Utilization of an amphipathic leucine zipper sequence to design antibacterial peptides with simultaneous modulation of toxic activity against human red blood cells

The toxicity of naturally occurring or designed antimicrobial peptides is a major barrier for converting them into drugs. To synthesize antimicrobial peptides with reduced toxicity, several amphipathic peptides were designed based on the leucine zipper sequence. The first one was a leucine zipper peptide (LZP); in others, leucine residues at the a- and/or d-position were substituted with single or double alanine residues. The results showed that LZP and its analogs exhibited appreciable and similar antibacterial activity against the tested gram-positive and gram-negative bacteria. However, the substitution of alanine progressively lowered the toxicity of LZP against human red blood cells (hRBCs). The substitution of leucine with alanine impaired the binding and localization of LZP to hRBCs, but had little effect on the peptide-induced damage of Escherichia coli cells. Although LZP and its analogs exhibited similar permeability, secondary structures, and localization in negatively charged membranes, significant differences were observed among these peptides in zwitterionic membranes. The results suggest a novel approach for designing antibacterial peptides with modulation of toxicity against hRBCs by employing the leucine zipper sequence. Also, to the best of our knowledge, the results demonstrate that this sequence could be utilized to design novel cell-selective molecules for the first time.     

Inducing toxicity by introducing a leucine-zipper-like motif in frog antimicrobial peptide, magainin 2

Cytotoxicity, a major obstacle in therapeutic application of antimicrobial peptides, is controlled by leucine-zipper-like sequences in melittin and other naturally occurring antimicrobial peptides. Magainin 2 shows significantly lower cytotoxicity than many naturally occurring antimicrobial peptides and lacks this structural element. To investigate the consequences of introducing a leucine zipper sequence in magainin 2, a novel analogue (Mag-mut) was designed by rearranging only the positions of its hydrophobic amino acids to include this structural element. Both magainin 2 and Mag-mut showed appreciable similarities in their secondary structures in the presence of negatively charged lipid vesicles, in localizing and permeabilizing the selected bacteria and exhibiting bactericidal activities. However, Mag-mut bound and localized strongly on to the mammalian cells tested and exhibited significantly higher cytotoxicity than magainin 2. Only Mag-mut, but not magainin 2, permeabilized human red blood cells and zwitterionic lipid vesicles. In contrast with magainin 2, Mag-mut self-assembled in an aqueous environment and bound co-operatively on to zwitterionic lipid vesicles. The peptides formed pores of different sizes on to a selected mammalian cell. The results of the present study indicate an important role of the leucine zipper sequence in the cytotoxicity of Mag-mut and demonstrate that its introduction into a non-toxic peptide, without altering the amino acid composition, can render cytotoxicity.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the α-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

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.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the alpha-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

Antimicrobial activity of leucine‐substituted decoralin analogs with lower hemolytic activity

Linear cationic α‐helical antimicrobial peptides are promising chemotherapeutics. Most of them act by different mechanisms, making it difficult to microorganisms acquiring resistance. Decoralin is an example of antimicrobial peptide; it was described by Konno et al. and presented activity against microorganisms, but with pronounced hemolytic activity. We synthesized leucine‐substituted decoralin analogs designed based on important physicochemical properties, which depend on the maintenance of the amphiphilic α‐helical tendency of the native molecule. Peptides were synthesized, purified, and characterized, and the conformational studies were performed. The results indicated that the analogs presented both higher therapeutic indexes, but with antagonistic behavior. While [Leu]¹⁰‐Dec‐NH₂ analog showed similar activity against different microorganisms (c.a. 0.4–0.8 μmol L^?1), helical structuration, and some hemolytic activity, [Leu]⁸‐Dec‐NH₂ analog did not tend to helical structure and presented antimicrobial activities two orders higher than the other two peptides analyzed. On the other hand, this analog showed to be the less hemolytic (MHC value = 50.0 μmol L^?1). This approach provided insight for understanding the effects of the leucine substitution in the amphiphilic balance. They led to changes on the conformational tendency, which showed to be important for the mechanism of action and affecting antimicrobial and hemolytic activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes

A number of research have proven that antimicrobial peptides are of greatest potential as a new class of antibiotics. Antimicrobial peptides and cell-penetrating peptides share some similar structure characteristics. In our study, a new peptide analog, APP (GLARALTRLLRQLTRQLTRA) from the cell-penetrating peptide ppTG20 (GLFRALLRLLRSLWRLLLRA), was identified simultaneously with the antibacterial mechanism of APP against Salmonella typhimurium and Streptococcus pyogenes. APP displayed potent antibacterial activity against Gram-negative and Gram-positive strains. The minimum inhibitory concentration was in the range of 2 to 4 μM. APP displayed higher cell selectivity (about 42-fold increase) as compared to the parent peptide for it decreased hemolytic activity and increased antimicrobial activity. The calcein leakage from egg yolk l-α-phosphatidylcholine (EYPC)/egg yolk l-α-phosphatidyl-dl-glycerol and EYPC/cholesterol vesicles demonstrated that APP exhibited high selectivity. The antibacterial mechanism analysis indicated that APP induced membrane permeabilization in a kinetic manner for membrane lesions allowing O-nitrophenyl-β-d-galactoside uptake into cells and potassium release from APP-treated cells. Flow cytometry analysis demonstrated that APP induced bacterial live cell membrane damage. Circular dichroism, fluorescence spectra, and gel retardation analysis confirmed that APP interacted with DNA and intercalated into the DNA base pairs after penetrating the cell membrane. Cell cycle assay showed that APP affected DNA synthesis in the cell. Our results suggested that peptides derived from the cell-penetrating peptide have the potential for antimicrobial agent development, and APP exerts its antibacterial activity by damaging bacterial cell membranes and binding to bacterial DNA to inhibit cellular functions, ultimately leading to cell death.     

Effects of dimerization of the cell‐penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action

The cell‐penetrating peptide Tat (48–60) (GRKKRRQRRRPPQ) derived from HIV‐1 Tat protein showed potent antibacterial activity (MIC: 2–8 µM ). To investigate the effect of dimerization of Tat (48–60) analog, [Tat(W): GRKKRRQRRRPWQ‐NH₂], on antimicrobial activity and mechanism of bactericidal action, its dimeric peptides, di‐Tat(W)‐C and di‐Tat(W)‐K, were synthesized by a disulfide bond linkage and lysine linkage of monomeric Tat(W), respectively. From the viewpoint of a weight basis and the monomer concentration, these dimeric peptides displayed almost similar antimicrobial activity against six bacterial strains tested but acted more rapidly against Staphylococcus aureus on kinetics of bactericidal activity, compared with monomeric Tat(W). Unlike monomeric Tat(W), these dimeric peptides significantly depolarized the cytoplasmic membrane of intact S. aureus cells at MIC and induced dye leakage from bacterial‐membrane‐mimicking egg yolk L ‐α‐phosphatidylethanolamine/egg yolk L ‐α‐phosphatidyl‐DL ‐glycerol (7:3, w/w) vesicles. Furthermore, these dimeric peptides were less effective to translocate across lipid bilayers than monomeric Tat(W). These results indicated that the dimerization of Tat analog induces a partial change in the mode of its bactericidal action from intracellular target mechanism to membrane‐targeting mechanism. Collectively, our designed dimeric Tat peptides with high antimicrobial activity and rapid bactericidal activity appear to be excellent candidates for future development as novel antimicrobial agents. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Identification and characterization of an amphipathic leucine zipper-like motif in Escherichia coli toxin hemolysin E. Plausible role in the assembly and membrane destabilization

Hemolysin E (HlyE) is a 34 kDa protein toxin, recently isolated from a pathogenic strain of Escherichia coli, which is believed to exert its toxic activity via formation of pores in the target cell membrane. With the goal of understanding the involvement of different segments of hemolysin E in the membrane interaction and assembly of the toxin, a conserved, amphipathic leucine zipper-like motif has been identified. In order to evaluate the possible structural and functional roles of this segment in HlyE, a 30-residue peptide (H-205) corresponding to the leucine zipper motif (amino acid 205-234) and two mutant peptides of the same size were synthesized and labeled by fluorescent probes at their N termini. The results show that the wild-type H-205 binds to both zwitterionic (PC/Chol) and negatively charged (PC/PG/Chol) phospholipid vesicles and also self-assemble therein. Detailed membrane-binding experiments revealed that this synthetic motif (H-205) formed large aggregates and inserted into the bilayer of only negatively charged lipid vesicles but not of zwitterionic membrane. Although both the mutants bound to zwitterionic and negatively charged lipid vesicles, neither of them inserted into the lipid bilayers nor assembled in any of these lipid vesicles. Furthermore, H-205 adopted a significant helical structure in membrane mimetic environments and induced the permeation of monovalent ions and release of entrapped calcein across the phospholipid vesicles more efficiently than the mutant peptides. The results presented here indicate that this H-205 (amino acid 205-234) segment may be an important structural element in hemolysin E, which could play a significant role in the binding and assembly of the toxin in the target cell membrane and its destabilization.     

