Ribosomally-synthesised cyclic peptides from plants as drug leads and pharmaceutical scaffolds

Owing to their exceptional stability and favourable pharmacokinetic properties, plant-derived cyclic peptides have recently attracted significant attention in the field of peptide-based drug design. This article describes the three major classes of ribosomally-synthesised plant peptides – the cyclotides, the PawS-derived peptides and the orbitides – and reviews their applications as leads or scaffolds in drug design. These ribosomally-produced peptides have a range of biological activities, including anti-HIV, cytotoxic and immunomodulatory activity. In addition, recent interest has focused on their use as scaffolds to stabilise bioactive peptide sequences, thereby enhancing their biopharmaceutical properties. There are now more than 30 published papers on such ‘grafting’ applications, most of which have been reported only in the last few years, and several such studies have reported in vivo activity of orally delivered cyclic peptides. In this article, we describe approaches to the synthesis of cyclic peptides and their pharmaceutically-grafted derivatives as well as outlining their biosynthetic routes. Finally, we describe possible bioproduction routes for pharmaceutically active cyclic peptides, involving plants and plant suspension cultures.     

Properties of apolipoprotein E derived peptide modulate their lipid-binding capacity and influence their anti-inflammatory function

Apolipoprotein-derived peptides are promising candidates for the treatment of various inflammatory conditions. The beneficial effects of these peptides are based on multiple mechanisms; prominent among them being high-affinity binding to pro-inflammatory oxidized phospholipids (Ox-PLs) and facilitating their sequestration/metabolism/clearance in the body. This indicates that peptides which can bind exclusively to Ox-PLs without recognizing normal, non-oxidized phospholipids (non-Ox-PLs) will be more potent anti-inflammatory agent than that of the peptides that bind to both Ox-PLs and non-Ox-PLs. In order to develop such Ox-PL-specific peptides, the knowledge about the properties (molecular determinants) of peptides that govern their Ox-PL preference is a must. In this study we have synthesized eleven peptides corresponding to the conserved regions of human apolipoprotein E and compared their biochemical properties, lipid-binding specificities, and anti-inflammatory properties. Our results show that these peptides exhibit considerably different specificities towards non-Ox-PL and different species of Ox-PLs. Some of these peptides bind exclusively to the Ox-PLs and inhibit the pro-inflammatory function of Ox-PLs in human blood. Biochemical characterization revealed that the peptides possess substantially different properties. Our results suggest that physicochemical properties of peptides play an important role in their lipid-binding specificity.     

Physicochemical properties of bacterial pro-inflammatory lipids influence their interaction with apolipoprotein-derived peptides

Apolipoprotein-derived peptides have emerged as a promising candidate for the treatment of various inflammatory disease conditions. Multiple mechanisms have been proposed to explain the beneficiary effects of these peptides and prominent among them being high-affinity binding of peptides to pro-inflammatory lipids and facilitating their sequestration/metabolism/clearance in the body. Pro-inflammatory lipids differ considerably in their molecular structures, chemical compositions and physicochemical properties. Importance of the properties of the pro-inflammatory lipids in their ability to bind to apolipoprotein-derived peptides is not studied in details. In this study, we have characterized the interaction of synthetic peptides derived from human apolipoprotein E with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), two potent bacterial pro-inflammatory lipids that differ considerably in their molecular structures and chemical compositions. Binding of the peptides to LPS and LTA was monitored by CD spectroscopy. Effect of the peptides on the biological activity of lipids was studied by monitoring the inhibition of LPS- or LTA-induced up-regulation of the inflammatory markers in the human blood cells. Physicochemical properties of lipid aggregates were determined by fluorescence spectroscopy and native PAGE. Our results show that physicochemical properties of LPS and LTA differ considerably and influence their interaction with apolipoprotein-derived peptides.     

Bioactive peptides from marine sources: pharmacological properties and isolation procedures

Marine organisms represent a valuable source of new compounds. The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new active substances in the field of the development of bioactive products. In this paper, the molecular diversity of different marine peptides is described as well as information about their biological properties and mechanisms of action is provided. Moreover, a short review about isolation procedures of selected bioactive marine peptides is offered. Novel peptides from sponges, ascidians, mollusks, sea anemones and seaweeds are presented in association with their pharmacological properties and obtainment methods.     

