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.     

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.     

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G¹LLKR⁵IKT⁸LL‐NH₂, it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly¹, Arg⁵, and Thr⁸ and lipophilic groups with different lengths in the N‐terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF⁵IKK⁸LL‐NH₂ exhibited high activity against Gram‐negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N‐terminus increased the antimicrobial activity against Gram‐positive and Gram‐negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α‐helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Effects of arginine and leucine substitutions on anti‐endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α‐amylase

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI‐1‐18 from rice α‐amylase (AmyI‐1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI‐1‐18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI‐1‐18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI‐1‐18. In the present study, anti‐inflammatory (anti‐endotoxic) activities of five AmyI‐1‐18 analogs containing arginine or leucine substitutions were investigated. Two single arginine‐substituted and two single leucine‐substituted AmyI‐1‐18 analogs inhibited the production of LPS‐induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI‐1‐18. These data indicate that enhanced cationic and hydrophobic properties of AmyI‐1‐18 are associated with improved anti‐endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC₅₀) of the three AmyI‐1‐18 analogs (G12R, D15R, and E9L) were 0.11–0.13 μm , indicating higher anti‐endotoxic activity than that of AmyI‐1‐18 (IC_50, 0.22 μm ), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI‐1‐18 analogs. In addition, AmyI‐1‐18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti‐inflammatory and LPS‐neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine‐substituted and leucine‐substituted AmyI‐1‐18 analogs with improved anti‐endotoxic and antimicrobial activities have clinical potential as dual‐function host defense agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

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

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.     

Conformations of helical Aib peptides containing a pair of l‐amino acid and d‐amino acid

A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, ¹H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 3₁₀‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 3₁₀‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial activity of human α‐defensin 6 analogs: insights into the physico‐chemical reasons behind weak bactericidal activity of HD6 in vitro

Human α‐defensin 6 (HD6), unlike other mammalian defensins, does not exhibit bactericidal activity, particularly against aerobic bacteria. Monomeric HD6 has a tertiary structure similar to other α‐defensins in the crystalline state. However, the physico‐chemical reasons behind the lack of antibacterial activity of HD6 are yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD6 analogs. A linear analog of HD6, in which the distribution of arginine residues was similar to active α‐defensins, shows broad‐spectrum antimicrobial activity, indicating that atypical distribution of arginine residues contributes to the inactivity of HD6. Peptides spanning the N‐terminal cationic segment were active against a wide range of organisms. Antimicrobial potency of these shorter analogs was further enhanced when myristic acid was conjugated at the N‐terminus. Cytoplasmic localization of the analogs without fatty acylation was observed to be necessary for bacterial killing, while they exhibited fungicidal activity by permeabilizing Candida albicans membranes. Myristoylated analogs and the linear full‐length arginine analog exhibited activity by permeabilizing bacterial and fungal membranes. Our study provides insights into the lack of bactericidal activity of HD6 against aerobic bacteria. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Structure–activity relationship of a novel pentapeptide with cancer cell growth‐inhibitory activity

We previously reported that yamamarin, a pentapeptide with an amidated C‐terminus (DILRG‐NH₂) isolated from larvae of the silkmoth, and its palmitoylated analog (C16‐DILRG‐NH₂) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure–activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C‐terminal part (‐RG‐NH₂) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG‐NH₂ and C16‐DILRG‐NH₂ revealed that the N‐terminal palmitoyl group of C16‐DILRG‐NH₂ did not affect the conformation of the C‐terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Intramolecular cyclization of the antimicrobial peptide Polybia‐MPI with triazole stapling: influence on stability and bioactivity

