibacterial activity and modulates hemolytic activity to different extents. Apart from hydrophobicity, distribution of cationic residues in MPhd1‐3 plays important roles for these activities.     

Structural and charge requirements for antimicrobial and hemolytic activity in the peptide PKLLETFLSKWIG,corresponding to the hydrophobic region of the antimicrobial protein bovine seminalplasmin

Several analogs of the 13-residue antimicrobial and hemolytic peptide PKLLETFLSKWIG (SPF), which is the most hydrophobic region of the 47-residue antimicrobial protein seminalplasmin [Sitaram, N. & Nagaraj, R. (1990) J. Biol. Chem. 265, 10438-104423 have been synthesized. The antimicrobial and hemolytic properties of the peptides were investigated with a view to gain an insight into the structural and charge requirements for these activities of SPF. Peptides in which E was replaced by K exhibited considerably improved antimicrobial activity with no concomitant increase in hemolytic activity. A peptide in which the aromatic amino acids were replaced by leucine exhibited antimicrobial activity like those of the peptides which had aromatic amino acids. Interchange in the positions of E and K and total replacement of K by E resulted in complete loss of activity. The peptides having antimicrobial activities showed appreciable helical content in a hydrophobic environment, whereas inactive peptides did not. Thus, by suitable‘engineering’ the biological activity of a short 13-residue peptide can be altered to yield peptides specifically having only antimicrobial activity with increased potency. © Munksgaard 1995.     

Effects of Hydrophobicity on the Antifungal Activity of α‐Helical Antimicrobial Peptides

We utilized a series of analogs of D‐V13K (a 26‐residue amphipathic α‐helical antimicrobial peptide, denoted D1) to compare and contrast the role of hydrophobicity on antifungal and antibacterial activity to the results obtained previously with Pseudomonas aeruginosa strains. Antifungal activity for zygomycota fungi decreased with increasing hydrophobicity (D‐V13K/A12L/A20L/A23L, denoted D4, the most hydrophobic analog was sixfold less active than D1, the least hydrophobic analog). In contrast, antifungal activity for ascomycota fungi increased with increasing hydrophobicity (D4, the most hydrophobic analog was fivefold more active than D1). Hemolytic activity is dramatically affected by increasing hydrophobicity with peptide D4 being 286‐fold more hemolytic than peptide D1. The therapeutic index for peptide D1 is 1569‐fold and 62‐fold better for zygomycota fungi and ascomycota fungi, respectively, compared with peptide D4. To reduce the hemolytic activity of peptide D4 and improve/maintain the antifungal activity of D4, we substituted another lysine residue in the center of the non‐polar face (V16K) to generate D5 (D‐V13K/V16K/A12L/A20L/A23L). This analog D5 decreased hemolytic activity by 13‐fold, enhanced antifungal activity to zygomycota fungi by 16‐fold and improved the therapeutic index by 201‐fold compared with D4 and represents a unique approach to control specificity while maintaining high hydrophobicity in the two hydrophobic segments on the non‐polar face of D5.     

Structure–biological activity relationships of 11-residue highly basic peptide segment of bovine lactoferrin

The antimicrobial peptide, lactoferricin, is generated upon the gastric pepsin cleavage of lactoferrin and has many basic and hydrophobic amino acid residues essential for its biological activity. To investigate the structure-antimicrobial activity relationships, the basic amino acid-rich region of bovine lactoferricin (BLFC), RRWQWRMKKLG, was selected. Using chemically synthesized BLFC and its substituted peptides, the antimicrobial activities of the peptides were tested by determining the minimal inhibitory concentration (MIC) of Escherichia coli and Bacillus subtilis and the disruption of the outer cell membrane of E. coli, and the peptide's toxicities were assayed by hemolysis. The short peptide (B3) composed of only 11 residues had similar antimicrobial activities while losing most of the hemolytic activities as compared with the 25 residue-long ones (B1 and B2). The short peptides (B3, B5 and B7) with double arginines at the N-termini had more potent antimicrobial activity than those (B4 and B6) with lysine. However, no antimicrobial and hemolytic activities were found in B8, in which all basic amino acids were substituted with glutamic acid, and in B9, in which all hydrophobic amino acids were substituted with alanine. The circular dichroism (CD) spectra of the short peptides in 30 MM SDS were correlated with their antimicrobial activities. These results suggested that the 11-residue peptide of BLFC is involved in the interaction with bacterial phospholipid membranes and plays an important role in antimicrobial activity with little or no hemolytic activity. © Munksgaard 1996.     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences,model membrane binding and biological activities

