Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Repurposing the scorpion venom peptide VmCT1 into an active peptide against Gram-negative ESKAPE pathogens

VmCT1 is a cationic antimicrobial peptide (AMP) from the venom of the scorpion Vaejovis mexicanus. VmCT1 and analogs were designed with single substitutions for verifying the influence of changes in physicochemical features described as important for AMPs antimicrobial and hemolytic activities, as well as their effect on VmCT1 analogs resistance against proteases action. The increase of the net positive charge by the introduction of an arginine residue in positions of the hydrophilic face of the helical structure affected directly the antimicrobial activity. Arg-substituted analogs presented activity against Gram-negative bacteria from the ESKAPE list of pathogens that were not observed for VmCT1. Additionally, peptides with higher net positive charge presented increased antimicrobial activity with values ranging from 0.39 to 12.5 μmol L⁻¹ against Gram-positive and Gram-negative bacteria and fungi. The phenylalanine substitution by glycine (position 1), and the valine substitution by a proline residue (position 8) led to analogs with lower hemolytic activity (at concentrations 50 and 100 μmol L⁻¹, respectively). These results revealed that it is possible to modulate the biological activities of VmCT1 derivatives by designing single substituted-analogs as prospective therapeutics against bacteria and fungi.     

Synthesis and antimicrobial activity of cysteine-free coprisin nonapeptides

Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. CopA3 (LLCIALRKK-NH₂), a 9-mer peptide containing a single free cysteine residue at position 3 of its sequence, was derived from the α-helical region of coprisin and exhibits potent antibacterial and anti-inflammatory activities. The single cysteine implies a tendency for dimerization; however, it remains unknown whether this cysteine residue is indispensible for CopA3’s antimicrobial activity. To address this issue, in the present study we synthesized eight cysteine-substituted monomeric CopA3 analogs and two dimeric analogs, CopA3 (Dimer) and CopIK (Dimer), and evaluated their antimicrobial effects against bacteria and fungi, as well as their hemolytic activity toward human erythrocytes. Under physiological conditions, CopA3 (Mono) exhibits a 6/4 (monomer/dimer) molar ratio in HPLC area percent, indicating that its effects on bacterial strains likely reflect a CopA3 (Mono)/CopA3 (Dimer) mixture. We also report the identification of CopW, a new cysteine-free nonapeptide derived from CopA3 that has potent antimicrobial activity with virtually no hemolytic activity. Apparently, the cysteine residue in CopA3 is not essential for its antimicrobial function. Notably, CopW also exhibited significant synergistic activity with ampicillin and showed more potent antifungal activity than either wild-type coprisin or melittin.     

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.     

Activity optimization of an undecapeptide analogue derived from a frog-skin antimicrobial peptide

While natural antimicrobial peptides are potential therapeutic agents, their physicochemical properties and bioactivity generally need to be enhanced for clinical and commercial development. We have previously developed a cationic, amphipathic α-helical, 11-residue peptide (named herein GA-W2: FLGWLFKWASK-NH₂) with potent antimicrobial and hemolytic activity, which was derived from a 24-residue, natural antimicrobial peptide isolated from frog skin. Here, we attempted to optimize peptide bioactivity by a rational approach to sequence modification. Seven analogues were generated from GA-W2, and their activities were compared with that of a 12-residue peptide, omiganan, which is being developed for clinical and commercial applications. Most of the modifications reported here improved antimicrobial activity. Among them, the GA-K4AL (FAKWAFKWLKK-NH₂) peptide displayed the most potent antimicrobial activity with negligible hemolytic activity, superior to that of omiganan. The therapeutic index of GA-K4AL was improved more than 53- and more than 31-fold against Gram-negative and Gram-positive bacteria, respectively, compared to that of the starting peptide, GA-W2. Given its relatively shorter length and simpler amino acid composition, our sequence-optimized GA-K4AL peptide may thus be a potentially useful antimicrobial peptide agent.     

The antimicrobial potential of a new derivative of cathelicidin from <Emphasis Type="Italic">Bungarus fasciatus</Emphasis> against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Cathelicidins are a family of antimicrobial peptides which exhibit broad antimicrobial activities against antibiotic-resistant bacteria. Considering the progressive antibiotic resistance, cathelicidin is a candidate for use as an alternative approach to treat and overcome the challenge of antimicrobial resistance. Cathelicidin-BF (Cath-BF) is a short antimicrobial peptide, which was originally extracted from the venom of Bungarus fasciatus. Recent studies have reported that Cath-BF and some related derivatives exert strong antimicrobial and weak hemolytic properties. This study investigates the bactericidal and cytotoxic effects of Cath-BF and its analogs (Cath-A and Cath-B). Cath-A and Cath-B were designed to increase their net positive charge, to have more activity against methicillin resistant S. aureus (MRSA). The results of this study show that Cath-A, with a +17-net charge, has the most noteworthy antimicrobial activity against MRSA strains, with minimum inhibitory concentration (MIC) ranging between 32–128 μg/ml. The bacterial kinetic analysis by 1 × MIC concentration of each peptide shows that Cath-A neutralizes the clinical MRSA isolate for 60 min. The present data support the notion that increasing the positive net charge of antimicrobial peptides can increase their potential antimicrobial activity. Cath-A also displayed the weakest cytotoxicity effect against human umbilical vein endothelial and H9c2 rat cardiomyoblast cell lines. Analysis of the hemolytic activity reveals that all three peptides exhibit minor hemolytic activity against human erythrocytes at concentrations up to 250 μg/ml. Altogether, these results suggest that Cath-A and Cath-B are competent candidates as novel antimicrobial compounds against MRSA and possibly other multidrug resistant bacteria.     

