Mammalian antimicrobial peptide protegrin‐4 self assembles and forms amyloid‐like aggregates: Assessment of its functional relevance

Protegrin‐4 (PG‐4) is a member of the porcine leukocyte protegrins family of cysteine‐rich antimicrobial peptides (AMPs) isolated from Sus scrofa. It consists of 18 amino acid residues and works as a part of innate immune system. In this study, we examined the intrinsic aggregation propensity of this AMP using multiple computational algorithms, namely, TANGO, AGGRESCAN, FOLDAMYLOID, AMYLPRED, and ZYGGREGATOR, and found that the peptide is predicted to have a high propensity for the β sheet formation that disposes this peptide to be amyloidogenic. Under in vitro conditions, PG‐4 formed visible aggregates and displayed the hallmark properties of typical amyloids such as enhanced binding of Congo red, increased fluorescence with Thioflavin‐T, and fibrillar morphology under transmission electron microscopy. Then we examined its antimicrobial activity against Bacillus subtilis and found that the aggregated peptide retained its antimicrobial activity. Additionally, the aggregates remain non‐toxic to the HEK293 and Caco2 cells. Our study suggests that the inherent aggregation properties of AMP can rationally be explored as a potential source of peptide‐based antimicrobials with enhanced stability.     

Stability of puroindoline peptides and effects on wheat rust

Peptides modelled on the tryptophan rich domain of puroindolines and the related grain softness protein-1 have a broad range of antibacterial and antifungal activities. With the aims of further investigating the activities of these antimicrobial peptides we studied their activity against wheat rust diseases and environmental stability. PINA-based peptides were found to have high pH and thermal stability in addition to being stable over long periods at room temperature. These properties could make them excellent candidates as preservatives in food. PuroA, Pina-R39G and PuroB peptides adversely affected the morphology of the stripe rust spores (Puccinia striiformis f. sp. tritici), while PuroA and PuroB showed moderate inhibition of their germination. Additionally, GSP-5D reduced the germination of leaf rust spores (P. triticina). PuroA and PuroB sprayed onto stripe rust infected plants effected a moderate reduction in the number of stripe rust uredinia on wheat seedlings, as did PuroB sprayed onto the seedlings and allowed to coat the leaves for 5 day prior to spore infection. The results suggest that the presence of the PIN-based peptides may lower frequency of initial infection foci.     

Improved anticancer potency by head‐to‐tail cyclization of short cationic anticancer peptides containing a lipophilic β2,2‐amino acid

We have recently reported a series of synthetic anticancer heptapeptides (H‐KKWβ^2,2WKK‐NH₂) containing a central achiral and lipophilic β^2,2‐amino acid that display low toxicity against non‐malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising β^2,2‐amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC‐5). The results demonstrated a considerable increase in anticancer potency following head‐to‐tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High‐resolution NMR studies and molecular dynamics simulations together with an annexin‐V‐FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Collection of antimicrobial peptides database and its derivatives: Applications and beyond

Collection of antimicrobial peptides (CAMP), CAMPSign, and ClassAMP are open‐access resources that have been developed to enhance research on antimicrobial peptides (AMPs). Comprehensive information on AMPs and machine learning‐based predictive models are made available for users through these resources. As of date, CAMP_R3 has 10,247 sequences, 757 structures, and 114 family‐specific signatures of AMPs along with associated tools for AMP sequence and structure analysis. CAMPSign uses family‐specific sequence conservation, in the form of patterns and hidden Markov models for identification of AMPs. ClassAMP can be used to classify AMPs as antibacterial, antifungal, or antiviral based on sequence information. Here we describe CAMP and its derivatives and illustrate, with a few examples, the contribution of these online resources to the advancement of our current understanding of AMPs.     

Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides

Pseudomonas aeruginosa is one of the major nosocomial pathogen that can causes a wide variety of acute and chronic infections P. aeruginosa is a dreaded bacteria not just because of the high intrinsic and acquired antibiotic resistance rates but also the biofilm formation and production of multiple virulence factors. We investigated the in vitro activities of antibiotics (ceftazidime, tobramycin, ciprofloxacin, doripenem, piperacillin and colistin) and antimicrobial cationic peptides (AMPs; LL-37, CAMA: cecropin(1–7)-melittin A(2–9) amide, melittin, defensin and magainin-II) alone or in combination against biofilms of laboratory strain ATCC 27853 and 4 clinical strains of P. aeruginosa. The minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentrations (MBEC) were determined by microbroth dilution technique. The MBEC values of antibiotics and AMPs were 80–>5120 and 640–>640 mg/L, respectively. When combined with the LL-37 or CAMA at 1/10× MBEC, the MBEC values of antibiotics that active against biofilms, were decreased up to 8-fold. All of the antibiotics, and AMPs were able to inhibit the attachment of bacteria at the 1/10× MIC and biofilm formation at 1× or 1/10× MIC concentrations. Time killing curve studies showed 3-log₁₀ killing against biofilms in 24 h with almost all studied antibiotics and AMPs. Synergism were seen in most of the studied combinations especially CAMA/LL-37 + ciprofloxacin against at least one or two strains’ biofilms. Since biofilms are not affected the antibiotics at therapeutic concentrations, using a combination of antimicrobial agents including AMPs, or inhibition of biofilm formation by blocking the attachment of bacteria to surfaces might be alternative methods to fight with biofilm associated infections.     

Biological consequences of improving the structural stability of hairpins that have antimicrobial activity

Designing new antimicrobial peptides (AMPs) focuses heavily on the activity of the peptide and less on the elements that stabilize the secondary structure of these peptides. Studies have shown that improving the structure of naturally occurring AMPs can affect activity and so here we explore the relationship between structure and activity of two non‐naturally occurring AMPs. We have used a backbone‐cyclized peptide as a template and designed an uncyclized analogue of this peptide that has antimicrobial activity. We focused on beta‐hairpin‐like structuring features. Improvements to the structure of this peptide reduced the activity of the peptide against gram‐negative, Escherichia coli but improved the activity against gram‐positive, Corynebacterium glutamicum. Distinctions in structuring effects on gram‐negative versus gram‐positive activity were also seen in a second peptide system. Structural improvements resulted in a peptide that was more active than the native against gram‐positive bacterium but less active against gram‐negative bacterium. Our results show that there is not always a correlation between improved hairpin‐structuring and activity. Other factors such as the type of bacteria being targeted as well as net positive charge can play a role in the potency of AMPs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

The ebb and flow of antimicrobial skin peptides defends northern leopard frogs (Rana pipiens) against chytridiomycosis

Many amphibian species are threatened with extinction by the emerging infectious disease, chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis. This unprecedented global crisis threatens to reduce the biodiversity of the entire amphibian class. The fungus invades the skin and impairs the uptake and retention of essential ions leading to cardiac arrest. Antimicrobial peptides (AMPs) secreted into the mucus of some amphibians are thought to be an important defense against chytridiomycosis. However, little is known about the quantities of AMPs secreted under natural conditions, whether they are sufficient to protect against this pathogen, and how they interact with commensal microbes. To understand how defensive peptides and skin microbes may interact, it is essential to know the precise quantities of AMPs present under natural conditions. Using matrix‐assisted laser desorption time‐of‐flight mass spectrometry and growth inhibition assays, we show that northern leopard frogs (Rana pipiens) at rest constitutively release low amounts of AMPs that inhibit B. dendrobatidis in vitro, and AMP defenses are elevated following a simulated predator attack. Using a synthetic peptide analogue of brevinin‐1Pb as an external control, we quantified the amounts of four previously described AMPs (brevinin 1Pa, brevinin‐1Pb, brevinin‐1Pd, and ranatuerin‐2P) at several time points after secretion. Once secreted onto the skin, the peptides are most active for 15 min, and small quantities persist for at least 2 h. Taken together, our data suggest that small amounts of AMPs are rapidly available and quite stable on the skin of R. pipiens. They are effective inhibitors of B. dendrobatidis at these low constitutive concentrations but degrade within 2 h, protecting the integrity of the skin and commensal bacteria.     