Addition of a small hydrophobic segment from the head region to an amphipathic leucine zipper like motif of E. coli toxin hemolysin E enhances the peptide-induced permeability of zwitterionic lipid vesicles

To find out the sequence requirement of the H-205 peptide, containing an amphipathic leucine zipper motif corresponding to the amino acid (a.a.) region 205-234 of hemolysin E (HlyE) to induce efficient permeation in zwitterionic lipid vesicles, the peptide was extended at the N-terminal after the addition of seven amino acids from the predicted transmembrane region in the head domain of the protein-toxin. The new peptide, H-198 (a.a. 198-234) and a scrambled mutant peptide of the same size were synthesized, fluorescently labeled and characterized functionally and structurally. The results showed that H-198 induced significantly higher permeation in the zwitterionic PC/Chol lipid vesicles than its shorter version, H-205. H-198 formed large aggregates in the PC/Chol vesicles unlike H-205 and also adopted more helical structure in the membrane mimetic environments compared to that of H-205. Fluorescence energy transfer experiments by flow cytometry indicated that only H-198 but not its mutant or H-205 oligomerized in the zwitterionic lipid vesicles, while in the negatively charged lipid vesicles both H-198 and H-205 formed oligomeric assembly. The results suggest a probable role of the hydrophobic residues of the head domain of HlyE in inducing permeability in the zwitterionic lipid vesicles by the peptide derived from the a.a. 198-234 of the toxin.     

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

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

Addition of a small hydrophobic segment from the head region to an amphipathic leucine zipper like motif of E. coli toxin hemolysin E enhances the peptide-induced permeability of zwitterionic lipid vesicles

To find out the sequence requirement of the H-205 peptide, containing an amphipathic leucine zipper motif corresponding to the amino acid (a.a.) region 205-234 of hemolysin E (HlyE) to induce efficient permeation in zwitterionic lipid vesicles, the peptide was extended at the N-terminal after the addition of seven amino acids from the predicted transmembrane region in the head domain of the protein-toxin. The new peptide, H-198 (a.a. 198-234) and a scrambled mutant peptide of the same size were synthesized, fluorescently labeled and characterized functionally and structurally. The results showed that H-198 induced significantly higher permeation in the zwitterionic PC/Chol lipid vesicles than its shorter version, H-205. H-198 formed large aggregates in the PC/Chol vesicles unlike H-205 and also adopted more helical structure in the membrane mimetic environments compared to that of H-205. Fluorescence energy transfer experiments by flow cytometry indicated that only H-198 but not its mutant or H-205 oligomerized in the zwitterionic lipid vesicles, while in the negatively charged lipid vesicles both H-198 and H-205 formed oligomeric assembly. The results suggest a probable role of the hydrophobic residues of the head domain of HlyE in inducing permeability in the zwitterionic lipid vesicles by the peptide derived from the a.a. 198-234 of the toxin.     

The introduction of l-phenylalanine into antimicrobial peptide protonectin enhances the selective antibacterial activity of its derivative phe-Prt against Gram-positive bacteria

