Showing papers in "Journal of Peptide Science in 2014"
TL;DR: The self‐assembly of several classes of amphiphilic peptides is reviewed, and selected applications are discussed, and the influence of environmental variables such as pH and temperature on aggregate nanostructure is discussed.
Abstract: The self-assembly of several classes of amphiphilic peptides is reviewed, and selected applications are discussed. We discuss recent work on the self-assembly of lipopeptides, surfactant-like peptides and amyloid peptides derived from the amyloid-β peptide. The influence of environmental variables such as pH and temperature on aggregate nanostructure is discussed. Enzyme-induced remodelling due to peptide cleavage and nanostructure control through photocleavage or photo-cross-linking are also considered. Lastly, selected applications of amphiphilic peptides in biomedicine and materials science are outlined. © 2014 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons, Ltd.
282 citations
TL;DR: Despite the highly efficient internalization of antibodies, enzymes, and other protein factors, as well as siRNA and RNA mimics, the uptake and stabile insertion of DNA into the genome of the host cells remain substantially challenging.
Abstract: The penetration of polar or badly soluble compounds through a cell membrane into live cells requires mechanical support or chemical helpers. Cell-penetrating peptides (CPPs) are very promising chemical helpers. Because of their low cytotoxicity and final degradation to amino acids, they are particularly favored in in vivo studies and for clinical applications. Clearly, the future of CPP research is bright; however, the required optimization studies for each drug require considerable individualized attention. Thus, CPPs are not the philosopher's stone. As of today, a large number of such transporter peptides with very different sequences have been identified. These have different uptake mechanisms and can transport different cargos. Intracellular concentrations of cargos can reach a low micromole range and are able to influence intracellular reactions. Internalized ribonucleic acids such as small interfering RNA (siRNA) and mimics of RNA such as peptide nucleic acids, morpholino nucleic acids, and triesters of oligonucleotides can influence transcription and translation. Despite the highly efficient internalization of antibodies, enzymes, and other protein factors, as well as siRNA and RNA mimics, the uptake and stabile insertion of DNA into the genome of the host cells remain substantially challenging.
This review describes a wide array of differing CPPs, cargos, cell lines, and tissues. The application of CPPs is compared with electroporation, magnetofection, lipofection, viral vectors, dendrimers, and nanoparticles, including commercially available products. The limitations of CPPs include low cell and tissue selectivity of the first generation and the necessity for formation of fusion proteins, conjugates, or noncovalent complexes to different cargos and of cargo release from intracellular vesicles. Furthermore, the noncovalent complexes require a strong molar excess of CPPs, and extensive experimentation is required to determine the most optimal CPP for any given cargo and cell type. Yet to predict which CPP is optimal for any given target remains a complex question. More recently, there have been promising developments: the enhancement of cell specificity using activatable CPPs, specific transport into cell organelles by insertion of corresponding localization sequences, and the transport of drugs through blood–brain barriers, through the conjunctiva of eyes, skin, and into nerve cells. Proteins, siRNA, and mimics of oligonucleotides can be efficiently transported into cells and have been tested for treatment of certain diseases. The recent state of the art in CPP research is discussed together with the overall scope, limitations, and some recommendations for future research directions. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
218 citations
TL;DR: The different pathways of biogenesis of DAACPs not only in bacteria but also in multicellular organisms are discussed, along with the description of the cellular specificity, the enzyme specificity and the substrate specificity of peptidyl aminoacyl l‐d isomerisation.
Abstract: Peptides and proteins are chiral molecules with their structure determined by the composition and configuration of the amino acids constituting them. Natural amino acids (except glycine) display two chiral types (l- and d-enantiomers). For example, the presence of octopine, a derivative of l-arginine and d-alanine in octopus, or peptidyl poly-d-glutamic acid in a bacterial cell wall was demonstrated in the 1920s and 1930s, respectively. Nevertheless, an old dogma in biology was that proteins (in a strict sense) are composed of amino acids in the l-configuration exclusively, until a d-alanyl residue was reported in a frog skin opioid peptide in the early 1980s, and since, numerous d-amino acid containing peptides (DAACPs) have been discovered in multicellular organisms. Several hypotheses may be formulated to explain the origin of a d-residue in the peptide/protein chain. It may result from different mechanisms such as incorporation of a d-amino acid, non-enzymatic racemisation associated with ageing or diseases and enzymatic posttranslational modification. In the last case, the DAACPs are synthesised via a ribosome-dependent manner, and a normal codon for l-amino acid is present in the mRNA at the position where the d-residue is processed in the mature peptide by peptidyl aminoacyl l-d isomerisation, a peculiar and subtle posttranslational modification. In this review, the different pathways of biogenesis of DAACPs not only in bacteria but also in multicellular organisms are discussed, along with the description of the cellular specificity, the enzyme specificity and the substrate specificity of peptidyl aminoacyl l-d isomerisation.