Studies on titin PEVK peptides and their interaction

Experiments were conducted on several synthetic and expressed peptides from the PEVK region of titin, the giant muscle protein. Different secondary structure prediction methods based on amino acid sequence gave estimates ranging from over 70% alpha helical to no helix (totally disordered) for the polyE peptide corresponding to human exon 115. Circular dichroism (CD) experiments demonstrated that both the positively charged PPAK modules and the negatively charged PolyE repeats had similar spectral properties with disordered secondary structure predominating. Gel permeation chromatography showed that both PPAK and polyE peptides had 2-4 times larger Stokes radii than expected from their molecular mass. Mixtures of the oppositely charged titin peptides caused no change in apparent secondary structure as observed by circular dichroism or migration properties using native gel electrophoresis. Similarly addition of calcium did not alter the CD spectra or peptide electrophoretic mobility of the individual peptides or their mixtures. The properties of both the PPAK and polyE type peptides suggest that both had most of the characteristic properties to be classified as intrinsically disordered proteins.     

Correlation between simulated physicochemical properties and hemolycity of protegrin-like antimicrobial peptides: predicting experimental toxicity

The therapeutic, antibiotic potential of antimicrobial peptides can be prohibitively diminished because of the cytotoxicity and hemolytic profiles they exhibit. Quantifying and predicting antimicrobial peptide toxicity against host cells is thus an important goal of AMP related research. In this work, we present quantitative structure activity relationships for toxicity of protegrin-like antimicrobial peptides against human cells (epithelial and red blood cells) based on physicochemical properties, such as interaction energies and radius of gyration, calculated from molecular dynamics simulations of the peptides in aqueous solvent. The hypothesis is that physicochemical properties of peptides, as manifest by their structure and interactions in a solvent and as captured by atomistic simulations, are responsible for their toxicity against human cells. Protegrins are beta-hairpin peptides with high activity against a wide variety of microbial species, but in their native state are toxic to human cells. Sixty peptides with experimentally determined toxicities were used to develop the models. We test the resulting relationships to determine their ability to predict the toxicity of several protegrin-like peptides. The developed QSARs provide insight into the mechanism of cytotoxic action of antimicrobial peptides. In a subsequent blind test, the QSAR correctly ranked four of five protegrin analogues newly synthesized and tested for toxicity.     

Relationships between the orientation and the structural properties of peptides and their membrane interactions

Physical properties of membranes, such as fluidity, charge or curvature influence their function. Proteins and peptides can modulate those properties and conversely, the lipids can affect the activity and/or the structure of the former. Tilted peptides are short hydrophobic protein fragments characterized by an asymmetric distribution of their hydrophobic residues when helical. They were detected in viral fusion proteins and in proteins involved in different biological processes that need membrane destabilization. Those peptides and non lamellar lipids such as PE or PA appear to cooperate in the lipid destabilization process by enhancing the formation of negatively-curved domains. Such highly bent lipidic structures could favour the formation of the viral fusion pore intermediates or that of toroidal pores. Structural flexibility appears as another crucial property for the interaction of peptides with membranes. Computational analysis on another kind of lipid-interacting peptides, i.e. cell penetrating peptides (CPP) suggests that peptides being conformationally polymorphic should be more prone to traverse the bilayer. Future investigations on the structural intrinsic properties of tilted peptides and the influence of CPP on the bilayer organization using the techniques described in this chapter should help to further understand the molecular determinants of the peptide/lipid inter-relationships.     