The effect of structural parameters and positive charge distance on the interaction free energy of antimicrobial peptides with membrane surface

Many attempts have been made to find hints explaining the relationship between physicochemical and structural properties of antimicrobial peptides (AMPs) which are relevant to their antimicrobial activities. We here found that there is a difference in the percentages of hydrophobic, hydrophilic, and charged residues between AMPs killing both bacteria and fungi (Group A) and AMPs that only kill bacteria (Group B). The percentage of charged residues in Group A AMPs is highly elevated, while in Group B the percentage of hydrophobic residues is increased. This result suggests a sequence-based mechanism of selectivity for AMPs. Moreover, we examined how the distance between basic residues affects the interaction free energy of AMPs with the membrane surface, since most of the known AMPs act by membrane perturbation. We measured the average distance between basic residues throughout the 3D structure of AMPs by defining D_pr parameter and calculated the interaction free energy for 10 AMPs that interacted with the DPPC membrane using molecular dynamics simulation. We found that the changes of the interaction free energy correlates with the change of D_pr by a linear regression coefficient of r^2?=?.47 and a cubic regression coefficient of r^2?=?.70.     

Ocellatin‐PT antimicrobial peptides: High‐resolution microscopy studies in antileishmania models and interactions with mimetic membrane systems

Although the mechanism of action of antimicrobial peptides (AMPs) is not clear, they can interact electrostatically with the cell membranes of microorganisms. New ocellatin‐PT peptides were recently isolated from the skin secretion of Leptodactylus pustulatus. The secondary structure of these AMPs and their effect on Leishmania infantum cells, and on different lipid surface models was characterized in this work. The results showed that all ocellatin‐PT peptides have an α‐helix structure and five of them (PT3, PT4, PT6 to PT8) have leishmanicidal activity; PT1 and PT2 affected the cellular morphology of the parasites and showed greater affinity for leishmania and bacteria‐mimicking lipid membranes than for those of mammals. The results show selectivity of ocellatin‐PTs to the membranes of microorganisms and the applicability of biophysical methods to clarify the interaction of AMPs with cell membranes.     

Photo‐inactivation of Bacillus endospores: inter‐specific variability of inactivation efficiency

The aims of this work were to (a) evaluate the susceptibility of endospores of Bacillus cereus, B. licheniformis, B. sphaericus and B. subtilis to photodynamic inactivation using a tricationic porphyrin as photosensitizer, (b) assess the efficiency of adsorption of the photosensitizer in endospore material as a determinant of the susceptibility of endospores of different Bacillus species to photo‐inactivation, (c) determine the value of B. cereus as a model organism for studies of antimicrobial photodynamic inactivation of bacterial endospores. The results of irradiation experiments with endospores of four species of Bacillus showed that B. cereus was the only species for which efficient endospore photo‐inactivation (> 3 log reduction) could be achieved. Endospores of B. licheniformis, B. sphaericus and B. subtilis were virtually resistant to photo‐inactivation with tricationic porphyrin. The amount of porphyrin bound to endospore material was not significantly different between species, regardless of the presence of an exosporium or exosporium‐like outer layer. The sensitivity of endospores to photodynamic inactivation with a tricationic porphyrin is highly variable among different species of the genus Bacillus. The presence of an exosporium in endospores of B. cereus and B. sphaericus, or an exosporium‐like glycoprotein layer in endospores of B. subtilis, did not affect the amount of bound photosensitizer and did not explain the inter‐species variability in susceptibility to photodynamic inactivation. The results imply that the use of B. cereus as a more amenable surrogate of the exosporium‐producing B. anthracis must be carefully considered when testing new photosensitizers for their antimicrobial photo‐inactivation properties.     

Antibacterial activity of different degree of hydrolysis of palm kernel expeller peptides against spore-forming and non-spore-forming bacteria