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt β-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Exploring relationships between mimic configuration,peptide conformation and biological activity in indolizidin‐2‐one amino acid analogs of gramicidin S*

Abstract: Indolizidin‐2‐one amino acids (I²aas, 6S‐ and 6R‐ 1 ) possessing 6S‐ and 6R‐ring‐fusion stereochemistry were introduced into the antimicrobial peptide gramicidin S (GS) to explore the relationships between configuration, peptide conformation and biological activity. Solution‐phase and solid‐phase techniques were used to synthesize three analogs with I²aa residues in place of the d ‐Phe‐Pro residues at the turn regions of GS: [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ), [Lys^2,2′,(6S)‐I²aa^{4?5,4′?5′}]GS ( 3 ) and [(6R)‐I²aa^{4?5,4′?5′}]GS ( 4 ). Although conformational analysis of [I²aa^{4?5,4′?5′}]GS analogs 2?4 indicated that both ring‐fusion stereoisomers of I²aa gave peptides with CD and NMR spectral data characteristic of GS, the (6S)‐I²aa analogs 2 and 3 exhibited more intense CD curve shapes, as well as greater numbers of nonsequential NOE between opposing Val and Leu residues, relative to the (6R)‐I²aa analog 4 , suggesting a greater propensity for the (6S)‐diastereomer to adopt the β‐turn/antiparallel β‐pleated sheet conformation. In measurements of antibacterial and antifungal activity, the (6S)‐I²aa analog 2 exhibited significantly better potency than the (6R)‐I²aa diastereomer 4 . Relative to GS, [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ) exhibited usually 1/2 to 1/4 antimicrobial activity as well as 1/4 hemolytic activity. In certain cases, antimicrobial and hemolytic activities of GS were shown to be dissociated through modification at the peptide turn regions with the (6S)‐I²aa diastereomer. The synthesis and evaluation of GS analogs 2?4 has furnished new insight into the importance of ring‐fusion stereochemistry for turn mimicry by indolizidin‐2‐one amino acids as well as novel antimicrobial peptides.     

Purification and Modeling Amphipathic Alpha Helical Antimicrobial Peptides from Skin Secretions of Euphlyctis cyanophlyctis

Antimicrobial peptides as ancient immune system are found in almost all types of living organisms. Amphibian's skin is an important source of bioactive peptides with strong antibacterial, antiviral, and antitumor properties. They have important role in inducing apoptosis as well as cancer therapy in vitro. In this study, we extracted and purified antimicrobial peptides from skin secretions of Euphlyctis cyanophlyctis and named them brevinin‐Eu and cyanophlyctin β. They showed favorable antibacterial properties on both Gram‐positive and Gram‐negative bacteria with ignorable hemolytic activity of <1.9% and 0.7% at very high concentrations of brevinin‐Eu and cyanophlyctin β, respectively. For antibacterial activity and MIC determination, two Gram‐positive (Staphylococcus aureus PTCC1431 and B. cereus PTCC1247) and two Gram‐negative bacteria (Escherichia coli HP101BA 7601c and Klebsiella pneumoniae PTCC1388) were assayed. MIC values of extracted peptides demonstrated that they can inhibit bacterial growth at very low concentration (17 and 12 μg/mL) for brevinin‐Eu and cyanophlyctin β, respectively. Structural prediction suggested that the brevinin‐Eu can efficiently bind and destroy bacterial membrane, but cyanophlyctin β uses a diverse mode of action.     

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

Abstract: Mastoparan B (MP‐B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic α‐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 α‐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.     

Rational Evolution of Antimicrobial Peptides Containing Unnatural Amino Acids to Combat Burn Wound Infections

Antimicrobial peptides have long been raised as a promising strategy to combat bacterial infection in burn wounds. Here, we attempted to rationally design small antimicrobial peptides containing unnatural amino acids by integrating in silico analysis and in vitro assay. Predictive quantitative sequence‐activity models were established and validated rigorously based on a large panel of nonamer antimicrobial peptides with known antibacterial activity. The best quantitative sequence‐activity model predictor was employed to guide genetic evolution of a peptide population. In the evolution procedure, a number of unnatural amino acids with desired physicochemical properties were introduced, resulting in a genetic evolution‐improved population, from which seven peptide candidates with top scores, containing 1–3 unnatural amino acids, and having diverse structures were successfully identified, and their antibacterial potencies against two antibiotic‐resistant bacterial strains isolated from infected burn wounds were measured using in vitro susceptibility test. Consequently, four (WL‐Orn‐LARKIV‐NH₂, ARKRWF‐Dab‐FL‐NH₂, KFI‐Hag‐IWR‐Orn‐R‐NH₂ and YW‐Hag‐R‐Cit‐RF‐Orn‐N‐NH₂) of the seven tested peptides were found to be more potent than reference Bac2A, the smallest naturally occurring broad spectrum antimicrobial peptide. Molecular dynamics simulations revealed that the designed peptides can fold into amphipathic helical structure that allows them to interact directly with microbial membranes.     