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.     

De novo generation of short antimicrobial peptides with simple amino acid composition

As potential therapeutic agents, antimicrobial peptides with shorter length and simpler amino acid composition can be better candidates for clinical and commercial development. Here, we attempted de novo design of short (5- to 11-residue) antimicrobial peptides with three kinds of amino acids. Amphipathic helical properties were conferred by using leucines and lysines and two tryptophan residues were positioned at the critical amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. According to this specified rule, 12 model peptides were generated and their helical propensity was confirmed by circular dichroism spectroscopy. Antimicrobial and hemolytic activities were compared with those of the known 12-residue peptide agent, omiganan, which is currently under therapeutic and commercial development. Antimicrobial activities against Gram-negative and Gram-positive bacteria, including a multi-drug resistant strain, were observed for certain 7- to 11-residue models. Among them, the most potent activity was found for a 9-residue peptide (L₅K₂W₂), although it also had severe hemolytic activity. Alternatively, an 11-residue peptide (L₄K₅W₂) with little hemolytic activity was potentially the most useful agent, as it showed higher antibacterial activity than omiganan. These results not only suggest useful candidates for novel antibiotic development, but also provide an efficient strategy to design such peptides.     

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.     

IsCT‐based analogs intending better biological activity

IsCT1‐NH₂ is a cationic antimicrobial peptide isolated from the venom of the scorpion Opisthacanthus madagascariensis that has a tendency to form an α‐helical structure and shows potent antimicrobial activity and also inopportunely shows hemolytic effects. In this study, five IsCT1 (ILGKIWEGIKSLF)‐based analogs with amino acid modifications at positions 1, 3, 5, or 8 and one analog with three simultaneous substitutions at the 1, 5, and 8 positions were designed. The net charge of each analog was between +2 and +3. The peptides obtained were characterized by mass spectrometry and analyzed by circular dichroism for their structure in different media. Studies of antimicrobial activity, hemolytic activity, and stability against proteases were also carried out. Peptides with a substitution at position 3 or 5 ([L]³‐IsCT1‐NH₂, [K]³‐IsCT1‐NH₂, or [F]⁵‐IsCT1‐NH₂) showed no significant change in an activity relative to IsCT1‐NH₂. The addition of a proline residue at position 8 ([P]⁸‐IsCT1‐NH₂) reduced the hemolytic activity as well as the antimicrobial activity (MIC ranging 3.13‐50 μmol L^?1), and the addition of a tryptophan residue at position 1 ([W]¹‐IsCT1‐NH₂) increased the hemolytic activity (MHC = 1.56 μmol L^?1) without an improvement in antimicrobial activity. The analog [A]¹[F]⁵[K]⁸‐IsCT1‐NH₂, which carries three simultaneous modifications, presented increasing or equivalent values in antimicrobial activity (MIC approximately 0.38 and 12.5 μmol L^?1) with a reduction in hemolytic activity. In addition, this analog presented the best resistance against proteases. This kind of strategy can find functional hotspots in peptide molecules in an attempt to generate novel potent peptide antibiotics.     

Rational design of cationic antimicrobial peptides by the tandem of leucine-rich repeat

Antimicrobial peptides represent ancient host defense effector molecules present in organisms across the evolutionary spectrum. Lots of antimicrobial peptides were synthesized based on well-known structural motif widely existed in a variety of lives. Leucine-rich repeats (LRRs) are sequence motifs present in over 60,000 proteins identified from viruses, bacteria, and eukaryotes. To elucidate if LRR motif possesses antimicrobial potency, two peptides containing one or two LRRs were designed. The biological activity and membrane–peptide interactions of the peptides were analyzed. The results showed that the tandem of two LRRs exhibited similar antibacterial activity and significantly weaker hemolytic activity against hRBCs than the well-known membrane active peptide melittin. The peptide with one LRR was defective at antimicrobial and hemolytic activity. The peptide containing two LRRs formed α-helical structure, respectively, in the presence of membrane-mimicking environment. LRR-2 retained strong resistance to cations, heat, and some proteolytic enzymes. The blue shifts of the peptides in two lipid systems correlated positively with their biological activities. Other membrane-peptide experiments further provide the evidence that the peptide with two LRRs kills bacteria via membrane-involving mechanism. The present study increases our new understanding of well-known LRR motif in antimicrobial potency and presents a potential strategy to develop novel antibacterial agents.     