Aims: The goal of this study was to determine inhibitory effect of palm kernel expeller (PKE) peptides of different degree of hydrolysis (DH %) against spore‐forming bacteria Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophillus, Bacillus subtilis, Bacillus thuringiensis, Clostridium perfringens; and non‐spore‐forming bacteria Escherichia coli, Lisinibacillus sphaericus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium and Staphylococcus aureus. Methods and Results: A range of DH % (50–100) of PKE peptides was prepared using alcalase, and hydrolysis conditions were determined using response surface methodology (RSM). The influence of pH (6·5–10·5), temperature (35–65°C), enzyme/substrate ratio (1–5%) and substrate concentration (1–2%) were studied on the response of the DH. The antibacterial activity of different DH % of PKE peptides was tested by using disc diffusion assay and micro‐broth dilution assay. According to the minimum inhibitory concentration (MIC) test on each of the PKE peptides of different DH %, the 70 DH % PKE peptide showed greater inhibitory effect compared to the 100 DH % PKE peptide against B. cereus, B. coagulans, B. megaterium, B. pumilus, B. stearothermophillus, B. subtilis, B. thuringiensis, Cl. perfringens, Lisinibacillus sphaericus and L. monocytogenes. Conclusions: The 70 DH % PKE peptides exhibited greatest overall antibacterial effect of the various peptides of PKE evaluated. Further research is needed to determine the mode of action of PKE peptides. Significance and Impact of the Study: Palm kernel expeller peptides, a natural plant product, effectively inhibited the growth of spore‐forming and non‐spore‐forming Gram‐positive bacteria. Potentially, PKE peptides could be used in food preservation and developed as antibacterial agent in the pharmaceutical industry.     

Epitope mapping of the N‐terminal portion of tissue transglutaminase protein antigen to identify linear epitopes in celiac disease

Celiac disease (CD) is an autoimmune mediated disease with complex and multifactorial etiology. Gluten intake triggers a composite immune response involving T‐cells and B‐cells and leading to the secretion of autoantibodies if a genetic predisposition is present. Untreated CD patients show high levels of circulating autoantibodies directed to different auto‐antigens present in the intestinal mucosa. The most important auto‐antigen is the endomysial enzyme tissue transglutaminase (tTG). Both IgA and IgG antibody isotypes to tTG are known, but only the IgA antibodies demonstrate the highest disease specificity and thus are considered disease biomarkers. Because the pathogenicity and exact tTG binding properties of these autoantibodies are still unclear, the characterization of tTG antigenic domains is a crucial step in understanding CD onset and the autoimmune pathogenesis. Overlapping peptide libraries can be used for epitope mapping of selected protein portions to determine antigenic fragments contributing to the immunological activity and possibly develop innovative peptide‐based tools with high specificity and sensitivity for CD. We performed an epitope mapping study to characterize putative linear auto‐antigenic epitopes present in the tTG N‐terminal portion (1–230). A library of 23 overlapping peptides spanning tTG(1–230) was generated by Fmoc/tBu solid‐phase peptide synthesis and screened by immunoenzymatic assays employing patients' sera. The results indicate that four synthetic peptides, that is, Ac‐tTG(1–15)‐NH₂, Ac‐tTG(41–55)‐NH₂, Ac‐tTG(51–65)‐NH₂, and Ac‐tTG(151–165)‐NH₂, are recognized by IgA autoantibodies circulating in CD patients' sera. These results offer important insight on the nature of the antigen‐antibody interaction. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Studies on the antileishmanial properties of the antimicrobial peptides temporin A,B and 1Sa

Given the paucity and toxicity of available drugs for leishmaniasis, coupled with the advent of drug resistance, the discovery of new therapies for this neglected tropical disease is recognised as being of the utmost urgency. As such antimicrobial peptides (AMPs) have been proposed as promising compounds against the causative Leishmania species, insect vector‐borne protozoan parasites. Here the AMP temporins A, B and 1Sa have been synthesised and screened for activity against Leishmania mexicana insect stage promastigotes and mammalian stage amastigotes, a significant cause of human cutaneous disease. In contrast to previous studies with other species the activity of these AMPs against L. mexicana amastigotes was low. This suggests that amastigotes from different Leishmania species display varying susceptibility to peptides from the temporin family, perhaps indicating differences in their surface structure, the proposed target of these AMPs. In contrast, insect stage L. mexicana promastigotes were sensitive to two of the screened temporins which clearly demonstrates the importance of screening AMPs against both forms of the parasite. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio

Eight peptides with differential growth–inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH₂) and temporin-1Gd (FILPLIASFLSKFL.NH₂) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC₅₀=2.4±0.1 μM for temporin-1Gb and 2.3±0.2 μM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.     