Cationic antimicrobial peptides have attracted increasing attention as a novel class of antibiotics to treat infectious diseases caused by pathogenic bacteria. However, susceptibility to protease is a shortcoming in their development. Cyclization is one approach to increase the proteolytic resistance of peptides. Therefore, to improve the proteolytic resistance of Polybia‐MPI, we have synthesized the MPI cyclic analogs C‐MPI‐1 (i‐to‐i+4) and C‐MPI‐2 (i‐to‐i+6) by copper(I)‐catalyzed azide–alkyne cycloaddition. Compared with MPI, C‐MPI‐1 displayed sustained antimicrobial activity and had enhanced anti‐trypsin resistance, while C‐MPI‐2 displayed no antimicrobial activity. The relationship between peptide structure and bioactivity was further investigated by probing the secondary structure of the peptides by circular dichroism. This showed that C‐MPI‐1 adopted an α‐helical structure in aqueous solution and, interestingly, had increased α‐helical conformation in 30 mM sodium dodecyl sulfate and 50% trifluoroethyl alcohol compared with MPI. C‐MPI‐2 that was not α‐helical in structure, suggesting that the propensity for α‐helix conformation may play an important role in cyclic peptide design. In addition, scanning electron microscopy, propidium iodide uptake, and membrane permeabilization assays indicated that MPI and the optimized analog C‐MPI‐1 had membrane‐active action modes, indicating that the peptides would not be susceptible to conventional resistance mechanisms. Our study provides additional insight into the influence of intramolecular cyclization at various positions on peptide structure and biological activity. In conclusion, the design and synthesis of cyclic analogs via click chemistry offer a new strategy for the development of stable antimicrobial agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Synthetic analogs of anoplin show improved antimicrobial activities

We present the antimicrobial and hemolytic activities of the decapeptide anoplin and 19 analogs thereof tested against methicillin‐resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), vancomycin‐resistant Enterococcus faecium (ATCC 700221) (VRE), and Candida albicans (ATCC 200955). The anoplin analogs contain substitutions in amino acid positions 2, 3, 5, 6, 8, 9, and 10. We use these peptides to study the effect of altering the charge and hydrophobicity of anoplin on activity against red blood cells and microorganisms. We find that increasing the charge and/or hydrophobicity improves antimicrobial activity and increases hemolytic activity. For each strain tested, we identify at least six anoplin analogs with an improved therapeutic index compared with anoplin, the only exception being Enterococcus faecium, against which only few compounds are more specific than anoplin. Both 2Nal⁶ and Cha⁶ show improved therapeutic index against all strains tested. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Micelle-bound structures and dynamics of the hinge deleted analog of melittin and its diastereomer: Implications in cell selective lysis by d-amino acid containing antimicrobial peptides

Melittin, the major component of the honey bee venom, is a 26-residue hemolytic and membrane active peptide. Structures of melittin determined either in lipid environments by NMR or by use of X-ray demonstrated two helical regions at the N- and C-termini connected by a hinge or a bend at the middle. Here, we show that deletion of the hinge residues along with two C-terminal terminal Gln residues (Q25 and Q26), yielding a peptide analog of 19-residue or Mel-H, did not affect antibacterial activity but resulted in a somewhat reduced hemolytic activity. A diastereomer of Mel-H or Mel-^dH containing d-amino acids [^dV5, ^dV8, ^dL11 and ^dK16] showed further reduction in hemolytic activity without lowering antibacterial activity. We have carried out NMR structures, dynamics (H-D exchange and proton relaxation), membrane localization by spin labeled lipids, pulse-field-gradient (PFG) NMR and isothermal titration calorimetry (ITC) in dodecylphosphocholine (DPC) micelles, as a mimic to eukaryotic membrane, to gain insights into cell selectivity of these melittin analogs. PFG-NMR showed Mel-H and Mel-^dH both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel-^dH interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel-^dH showed multiple β-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D₂O demonstrated a large difference in dynamics between Mel-H and Mel-^dH, whereby almost all backbone protons of Mel-^dH showed a much faster rate of exchange as compared to Mel-H. Proton T₁ relaxation had suggested a mobile backbone of Mel-^dH peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel-^dH is largely located at the surface of the micelle. Taken together, results presented in this study demonstrated that the poor hemolytic activity of the d-amino acid containing analogs of antimicrobial peptides may be correlated with their flexible dynamics at the membrane surface.     