100 citations
TL;DR: Experimental and theoretical data is presented on handedness preference and switches between right‐handed and left‐handed helical peptide structures generated by protein amino acids or appropriately designed, side‐chain modified analogs, and three categories of screw‐sense issues.
Abstract: In this article, we review the relevant results obtained during almost 60 years of research on a specific aspect of stereochemistry, namely handedness preference and switches between right-handed and left-handed helical peptide structures generated by protein amino acids or appropriately designed, side-chain modified analogs. In particular, we present and discuss here experimental and theoretical data on three categories of those screw-sense issues: (i) right-handed/left-handed α-helix transitions underwent by peptides rich in Asp, specific Asp β-esters, and Asn; (ii) comparison of the preferred conformations adopted by helical host-guest peptide series, each characterized by an amino acid residue (e.g. Ile or its diastereomer aIle) endowed with two chiral centers in its chemical structure; and (iii) right-handed (type I)/left-handed (type II) poly-(Pro)n helix transitions monitored for peptides rich in Pro itself or its analogs with a pyrrolidine ring substitution, particularly at the biologically important position 4. The unique modular and chiral properties of peptides, combined with their relatively easy synthesis, the chance to shape them into the desired conformation, and the enormous chemical diversity of their coded and non-coded α-amino acid building blocks, offer a huge opportunity to structural chemists for applications to bioscience and nanoscience problems.
84 citations
TL;DR: While genome‐wide association studies as well as candidate gene studies have revealed a great deal of insight into the contribution of genetics to obesity development and susceptibility, advances in adipose tissue research have substantially changed the understanding of adipose tissues function.
Abstract: While genome-wide association studies as well as candidate gene studies have revealed a great deal of insight into the contribution of genetics to obesity development and susceptibility, advances in adipose tissue research have substantially changed the understanding of adipose tissue function. Its perception has changed from passive lipid storage tissue to active endocrine organ regulating and modulating whole-body energy homeostasis and metabolism and inflammatory and immune responses by secreting a multitude of bioactive molecules, termed adipokines. The expression of human vaspin (serpinA12) is positively correlated to body mass index and insulin sensitivity and increases glucose tolerance in vivo, suggesting a compensatory role in response to diminished insulin signaling in obesity. Recently, considerable insight has been gained into vaspin structure, function, and specific target tissue-dependent effects, and several lines of evidence suggest vaspin as a promising candidate for drug development for the treatment of obesity-related insulin resistance and inflammation. These will be summarized in this review with a focus on molecular mechanisms and pathways.
81 citations
TL;DR: Analysis of the relationship between the R5 amino acid sequence and its activity in silica precipitation emphasizes the essential role of the lysine residues in mediating silica polycondensation.
Abstract: The R5 peptide is derived from silaffin peptides naturally occurring in the diatom Cylindrotheca fusiformis and exhibits outstanding activity in silica precipitation. Because of its ability to cause silicification under mild conditions, several biotechnological applications based on R5-mediated biomimetic silica formation have already been reported. Yet a more detailed understanding of the R5 peptide and its intrinsic silica precipitation activity will help the rational design of R5 peptide variants as efficient agents for defined silica precipitation. The herein presented analysis of the relationship between the R5 amino acid sequence and its activity in silica precipitation emphasizes the essential role of the lysine residues in mediating silica polycondensation. Furthermore, a tetra amino acid motif (RRIL) has to be present within the R5 sequence, but in contrast to previous reports, we demonstrate that localization of the RRIL motif shows minor impact on silica precipitation activity but rather on morphology of the resulting silica material. The amino acid sequence of silaffin peptides is a well-balanced arrangement in terms of charges, functional groups and distances. The impact of this pattern of charges and functionalities was highlighted by the disturbed morphology of silica spheres resulting from R5 variants with scrambled sequences. A detailed understanding of the highly evolved silaffin sequence(s) will contribute to unravel the intriguing process of silica biomineralization in diatoms.