Effect of hydrocarbon stapling on the properties of α-helical antimicrobial peptides isolated from the venom of hymenoptera

The impact of inserting hydrocarbon staples into short α-helical antimicrobial peptides lasioglossin III and melectin (antimicrobial peptides of wild bee venom) on their biological and biophysical properties has been examined. The stapling was achieved by ring-closing olefin metathesis, either between two S-2-(4′-pentenyl) alanine residues (S ₅) incorporated at i and i + 4 positions or between R-2-(7′-octenyl) alanine (R ₈) and S ₅ incorporated at the i and i + 7 positions, respectively. We prepared several lasioglossin III and melectin analogs with a single staple inserted into different positions within the peptide chains as well as analogs with double staples. The stapled peptides exhibited a remarkable increase in hemolytic activity, while their antimicrobial activities decreased. Some single stapled peptides showed a higher resistance against proteolytic degradation than native ones, while the double stapled analogs were substantially more resistant. The CD spectra of the singly stapled peptides measured in water showed only a slightly better propensity to form α-helical structure when compared to native peptides, whereas the doubly stapled analogs exhibited dramatically enhanced α-helicity.     

Cationic amphipathic peptide analogs of cathelicidin LL‐37 as a probe in the development of antimicrobial/anticancer agents

Cathelicidin LL‐37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL‐37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13‐amino acid cathelicidin 17‐29 amide segment F¹⁷KRIV²¹QR²³IK²⁵DF²⁷LR‐NH₂ were prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were performed on residues R²³ and K²⁵ in the hydrophilic side, V²¹and F²⁷ in the hydrophobic side of the interphase, and F¹⁷ that interacts with cell membranes. Specific motifs IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals were also inserted into the 17‐29 fragment to investigate how they affect activity. Substitution of the amino‐terminal positive charge by acetylation and replacement of lysine by the aliphatic leucine in the peptide analog Ac‐FKRIVQRIL²⁵DFLR‐NH₂ resulted in significant cytotoxicity against A549 cancer cells with an IC₅₀ value 3.90 μg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac‐FKRIVQI²³IKK²⁶FLR‐NH₂, which incorporates the IIKK motif and the peptides FKRIVQL²³L²⁴KK²⁶L²⁷LR‐NH₂ and Ac‐FKRIVQL²³L²⁴KK²⁶L²⁷LR‐NH₂, which incorporate the LLKKL motif, displayed potent antimicrobial activity against gram‐negative bacteria (MIC 3–7.5 μg/mL) and substantial cytotoxicity against bronchial epithelial cancer cells, (IC₅₀ 12.9–9.8 μg/mL), with no cytotoxic activity for human erythrocytes. The helical conformation of the synthetic peptides was confirmed by circular dichroism. Our study shows that appropriate substitutions, mainly in positions of the interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin 17‐29 segment, may lead to the preparation of effective biological compounds.     

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.     

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.     

Antibacterial and leishmanicidal activities of temporin-SHd,a 17-residue long membrane-damaging peptide

Temporins are a family of short antimicrobial peptides (8–17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF_amide), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.     

Identification and target-modifications of temporin-PE: A novel antimicrobial peptide in the defensive skin secretions of the edible frog, Pelophylax kl. esculentus

A potent natural antimicrobial peptide named temporin-PE was identified and encoded from the skin secretions of Pelophylax kl. esculentus via “shotgun” cloning and LC-MS/MS fragmentation analysis. Target-modifications were carried out to further enhance the antimicrobial and anti-proliferative bioactivities, whilst decreasing the hemolytic effect. A range of bioassays demonstrated that replacing a proline with a tyrosine residue resulted in a loss of the bioactivity against Gram-negative bacteria, but dramatically improved the hemolytic and anti-proliferative activity, indicating the FLP- motif influences the hemolytic activity of temporins. Moreover, the coupling of TAT to the peptide dramatically improved its antimicrobial activity, indicating coupling TAT to these peptides could be considered as a potential tool to improve their antimicrobial activity. Overall, we have shown that targeted modifications of this natural antimicrobial peptide can adjust its bioactivities to help its development as an antibiotic or anti-proliferative agent.     

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.     

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.     

Effects of two glycine residues in positions 13 and 17 of pleurocidin on structure and bacterial cell selectivity

Pleurocidin (Ple), a 25-residue alpha-helical antimicrobial peptide, isolated from skin mucosa of the winter flounder, shows potent bacterial cell selectivity. In this study, the effect of two glycine residues in positions 13 and 17 of Ple on structure and bacterial cell selectivity was investigated by Gly-->Ala substitution. Ala-substitution (Gly(13, 17)-->Ala, Gly13-->Ala and Gly17-->Ala) in positions 13 and 17 of Ple did not induce a significant change in antibacterial activity, but increased hemolytic activity. Both Gly(13, 17)-->Ala and Gly17-->Ala substitution did not cause a remarkable change in alpha-helical content in SDS micelles, while Gly(13, 17)-->Ala substitution caused a drastic increase in alpha-helical content. These results suggest that the hinge region from Gly13 to Gly17 of Ple is assumed to provide its conformational flexibility and bacterial cell selectivity.