Antimicrobial and hemolytic activities of crabrolin,a 13-residue peptide from the venom of the European hornet,Vespa crabro,and its analogs

The venom of insects like bee, hornet and wasp contain peptides that exhibit potent biological activities. Many of these peptides are composed of 13-26 residues and are thus accessible through chemical synthesis as well as amenable to studies directed toward structure-function correlations. In this report, we describe antibacterial and hemolytic activities of crabrolin: FLPLILRKIVTAL-NH₂, a 13-residue peptide present in the venom of the hornet Vespa crabro and related peptides. The analogs were chosen so that the role of proline and positively charged amino acids in modulating biological activities could be evaluated. Our results indicate that, although helical conformation is necessary for hemolytic activity, it is not a prerequisite for antibacterial activity. Appropriately positioned, charged and hydrophobic residues and overall hydrophobicity appear to determine antibacterial activity. The discovery of a large number of host-defense peptides in a variety of species in recent years offers a large repertoire of molecules that can be “engineered” based on biophysical principles to yield molecules with specific activities.     

Isolation of peptides of the brevinin‐1 family with potent candidacidal activity from the skin secretions of the frog Rana boylii

Abstract: The emergence of strains of the human pathogen Candida albicans with resistance to commonly used antibiotics has necessitated a search for new types of antifungal agents. Six peptides with antimicrobial activity were isolated from norepinephrine‐stimulated skin secretions from the foothill yellow‐legged frog Rana boylii. Brevinin‐1BYa (FLPILASLAA¹⁰KFGPKLF CLV²⁰TKKC) was particularly potent against C. albicans [minimal inhibitory concentration (MIC) = 3 μm ] and also active against Escherichia coli (MIC = 17 μm ) and Staphylococcus aureus (MIC = 2 μm ), but its therapeutic potential for systemic use is limited by its strong hemolytic activity (HC₅₀ = 4 μm ). The single amino acid substitution (Phe¹² → Leu) in brevinin‐1BYb resulted in a fourfold lower potency against C. albicans and the additional amino acid substitutions (Lys¹¹ → Thr, Phe¹⁷ → Leu and Val²⁰ → Ile) in brevinin‐1BYc resulted in a ninefold decrease in activity. Two members of the ranatuerin‐2 family and one member of the temporin family were also isolated from the secretions but showed relatively low potency against the three microorganisms tested.     

Use of the excluded protecting group (EPG) method for peptide synthesis

Abstract: The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin‐1, [Ala¹⁹, Asn²²]magainin‐1. In the approach reported, the C‐terminal amino acid is esterified to the 2‐position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl‐steroid as the Fmoc‐pentafluorophenyl‐ester. The Fmoc group is removed with Et₂NH/DMF (～15% v/v) and, after evaporation to ～10 mL, the solution chromatographed on Sephadex LH‐20 in DMF. The dipeptidyl‐steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc‐amino acid‐pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn°/AcOH to yield the peptide with intact α‐amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C‐terminal amino acids in other EPG syntheses.     

Gramicidin S is active against both gram-positive and gram-negative bacteria

Four linear and four cyclic analogs of gramicidin S (GS) in which d -Phe was replaced with either d -His, d -Ser, d -Tyr or D-Asn have been prepared by solid-phase peptide synthesis and characterized with respect to antibacterial, antifungal and hemolytic activity. Unlike previous reports, GS and a number of cyclic analogs were found to be active against gram-positive as well as gram-negative bacteria. GS showed MICs ranging from 3 to 12.5 μg/mL for gram-negative bacteria, compared to MICs of 3 μg/mL for gram-positive bacteria. Furthermore, these analogs were also found to exhibit antifungal activity. Unlike the cyclic analogs, all linear analogs were found to be inactive against a wide range of microorganisms tested, and showed low levels of hemolytic activity. The antibacterial activity was found to be highly dependent on the type of assay used, with solution-based assays showing greater activity against gram-negative bacteria than agar-based assays. The GS cyclic analogs were all less toxic than GS itself, with the analog containing the d -Phe to d -Tyr substitution showing the greatest activity of the synthetic analogs. Hemolytic activity in solution against human and sheep red blood cells paralleled antibiotic activity, with those peptides exhibiting greater antibiotic activity generally showing greater hemolytic activity. Membrane destabilization as monitored using the hydrophobic probe N-phenyl-1-naphthylamine was also found to parallel antibacterial and hemolytic activity of cyclic and linear analogs. These results indicate that GS and certain related analogs may have applications as broad-spectrum antibiotics and should be reevaluated for such purposes. © Munksgaard 1996.     