Synthetic antimicrobial β‐peptide in dual‐treatment with fluconazole or ketoconazole enhances the in vitro inhibition of planktonic and biofilm Candida albicans

Fungal infections are a pressing concern for human health worldwide, particularly for immunocompromised individuals. Current challenges such as the elevated toxicity of common antifungal drugs and the emerging resistance towards these could be overcome by multidrug therapy. Natural antimicrobial peptides, AMPs, in combination with other antifungal agents are a promising avenue to address the prevailing challenges. However, they possess limited biostability and susceptibility to proteases, which has significantly hampered their development as antifungal therapies. β‐peptides are synthetic materials designed to mimic AMPs while allowing high tunability and increased biostability. In this work, we report for the first time the inhibition achieved in Candida albicans when treated with a mixture of a β‐peptide model and fluconazole or ketoconazole. This combination treatment enhanced the biological activity of these azoles in planktonic and biofilm Candida, and also in a fluconazole‐resistant strain. Furthermore, the in vitro cytotoxicity of the dual treatment was evaluated towards the human hepatoma cell line, HepG2, a widely used model derived from liver tissue, which is primarily affected by azoles. Analyses based on the LA‐based method and the mass‐action law principle, using a microtiter checkerboard approach, revealed synergism of the combination treatment in the inhibition of planktonic C. albicans. The dual treatment proved to be fungicidal at 48 and 72 h. Interestingly, it was also found that the viability of HepG2 was not significantly affected by the dual treatments. Finally, a remarkable enhancement in the inhibition of the highly azole‐resistant biofilms and fluconazole resistant C. albicans strain was obtained. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of two novel antimicrobial peptides from the cuticular extracts of the ant Trichomyrmex criniceps (Mayr), (Hymenoptera: Formicidae)

Antimicrobial peptides (AMPs) from cuticular extracts of worker ants of Trichomyrmex criniceps (Mayr, Hymenoptera: Formicidae) were isolated and evaluated for their antimicrobial activity. Eight peptides ranging in mass from 804.42 to 1541.04 Da were characterized using a combination of analytical and bioinformatics approach. All the eight peptides were novel with no similarity to any of the AMPs archived in the Antimicrobial Peptide Database. Two of the eight novel peptides, the smallest and the largest by mass were named Crinicepsin‐1 and Crinicepsin‐2 and were chemically synthesized by solid phase peptide synthesis. The two synthetic peptides had antibacterial and weak hemolytic activity.     

The Antimicrobial Domains of Wheat Puroindolines Are Cell-Penetrating Peptides with Possible Intracellular Mechanisms of Action

The puroindoline proteins (PINA and PINB) of wheat display lipid-binding properties which affect the grain texture, a critical parameter for wheat quality. Interestingly, the same proteins also display antibacterial and antifungal properties, attributed mainly to their Tryptophan-rich domain (TRD). Synthetic peptides based on this domain also display selectivity towards bacterial and fungal cells and do not cause haemolysis of mammalian cells. However, the mechanisms of these activities are unclear, thus limiting our understanding of the in vivo roles of PINs and development of novel applications. This study investigated the mechanisms of antimicrobial activities of synthetic peptides based on the TRD of the PINA and PINB proteins. Calcein dye leakage tests and transmission electron microscopy showed that the peptides PuroA, Pina-M and Pina-W→F selectively permeabilised the large unilamellar vesicles (LUVs) made with negatively charged phospholipids mimicking bacterial membranes, but were ineffective against LUVs made with zwitterionic phospholipids mimicking eukaryotic membranes. Propidium iodide fluorescence tests of yeast (Saccharomyces cerevisiae) cells showed the peptides were able to cause loss of membrane integrity, PuroA and Pina-M being more efficient. Scanning electron micrographs of PINA-based peptide treated yeast cells showed the formation of pits or pores in cell membranes and release of cellular contents. Gel retardation assays indicated the peptides were able to bind to DNA in vitro, and the induction of filamental growth of E. coli cells indicated in vivo inhibition of DNA synthesis. Together, the results strongly suggest that the PIN-based peptides exert their antimicrobial effects by pore formation in the cell membrane, likely by a carpet-like mechanism, followed by intracellular mechanisms of activity.     

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Dermaseptin 01 as antimicrobial peptide with rich biotechnological potential: study of peptide interaction with membranes containing Leishmania amazonensis lipid‐rich extract and membrane models

This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA‐ KAAGQAALGAL‐NH₂, DS 01) with phospholipid (PL) monolayers comprising (i) a lipid‐rich extract of Leishmania amazonensis (LRE‐La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid‐air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE‐La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE‐La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.