62 citations
TL;DR: The use of selenocysteine in ligation chemistry as well as recent investigations of chemoselective ligation–deselenization chemistry at other selenol‐derived amino acids that have the potential to greatly expand the number of targets that can be accessed by chemical synthesis are outlined.
Abstract: The convergent assembly of peptide fragments by native chemical ligation has revolutionized the way in which proteins can be accessed by chemical synthesis A variation of native chemical ligation involves the reaction of peptides bearing an N-terminal selenocysteine residue with peptide thioesters, which proceeds through the same mechanism as the parent reaction This transformation was first investigated in 2001 for the installation of selenocysteine into peptides and proteins via ligation chemistry The recent discovery that selenocysteine residues within peptides can be chemoselectively deselenized without the concomitant desulfurization of cysteine residues has led to renewed interest in ligation chemistry at selenocysteine This review outlines the use of selenocysteine in ligation chemistry as well as recent investigations of chemoselective ligation-deselenization chemistry at other selenol-derived amino acids that have the potential to greatly expand the number of targets that can be accessed by chemical synthesis
60 citations
TL;DR: The ELISA and in vivo near‐infrared fluorescence imaging revealed that peptide‐nanosheets did not activate the immune system despite the same hydrophilic block being used, suggesting the high surface density of poly(sarcosine) chains on the peptide-nanosheet may be one of the causes of the suppressive immune response.
Abstract: Nanoparticles are expected to be applicable for the theranostics as a carrier of the diagnostic and therapeutic agents. Lactosome is a polymeric micelle composed of amphiphilic polydepsipeptide, poly(sarcosine)64-block-poly(L-lactic acid)30, which was found to accumulate in solid tumors through the enhanced permeability and retention effect. However, lactosome was captured by liver on the second administration to a mouse. This phenomenon is called as the accelerated blood clearance phenomenon. On the other hand, peptide-nanosheet composed of amphiphilic polypeptide, poly(sarcosine)60-block-(L-Leu-Aib)6, where the poly(L-lactic acid) block in lactosome was replaced with the (L-Leu-Aib)6 block, abolished the accelerated blood clearance phenomenon. The ELISA and in vivo near-infrared fluorescence imaging revealed that peptide-nanosheets did not activate the immune system despite the same hydrophilic block being used. The high surface density of poly(sarcosine) chains on the peptide-nanosheet may be one of the causes of the suppressive immune response.
53 citations
47 citations
TL;DR: It is shown that observed blue PL peak is similar in these supramolecular nanofibrillar structures and is excited by the network of non‐covalent hydrogen bonds that link newly thermally induced neighboring cyclic dipeptide strands to final extended β‐sheet structure of amyloid‐like nan ofibrils.
Abstract: Thermally induced phase transformation in bioorganic nanotubes, which self-assembled from two ultrashort dipeptides of different origin, aromatic diphenylalanine (FF) and aliphatic dileucine (LL), is studied. In both FF and LL nanotubes, irreversible phase transformation found at 120-180 °C is governed by linear-to-cyclic dipeptide molecular modification followed by formation of extended β-sheet structure. As a result of this process, native open-end FF and LL nanotubes are transformed into ultrathin nanofibrils. Found deep reconstructions at all levels from macroscopic (morphology) and structural space symmetry to molecular give rise to new optical properties in both aromatic FF and aliphatic LL nanofibrils and generation of blue photoluminescence (PL) emission. It is shown that observed blue PL peak is similar in these supramolecular nanofibrillar structures and is excited by the network of non-covalent hydrogen bonds that link newly thermally induced neighboring cyclic dipeptide strands to final extended β-sheet structure of amyloid-like nanofibrils. The observed blue PL peak in short dipeptide nanofibrils is similar to the blue PL peak that was recently found in amyloid fibrils and can be considered as the optical signature of β-sheet structures. Nanotubular structures were characterized by environmental scanning electron microscope, ToF-secondary ion mass spectroscopy, CD and fluorescence spectroscopy.