Antimicrobial activity of arginine‐ and tryptophan‐rich hexapeptides: the effects of aromatic clusters,d‐amino acid substitution and cyclization

Abstract: Many antimicrobial peptides bear arginine (R)‐ and tryptophan (W)‐rich sequence motifs. Based on the sequence Ac‐RRWWRF‐NH₂, sets of linear and cyclic peptides were generated by changes in the amino acid sequence, l ‐d ‐amino acid exchange and naphthylalanine substituted for tryptophan. Linear RW‐peptides displayed moderate activity towards Gram‐positive Bacillus subtilis (15 < MIC < 31 μm ) and were inactive against Gram‐negative Escherichia coli at peptide concentrations <100 μm . Cyclization induced high antimicrobial activity. The effect of cyclization was most pronounced for peptides with three adjacent aromatic residues. Incorporation of d ‐amino acid residues had minor influence on the biological activity. The haemolytic activity of all RW‐peptides at 100 μm concentration was low (<7% lysis for linear R/W‐rich peptides and <28% for the cyclic analogues). Introduction of naphthylalanine enhanced the biological activities of both the linear and cyclic peptides. All peptides induced permeabilization of large unilamellar vesicles (LUVs) composed of lipids of the membrane of B. subtilis and erythrocytes, but surprisingly had no effect on LUVs composed of lipids of the E. coli inner membrane. The profiles of peptide activity against B. subtilis and red blood cells correlated with the permeabilizing effects on the corresponding model membranes and were related to hydrophobicity parameters as derived from reversed phase high‐performance liquid chromatography (HPLC). The results underlined the importance of amphipathicity as a driving force for cell lytic activity and suggest that conformational constraints and an appropriate position of aromatic residues allowing the formation of hydrophobic clusters are highly favourable for antimicrobial activity and selectivity.     

Biophysical studies and anti‐growth activities of a peptide,a certain analog and a fragment peptide derived from α‐fetoprotein

Abstract: A chemically synthesized 34‐amino acid peptide, an analog, and a fragment of the peptide have been purified and studied. Biophysical studies were carried out to determine some of the metal ion binding properties of the original peptide and an analog of this parent peptide, in which the two histidine residues were replaced by alanines. As shown by visible absorption spectroscopy, Co (II) forms a complex with the parent peptide, but not with the analog peptide, and one or two histidines in the parent peptide are ligands for Co (II) ion binding. The effects on disulfide bond formation in the peptide by Zn (II) and Co (II) ions were also examined for this analog. Anti‐growth assays were performed using the original cysteine‐containing peptide with Zn (II) ion complexed to the peptide through the two cysteine residues. These rat uterine growth assays showed that the complexing of Zn (II) ion to the peptide maintained the anti‐growth activity of the peptide, while gel‐filtration experiments showed the zinc ions maintained the peptide in its anti‐growth form indefinitely in solution. A saliently important part of this research was the discovery that a fragment of the peptide consisting of a middle sequence of 14 amino acids was found to have significant anti‐growth activity in the rat uterine assay. Its activity suggested that this fragment might be considered a viable candidate for testing in anti‐cancer protocols.     

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

Abstract: 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‐1Oa FLPLLASLFSRLL.NH₂, 13 nmol/g; temporin‐1Ob FLPLIGKILGTI L.NH₂, 350 nmol/g; temporin‐1Oc FLPLLASLFSRLF.NH₂, 14 nmol/g; and temporin‐1Od 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 α‐helical conformation but it is significant that temporin‐1Od, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 µm compared with 2 µm for temporin‐1Oa). 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.     

Influence of preformed α-helix and α-helix induction on the activity of cationic antimicrobial peptides

One prominent class of cationic antibacterial peptides comprises the α-helical class, which is unstructured in free solution but folds into an amphipathic α-helix upon insertion into the membranes of target cells. To investigate the importance of α-helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1-8) and bee melittin (amino acids 1-18) peptides. The new peptides were predicted to have a high tendency to form α-helices or to have preformed α-helices by virtue of construction of a lactam bridge between glutamate and lysine side-chains at positions i and i+ 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized α-helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of α-helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase α-helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r²= 0.88) with a helican parameter which was calculated as the induction of α-helix in α membrane-mimicking environment divided by the α-helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.