Melectin MAPs: the influence of dendrimerization on antimicrobial and hemolytic activity

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH₂) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O₂) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.     

Effects of fluidity on the ensemble structure of a membrane embedded α‐helical peptide

Melittin, the main hemolytic component of honeybee venom, is unfolded in an aqueous environment and folds into an α‐helical conformation in a lipid environment. Membrane fluidity is known to affect the activity and structure of melittin. By combining two structurally sensitive optical methods, circular dichroism (CD) and deep‐ultraviolet resonance Raman spectroscopy (dUVRR), we have identified distinct structural fluctuations in melittin correlated with increased and decreased 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine bilayer fluidities. CD spectra have reduced intensity at temperatures above 22°C and high concentrations of the cholesterol analog 5α‐cholestan‐3β‐ol indicating distortions in the α‐helical structure under these conditions. No increase in the amide S is observed in the temperature‐dependent dUVRR spectra, suggesting an increase in 3₁₀‐helical structure with increasing temperatures above 22°C. However, incorporation of 25 mol% 5α‐cholestan‐3β‐ol resulted in a small increase in the amide S intensity indicating partial unfolding of melittin. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 895–902, 2014.     

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.     

Design of novel analogues of short antimicrobial peptide anoplin with improved antimicrobial activity

Currently, novel antibiotics are urgently required to combat the emergence of drug‐resistant bacteria. Antimicrobial peptides with membrane‐lytic mechanism of action have attracted considerable interest. Anoplin, a natural α‐helical amphiphilic antimicrobial peptide, is an ideal research template because of its short sequence. In this study, we designed and synthesized a group of analogues of anoplin. Among these analogues, anoplin‐4 composed of d ‐amino acids displayed the highest antimicrobial activity due to increased charge, hydrophobicity and amphiphilicity. Gratifyingly, anoplin‐4 showed low toxicity to host cells, indicating high bacterial selectivity. Furthermore, the mortality rate of mice infected with Escherichia coli was significantly reduced by anoplin‐4 treatment relative to anoplin. In conclusion, anoplin‐4 is a novel anoplin analogue with high antimicrobial activity and enzymatic stability, which may represent a potent agent for the treatment of infection. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Dissection of antibacterial and toxic activity of melittin: a leucine zipper motif plays a crucial role in determining its hemolytic activity but not antibacterial activity

Melittin, a naturally occurring antimicrobial peptide, exhibits strong lytic activity against both eukaryotic and prokaryotic cells. Despite a tremendous amount of work done, very little is known about the amino acid sequence, which regulates its toxic activity. With the goal of understanding the basis of toxic activity and poor cell selectivity in melittin, a leucine zipper motif has been identified. To evaluate the possible structural and functional roles of this motif, melittin and its two analogs, after substituting the heptadic leucine by alanine, were synthesized and characterized. Functional studies indicated that alanine substitution in the leucine zipper motif resulted in a drastic reduction of the hemolytic activity of melittin. However, interestingly, both the designed analogs exhibited antibacterial activity comparable to melittin. Mutations caused a significant decrease in the membrane permeability of melittin in zwitterionic but not in negatively charged lipid vesicles. Although both the analogs exhibited similar secondary structures in the presence of negatively charged lipid vesicles as melittin, they failed to adopt a significant helical structure in the presence of zwitterionic lipid vesicles. Results suggest that the substitution of heptadic leucine by alanine impaired the assembly of melittin in an aqueous environment and its localization only in zwitterionic but not in negatively charged membrane. Altogether, the results suggest the identification of a structural element in melittin, which probably plays a prominent role in regulating its toxicity but not antibacterial activity. The results indicate that cell selectivity in some antimicrobial peptides can probably be introduced by modulating their assembly in an aqueous environment.