44 citations
TL;DR: The results clearly show that the protocol developed minimises the potential detrimental effect that thermolysin can have on the cells and that these enzymatically triggered hydrogels can be used for the 3D encapsulation and culture of cells.
Abstract: We have investigated the possibility of using enzymatically triggered peptide hydrogels for the encapsulation and culture of cells Based on recent work done on the enzymatically triggered gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) using thermolysin, a protease enzyme from Bacillus Thermoproteolyticus Rokko, we have investigated the possibility of using this gelation triggering mechanism to encapsulate cells within a 3D hydrogel matrix First, the properties of enzymatically triggered hydrogels prepared in phosphate buffer solution were investigated and compared with the properties of hydrogels prepared in HPLC grade water from our previous work We showed that the use of phosphate buffer solution allowed the production of hydrogels with very high shear moduli (>1 MPa) The gelation kinetics was also investigated, and the mechanical properties of the system were shown to closely follow the synthesis of the octapeptide by the enzyme through reverse hydrolysis In a second phase, we developed, on the basis of information acquired, a facile protocol for the encapsulation of cells and plating of the hydrogel Human dermal fibroblasts were then used to exemplify the use of these materials FEFEFKFK octapeptide hydrogels prepared under the same conditions and with the same mechanical properties were used as a control We showed that no significant differences were observed between the two systems and that after a decrease in cell number on day 1, cells start to proliferate After 5 days of culture, the cells can be seen to start to adopt a stretched morphology typical of fibroblasts The results clearly show that the protocol developed minimises the potential detrimental effect that thermolysin can have on the cells and that these enzymatically triggered hydrogels can be used for the 3D encapsulation and culture of cells
TL;DR: The rational design of potent monocyclic and bicyclic inhibitors of human matriptase‐1 is reported, finding that the presence of positive charge and lack of bulky residues at the peptide N‐terminus is required for the maintenance of inhibitory activity.
Abstract: Sunflower trypsin inhibitor-1 (SFTI-1), a bicyclic tetradecapeptide, has become a versatile tool as a scaffold for the development of the inhibitors of therapeutically relevant serine proteases, among them matriptase and kallikreins. Herein, we report the rational design of potent monocyclic and bicyclic inhibitors of human matriptase-1. We found that the presence of positive charge and lack of bulky residues at the peptide N-terminus is required for the maintenance of inhibitory activity. Replacement of the N-terminal glycine residue by lysine allowed for the chemical conjugation with a fluorophor via the e-amino group without significant loss of inhibitory activity. Head-to-tail and side-chain-to-tail cyclization resulted in potent inhibitors with comparable activities against matriptase-1. The most potent synthetic bicyclic inhibitor found in this study (Ki=2.6nM at pH 7.6) is a truncated version of SFTI-1 (cyclo-KRCTKSIPPRCH) lacking a C-terminal proline and aspartate residue. It combines an internal disulfide bond with a peptide macrocycle that is formed through side-chain-to-tail cyclization of the e-amino group of an N-terminal lysine and a C-terminal proline. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site.
TL;DR: In this paper, the authors reported the first drug conjugate with a negatively charged amphipathic cell-penetrating peptide and compared two different doxorubicin cellpenetrated peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH.
Abstract: We report the first drug conjugate with a negatively charged amphipathic cell-penetrating peptide. Furthermore, we compare two different doxorubicin cell-penetrating peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH, namely the positively charged octaarginine and the negatively charged proline-rich amphipathic peptide. These conjugates were prepared exploiting a novel heterobifunctional crosslinker to join the N-terminal cysteine residue of the peptides with the aliphatic ketone of doxorubicin. This small linker contains an activated thiol as well as aminooxy functionality, capable of generating a stable oxime bond with the C-13 carbonyl group of doxorubicin. The disulfide bond formed between the peptide and doxorubicin enables the release of the drug in the cytosol, as confirmed by drug-release studies performed in the presence of glutathione. Additionally, the cytotoxicity as well as the cellular uptake and distribution of this tripartite drug delivery system was investigated in MCF-7 and HT-29 cell lines.