Protonectin was a typical amphiphilic antimicrobial peptide with potent antimicrobial activity against Gram-positive and Gram-negative bacteria. In the present study, when its eleventh amino acid in the sequence was substituted by phenylalanine, the analog named phe-Prt showed potent antimicrobial activity against Gram-positive bacteria, but no antimicrobial activity against Gram-negative bacteria, indicating a significant selectivity between Gram-positive bacteria and Gram-negative bacteria. However, when Gram-negative bacteria were incubated with EDTA, the bacteria were susceptible to phe-Prt. Next, the binding effect of phe-Prt with LPS was determined. Our result showed that LPS could hamper the bactericidal activity of phe-Prt against Gram-positive bacteria. The result of zeta potential assay further confirmed the binding effect of phe-Prt with LPS for it could neutralize the surface charge of E. coli and LPS. Then, the effect of phe-Prt on the integrity of outer membrane of Gram-negative bacteria was determined. Our results showed that phe-Prt had a much weaker disturbance to the outer membrane of Gram-negative bacteria than the parent peptide protonectin. In summary, the introduction of l-phenylalanine into the sequence of antimicrobial peptide protonectin made phe-Prt show significant selectivity against Gram-positive bacteria, which could partly be attributed to the delay effect of LPS for phe-Prt to access to cell membrane. Although further study is still needed to clarify the exact mechanism of selectivity, the present study provided a strategy to develop antimicrobial peptides with selectivity toward Gram-positive and Gram-negative bacteria.

     

Structures of the dimeric and monomeric variants of magainin antimicrobial peptides (MSI-78 and MSI-594) in micelles and bilayers, determined by NMR spectroscopy

Magainins are antimicrobial peptides that selectively disrupt bacterial cell membranes. In an effort to determine the propensity for oligomerization of specific highly active magainin analogues in membrane mimetic systems, we studied the structures and lipid interactions of two synthetic variants of magainins (MSI-78 and MSI-594) originally designed by Genaera Corp. Using NMR experiments on these peptides solubilized in dodecylphosphocholine (DPC) micelles, we found that the first analogue, MSI-78, forms an antiparallel dimer with a "phenylalanine zipper" holding together two highly helical protomers, whereas the second analogue, MSI-594, whose phenylalanines 12 and 16 were changed into glycine and valine, respectively, does not dimerize under our experimental conditions. In addition, magic angle spinning solid-state NMR experiments carried out on multilamellar vesicles were used to corroborate the helical conformation of the peptides found in detergent micelles and support the existence of a more compact structure for MSI-78 and a pronounced conformational heterogeneity for MSI-594. Since magainin activity is modulated by oligomerization within the membrane bilayers, this study represents a step forward in understanding the role of self-association in determining magainin function.     

Consequences of alteration in leucine zipper sequence of melittin in its neutralization of lipopolysaccharide-induced proinflammatory response in macrophage cells and interaction with lipopolysaccharide

The bee venom antimicrobial peptide, melittin, besides showing versatile activity against microorganisms also neutralizes lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells. However, how the amino acid sequence of melittin contributes in its anti-inflammatory properties is mostly unknown. To determine the importance of the leucine zipper sequence of melittin in its neutralization of LPS-induced inflammatory responses in macrophages and interaction with LPS, anti-inflammatory properties of melittin and its three analogues and their interactions with LPS were studied in detail. Two of these analogues, namely melittin Mut-1 (MM-1) and melittin Mut-2 (MM-2), possess leucine to alanine substitutions in the single and double heptadic leucine residue(s) of melittin, respectively, whereas the third analogue is a scrambled peptide (Mel-SCR) that contains the amino acid composition of melittin with minor rearrangement in its leucine zipper sequence. Although MM-1 partly inhibited the production of proinflammatory cytokines in RAW 264.7 and rat primary macrophage cells in the presence of LPS, MM-2 and Mel-SCR were negligibly active. A progressive decrease in interaction of melittin with LPS, aggregation in LPS, and dissociation of LPS aggregates with alteration in the leucine zipper sequence of melittin was observed. Furthermore, with alteration in the leucine zipper sequence of melittin, these analogues failed to exhibit cellular responses associated with neutralization of LPS-induced inflammatory responses in macrophage cells by melittin. The data indicated a probable important role of the leucine zipper sequence of melittin in neutralizing LPS-induced proinflammatory responses in macrophage cells as well as in its interaction with LPS.     

Promotion of peptide antimicrobial activity by fatty acid conjugation

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

Inhibition of HIV-1 entry before gp41 folds into its fusion-active conformation

HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). The core of the activated conformation of gp41 consists of a trimer of heterodimers comprising a leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation was found between the action of DP178, which is a potent inhibitor of HIV-1 entry into its host cell, and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was further tested and confirmed by circular dichroism spectroscopy. We found that whereas DP178 perturbs the partial alpha-helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it cannot perturb the trimer of heterodimers conformation, modeled by the complex of N36 or N51 with C34. Therefore, we suggest that the already formed trimer of heterodimers is not the target of inhibition by DP178. Our results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation.     