TL;DR: It is shown how templates can be used to control the reactivity and chemoselectivity of NCL reactions and potential applications in nucleic acid diagnosis, origin‐of‐life studies and gene‐expression‐specific therapies are discussed.
Abstract: Native chemical ligation (NCL) is a powerful method for the convergent synthesis of proteins and peptides. In its original format, NCL between a peptide containing a C-terminal thioester and another peptide offering an N-terminal cysteine has been used to enable protein synthesis of unprotected peptide fragments. However, the applications of NCL extend beyond the scope of protein synthesis. For instance, NCL can be put under the control of template molecules. In such a scenario, NCL enables the design of conditional reaction systems in which, peptide bond formation occurs only when a specific third party molecule is present. In this review, we will show how templates can be used to control the reactivity and chemoselectivity of NCL reactions. We highlight peptide and nucleic-acid-templated NCL reactions and discuss potential applications in nucleic acid diagnosis, origin-of-life studies and gene-expression-specific therapies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
TL;DR: A method for the synthesis of side‐ chain to side‐chain cyclic peptides based on the intermolecular reaction between a linear peptide functionalized with two aminooxy or hydrazide side chains and an organic dialdehyde linker is reported.
Abstract: Peptide cyclization via chemoselective reactions between side chains has proven a useful strategy to control folded structure. We report here a method for the synthesis of side-chain to side-chain cyclic peptides based on the intermolecular reaction between a linear peptide functionalized with two aminooxy or hydrazide side chains and an organic dialdehyde linker. A family of oxime-based and hydrazone-based cyclic products is prepared in a modular and convergent fashion by combination of unprotected linear peptide precursors and various small molecule linkers in neutral aqueous buffer. The side-chain to side-chain linkages that result can alter peptide folding behavior. The dynamic covalent nature of the Schiff bases in the cyclic products can be utilized to create mixtures where product composition changes in response to experimental conditions. Thus, a linear peptide precursor can select one organic linker from a mixture, and a cyclic product can dynamically exchange the small molecule component of the macrocycle.
TL;DR: 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.
Abstract: 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 -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 (c) 2014 European Peptide Society and John Wiley & Sons, Ltd.
TL;DR: The results of the present study reveals that cyclo (d‐Tyr‐d‐Phe) is more bioactive than cyclo(l‐T Tyr‐l‐phe), the standard antioxidant agent, which is almost equal to that of butylated hydroxyanisole, the standard antioxidants agent.
Abstract: A new microbial cyclic dipeptide (diketopiperazine), cyclo(d-Tyr-d-Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d-Tyr-d-Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d-Tyr-d-Phe) recorded significant antitumor activity against A549 cells (IC50 value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d-Tyr-d-Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d-Tyr-d-Phe). Flow cytometry analysis showed that the cyclo(d-Tyr-d-Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase-3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d-Tyr-d-Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d-Tyr-d-Phe) with synthetic cyclo(d-Tyr-d-Phe) and cyclo(l-Tyr-l-Phe). Natural and synthetic cyclo(d-Tyr-d-Phe) recorded similar pattern of activity. Although synthetic cyclo(l-Tyr-l-Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l-Tyr-l-Phe) recorded significant activity than natural and synthetic cyclo(d-Tyr-d-Phe). The results of the present study reveals that cyclo(d-Tyr-d-Phe) is more bioactive than cyclo(l-Tyr-l-Phe). To the best of our knowledge, this is the first time that cyclo(d-Tyr-d-Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d-Tyr-d-Phe) is also reported for the first time. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
TL;DR: This study isolated and characterized SsmTx‐I, a novel neurotoxin from the venom of S. subspinipes mutilans that contains 36 residues with four cysteines forming two disulfide bonds and reported a specific Kv2.1‐blocker from centipede venom and provided a basis for future investigations of Ssmtx‐I.
Abstract: Scolopendra subspinipes mutilans, also known as Chinese red-headed centipede, is a venomous centipede from East Asia and Australasia. Venom from this animal has not been researched as thoroughly as venom from snakes, snails, scorpions, and spiders. In this study, we isolated and characterized SsmTx-I, a novel neurotoxin from the venom of S. subspinipes mutilans. SsmTx-I contains 36 residues with four cysteines forming two disulfide bonds. It had low sequence similarity (<10%) with other identified peptide toxins. By whole-cell recording, SsmTx-I significantly blocked voltage-gated K+ channels in dorsal root ganglion neurons with an IC50 value of 200 nM, but it had no effect on voltage-gated Na+ channels. Among the nine K+ channel subtypes expressed in human embryonic kidney 293 cells, SsmTx-I selectively blocked the Kv2.1 current with an IC50 value of 41.7 nM, but it had little effect on currents mediated by other K+ channel subtypes. Blockage of Kv2.1 by SsmTx-I was not associated with significant alteration of steady-state activation, suggesting that SsmTx-I might act as a simple inhibitor or channel blocker rather than a gating modifier. Our study reported a specific Kv2.1-blocker from centipede venom and provided a basis for future investigations of SsmTx-I, for example on structure–function relationships, mechanism of action, and pharmacological potential. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
TL;DR: Results revealed that temporin‐SHa retains its antimicrobial activity after covalent grafting, and represents an attractive candidate as antimicrobial coating agent.
Abstract: Development of resistant bacteria onto biomaterials is a major problem leading to nosocomial infections. Antimicrobial peptides are good candidates for the generation of antimicrobial surfaces because of their broad-spectrum activity and their original mechanism of action (i.e. rapid lysis of the bacterial membrane) making them less susceptible to the development of bacterial resistance. In this study, we report on the covalent immobilisation of temporin-SHa on a gold surface modified by a thiolated self-assembled monolayer. Temporin-SHa (FLSGIVGMLGKLFamide) is a small hydrophobic and low cationic antimicrobial peptide with potent and very broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts and parasites. We have analysed the influence of the binding mode of temporin-SHa on the antibacterial efficiency by using a covalent binding either via the peptide NH2 groups (random grafting of α- and e-NH2 to the surface) or via its C-terminal end (oriented grafting using the analogue temporin-SHa-COOH). The surface functionalization was characterised by IR spectroscopy (polarisation modulation reflection absorption IR spectroscopy) while antibacterial activity against Listeria ivanovii was assessed by microscopy techniques, such as atomic force microscopy and scanning electron microscopy equipped with a field emission gun. Our results revealed that temporin-SHa retains its antimicrobial activity after covalent grafting. A higher amount of bound temporin-SHa is observed for the C-terminally oriented grafting compared with the random grafting (NH2 groups). Temporin-SHa therefore represents an attractive candidate as antimicrobial coating agent. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
TL;DR: This work provides a detailed protocol for the preparation of bis(2‐sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C‐terminal bis(3‐mercaptopropionic acid)amido peptides and of peptide thioesters derived from 3‐mer Captoprostionic acid.
Abstract: Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
TL;DR: This paper describes a versatile convergent strategy for the construction of protein mimics presenting three different cyclic peptides, using an orthogonal alkyne protection strategy and peptide loops were introduced successively onto a triazacyclophane scaffold via Cu(I)‐catalyzed azideAlkyne cycloaddition.
Abstract: Synthetic mimics of discontinuous epitopes may have a wide range of potential applications, including synthetic vaccines and inhibition of protein-protein interactions. However, synthetic access to these relatively complex peptide molecular constructs is limited. This paper describes a versatile convergent strategy for the construction of protein mimics presenting three different cyclic peptides. Using an orthogonal alkyne protection strategy, peptide loops were introduced successively onto a triazacyclophane scaffold via Cu(I)-catalyzed azide alkyne cycloaddition. This method provides rapid access to protein mimics requiring different peptide segments for their interaction and activity.
TL;DR: The solid‐phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide‐capped derivative are presented, opening new perspectives toward the design of new sporozoiticidal compounds.
Abstract: Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid-phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide-capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile5 or the His6 residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala6]-AII (79%), and [Ala5]-AII (75%). Analogs [Ala3]-AII, [Ala1]-AII, and AII-NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala8]-AII, [Ala7]-AII, [Ala4]-AII, and [Ala2]-AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a β-fold conformation in different solutions. All AII analogs, except [Ala4]-AII and [Ala8]-AII, show contractile responses and interact with the AT1 receptor, [Ala5]-AII and [Ala6]-AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
TL;DR: It was found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli, and treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells.