Structure-function study of cathelicidin-derived bovine antimicrobial peptide BMAP-28: Design of its cell-selective analogs by amino acid substitutions in the heptad repeat sequences

Although BMAP-28 is a potent cathelicidin-derived bovine antimicrobial peptide, its cytotoxic activity against the human and other mammalian cells is of concern for converting it into a novel antimicrobial drug. We have identified a short leucine and isoleucine zipper sequences at the N- and C-terminals of BMAP-28, respectively. To understand the possible role of these structural elements in BMAP-28, a number of alanine-substituted analogs were designed, synthesized and characterized along with the wild-type peptide. The substitution of amino acids at single or multiple ‘a’ position(s) of these structural motifs by alanine showed significant effects on the cytotoxic activity of the molecule on the human red blood cells (hRBCs) and 3T3 cells without showing much effects on their MIC values against the selected bacteria. BMAP-28 and all its analogs depolarized the Escherichia coli cells with almost equal efficacy. In contrast, the alanine-substituted analogs of BMAP-28 depolarized hRBCs much less efficiently than the parent molecule. Results further showed that BMAP-28 assembled appreciably onto the live E. coli and hRBC. However, the selected less toxic analogs of BMAP-28 although assembled as good as the parent molecule onto the live E. coli cells, their assembly onto the live mammalian hRBCs was much weaker as compared to that of the wild-type molecule. Looking at the remarkable similarity with the data presented in our previous work on melittin, it appears that probably the heptad repeat sequence possesses a general role in maintaining the cytotoxicity of the antimicrobial peptides against the mammalian cells and assembly therein.     

Properties and structure-activity studies of cyclic beta-hairpin peptidomimetics based on the cationic antimicrobial peptide protegrin I

The properties and structure-activity relationships (SAR) of a macrocyclic analogue of porcine protegrin I (PG-I) have been investigated. The lead compound, having the sequence cyclo-(-Leu-Arg-Leu-Lys-Lys-Arg-Arg-Trp-Lys-Tyr-Arg-Val-d-Pro-Pro-), shows antimicrobial activity against Gram-positive and -negative bacteria, but a much lower haemolytic activity and a much reduced ability to induce dye release from phosphatidylcholine/phosphatidylglycerol liposomes, when compared to PG-I. The enantiomeric form of the lead peptide shows comparable antimicrobial activity, a property shared with other cationic antimicrobial peptides acting on cell membranes. SAR studies involving the synthesis and biological profiling of over 100 single site substituted analogues, showed that the antimicrobial activity was tolerant to a large number of the substitutions tested. Some analogues showed slightly improved antimicrobial activities (2-4-fold lowering of MICs), whereas other substitutions caused large increases in haemolytic activity on human red blood cells.     

Using fluorous amino acids to probe the effects of changing hydrophobicity on the physical and biological properties of the beta-hairpin antimicrobial peptide protegrin-1

Protegrins are potent members of the beta-hairpin-forming class of antimicrobial peptides. Key to their antimicrobial activity is their assembly into oligomeric structures upon binding to the bacterial membrane. To examine the relationship between the physicochemical properties of the peptide and its biological activity, we have synthesized variants of protegrin-1 in which key residues in the hydrophobic core, valine-14 and -16, are changed to leucine and to the extensively fluorinated analogue hexafluoroleucine. These substitutions have the effect of making the peptide progressively more hydrophobic while minimally perturbing the secondary structure. The leucine-containing peptide was significantly more active than wild-type protegrin against several common pathogenic bacterial strains, whereas the hexafluoroleucine-substituted peptide, in contrast, showed significantly diminished activity against several bacterial strains. Isothermal titration calorimetry measurements revealed significant changes in the interaction of the peptides binding to small unilamelar vesicles that mimic the lipid composition of the bacterial membrane. The binding isotherms for wild-type and leucine-substituted protegrins indicate that electrostatic interactions dominate the membrane-peptide interaction, whereas the isotherm for the hexafluoroleucine-substituted protegrin suggests a diminished electrostatic component to binding. Notably both of these substitutions appear to alter the stoichiometry of the lipid-peptide interaction, suggesting that these substitutions may stabilize oligomerized forms of protegrin that are postulated to be intermediates in the assembly of the beta-barrel membrane pore structure.