Abstract: The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletesdaviesanus (Hymenoptera:Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In ourinvestigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candidaalbicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for themechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterialmembrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage wasobserved from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipidswasalsoestablishedbytheblueshiftinthetryptophanemissionspectramaximawhentheinteractionsoftryptophan-containingCOD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities ofCODs.Moreover, we found thatthe studied peptidespermeated both the outer and inner cytoplasmic membranes ofEscherichiacoli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detectingcytoplasmic β-galactosidase released duringthe interaction of peptides withE.coli cells. Transmission electron microscopyrevealed that treatment of E.coli with one of the COD analogs caused leakage of bacterial content mainly from the septalareas of the cells. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.Additional supporting information may be found in the online version of this article at the publisher’s web site.Keywords: antimicrobial peptides; wild-bee venom; CD spectroscopy; large unilamellar vesicles; membrane permeabilization; electronmicroscopy
TL;DR: Viruses provide attracting suggestions for the development of targeted drug carriers as they have evolved naturally to deliver their genomes to host cells with high fidelity.
Abstract: Nanotechnology is an expanding area of study with potentially pivotal applications in a discipline as medicine where new biomedical active molecules or strategies are continuously developing. One of the principal drawbacks for the application of new therapies is the difficulty to cross membranes that represent the main physiological barrier in our body and in all living cells. Membranes are selectively permeable and allow the selective internalization of substances; generally, they form a highly impermeable barrier to most polar and charged molecules, and represent an obstacle for drug delivery, limiting absorption to specific routes and mechanisms. Viruses provide attracting suggestions for the development of targeted drug carriers as they have evolved naturally to deliver their genomes to host cells with high fidelity.
A detailed understanding of virus structure and their mechanisms of entry into mammalian cells will facilitate the development and analysis of virus-based materials for medical applications. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
TL;DR: Considering the increase in multidrug‐resistant bacterial infections, CPF‐C1 may offer a new strategy that can be considered a potential therapeutic agent for the treatment of diseases caused by multidrog‐resistant bacteria.
Abstract: Hospital-acquired infections caused by multidrug-resistant bacteria pose significant challenges for treatment, which necessitate the development of new antibiotics. Antimicrobial peptides are considered potential alternatives to conventional antibiotics. The skin of Anurans (frogs and toads) amphibians is an extraordinarily rich source of antimicrobial peptides. CPF-C1 is a typical cationic antimicrobial peptide that was originally isolated from the tetraploid frog Xenopus clivii. Our results showed that CPF-C1 has potent antimicrobial activity against both sensitive and multidrug-resistant bacteria. It disrupted the outer and inner membranes of bacterial cells. CPF-C1 induced both propidium iodide uptake into the bacterial cell and the leakage of calcein from large liposome vesicles, which suggests a mode of action that involves membrane disturbance. Scanning electron microscopy and transmission electron microscopy verified the morphologic changes of CPF-C1-treated bacterial cells and large liposome vesicles. The membrane-dependent mode of action signifies that the CPF-C1 peptide functions freely and without regard to conventional resistant mechanisms. Additionally, it is difficult for bacteria to develop resistance against CPF-C1 under this action mode. Other studies indicated that CPF-C1 had low cytotoxicity against mammalian cell. In conclusion, considering the increase in multidrug-resistant bacterial infections, CPF-C1 may offer a new strategy that can be considered a potential therapeutic agent for the treatment of diseases caused by multidrug-resistant bacteria.
TL;DR: Conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half‐life, which maintains full potency at both the peripheral and central NMU receptors, and reinforces the notion that NMU‐based therapeutics are promising candidates for the treatment of obesity and diabetes.
Abstract: Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.
TL;DR: The study elucidates the structure–activity relationship for the effect of d‐amino acid substitutions in MAC‐1 using NMR spectroscopy and suggests a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate.
Abstract: 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.
TL;DR: This study indicates that peptides that are known to disrupt the membrane by different mechanisms show very distinct trends of the order parameter as a function of peptide concentration, and spin‐labeled bicelles proved to be a good system to evaluate the membrane disruption mechanism of new AMPs.
Abstract: Bicelles are model membrane systems that can be macroscopically oriented in a magnetic field at physiological temperature. The macroscopic orientation of bicelles allows to detect, by means of magnetic resonance spectroscopies, small changes in the order of the bilayer caused by solutes interacting with the membrane. These changes would be hardly detectable in isotropic systems such as vesicles or micelles. The aim of this work is to show that bicelles represent a convenient tool to investigate the behavior of antimicrobial peptides (AMPs) interacting with membranes, using electron paramagnetic resonance (EPR) spectroscopy. We performed the EPR experiments on spin-labeled bicelles using various AMPs of different length, charge, and amphipathicity: alamethicin, trichogin GA IV, magainin 2, HP(2-20), and HPA3. We evaluated the changes in the order parameter of the spin-labeled lipids as a function of the peptide-to-lipid ratio. We show that bicelles labeled at position 5 of the lipid chains are very sensitive to the perturbation induced by the AMPs even at low peptide concentrations. Our study indicates that peptides that are known to disrupt the membrane by different mechanisms (i.e., alamethicin vs magainin 2) show very distinct trends of the order parameter as a function of peptide concentration. Therefore, spin-labeled bicelles proved to be a good system to evaluate the membrane disruption mechanism of new AMPs.
TL;DR: Interestingly, the antimicrobial and anti‐inflammatory activity of the peptide TB_KKG6A against Pseudomonas aeruginosa is found, being able to downregulate the pro‐inflammatory chemokines and cytokines interleukin (IL)‐8,IL‐1β, IL‐6 and tumor necrosis factor‐α produced downstream infected human bronchial epithelial cells.
Abstract: Natural peptides with antimicrobial properties are deeply investigated as tools to fight bacteria resistant to common antibiotics. Small peptides, as those belonging to the temporin family, are very attractive because their activity can easily be tuned after small modification to their primary sequence. Structure-activity studies previously reported by us allowed the identification of one peptide, analogue of temporin B, TB_KKG6A, showing, unlike temporin B, antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigated the antimicrobial and anti-inflammatory activity of the peptide TB_KKG6A against Pseudomonas aeruginosa. Interestingly, we found that the peptide exhibits antimicrobial activity at low concentrations, being able to downregulate the pro-inflammatory chemokines and cytokines interleukin (IL)-8, IL-1β, IL-6 and tumor necrosis factor-α produced downstream infected human bronchial epithelial cells. Experiments were carried out also with temporin B, which was found to show pro-inflammatory activity. Details on the interaction between TB_KKG6A and the P. aeruginosa LPS were obtained by circular dichroism and fluorescence studies.
TL;DR: The antitumor effect induced by systemic administration of a peptide that targets COMMD1 for stabilization is described, and biological characterization of the main metabolites of the peptide CIGB‐552 suggests that the cell‐penetrating capacity plays an important role in the cytotoxic activity.
Abstract: Accumulation of the COMMD1 protein as a druggable pharmacology event to target cancer cells has not been evaluated so far in cancer animal models. We have previously demonstrated that a second-generation peptide, with cell-penetrating capacity, termed CIGB-552, was able to induce apoptosis mediated by stabilization of COMMD1. Here, we explore the antitumor effect by subcutaneous administration of CIGB-552 in a therapeutic schedule. Outstandingly, a significant delay of tumor growth was observed at 0.2 and 0.7 mg/kg (p < 0.01) or 1.4 mg/kg (p < 0.001) after CIGB-552 administration in both syngeneic murine tumors and patient-derived xenograft models. Furthermore, we evidenced that 131I-CIGB-552 peptide was actually accumulated in the tumors after administration by subcutaneous route. A typical serine-proteases degradation pattern for CIGB-552 in BALB/c mice serum was identified. Further, biological characterization of the main metabolites of the peptide CIGB-552 suggests that the cell-penetrating capacity plays an important role in the cytotoxic activity. This report is the first in describing the antitumor effect induced by systemic administration of a peptide that targets COMMD1 for stabilization. Moreover, our data reinforce the perspectives of CIGB-552 for cancer targeted therapy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.