Artificial peptides to induce membrane denaturation and disruption and modulate membrane composition and fusion

Membranes consisting of phospholipid bilayers are an essential constituent of eukaryotic cells and their compartments. The alteration of their composition, structure, and morphology plays an important role in modulating physiological processes, such as transport of molecules, cell migration, or signaling, but it can also lead to lethal effects. The three main classes of membrane-active peptides that are responsible for inducing such alterations are cell-penetrating peptides (CPPs), antimicrobial peptides (AMPs), and fusion peptides (FPs). These peptides are able to interact with lipid bilayers in highly specific and tightly regulated manners. They can either penetrate the membrane, inducing nondestructive, transient alterations, or disrupt, permeabilize, or translocate through it, or induce membrane fusion by generating attractive forces between two bilayers. Because of these properties, membrane-active peptides have attracted the attention of the pharmaceutical industry, and naturally occurring bioactive structures have been used as a platform for synthetic modification and the development of artificial analogs with optimized therapeutic properties to transport biologically active cargos or serve as novel antimicrobial agents. In this review, we focus on synthetic membrane interacting peptides with bioactivity comparable with their natural counterparts and describe their mechanism of action.     

Differential interaction of peptides derived from C-terminal domain of human apolipoprotein E with platelet activating factor analogs

Apolipoprotein-derived peptides are promising candidates for the treatment of various inflammatory conditions and the main mechanism proposed for the protective action of these peptides includes binding to pro-inflammatory lipid mediators with high affinity and facilitating their sequestration/metabolism/clearance in the body. Molecules that act as pro-inflammatory lipid mediators differ considerably in their molecular structures, chemical compositions and physicochemical properties. Importance of the properties of pro-inflammatory lipid mediators on the biological activity of apolipoprotein-derived peptides has not been studied in detail. In this study, we characterized the physicochemical properties of aggregates containing lyso-PAF, acetyl-PAF and butanoyl-PAF, three closely related pro-inflammatory lipid mediators, and studied their interaction with peptides derived from the C-terminal domains of human apolipoprotein E. These PAF-analogs differ only in the chemical composition of the functional groups they carry at the sn-2 positions. Our results show that physicochemical properties of aggregates containing lyso-PAF, acetyl-PAF and butanoyl-PAF differ considerably and affect their apolipoprotein-derived peptides-binding capacity.     

Ponericins, new antibacterial and insecticidal peptides from the venom of the ant Pachycondyla goeldii

The antimicrobial, insecticidal, and hemolytic properties of peptides isolated from the venom of the predatory ant Pachycondyla goeldii, a member of the subfamily Ponerinae, were investigated. Fifteen novel peptides, named ponericins, exhibiting antibacterial and insecticidal properties were purified, and their amino acid sequences were characterized. According to their primary structure similarities, they can be classified into three families: ponericin G, W, and L. Ponericins share high sequence similarities with known peptides: ponericins G with cecropin-like peptides, ponericins W with gaegurins and melittin, and ponericins L with dermaseptins. Ten peptides were synthesized for further analysis. Their antimicrobial activities against Gram-positive and Gram-negative bacteria strains were analyzed together with their insecticidal activities against cricket larvae and their hemolytic activities. Interestingly, within each of the three families, several peptides present differences in their biological activities. The comparison of the structural features of ponericins with those of well-studied peptides suggests that the ponericins may adopt an amphipathic alpha-helical structure in polar environments, such as cell membranes. In the venom, the estimated peptide concentrations appear to be compatible with an antibacterial activity in vivo. This suggests that in the ant colony, the peptides exhibit a defensive role against microbial pathogens arising from prey introduction and/or ingestion.     

A colorimetric assay for rapid screening of antimicrobial peptides

The increased resistance of various bacteria toward available antibiotic drugs has initiated intensive research efforts into identifying new sources of antimicrobial substances. Short antibiotic peptides (10-30 residues) are prevalent in nature as part of the intrinsic defense mechanisms of most organisms and have been proposed as a blueprint for the design of novel antimicrobial agents. Antimicrobial peptides are generally believed to kill bacteria through membrane permeabilization and extensive pore-formation. Assays providing rapid and easy evaluation of interactions between antimicrobial membrane peptides and lipid bilayers could significantly improve screening for substances with effective antibacterial properties, as well as contribute to the elucidation of structural and functional properties of antimicrobial peptides. Here we describe a colorimetric sensor in which particles composed of phospholipids and polymerized polydiacetylene (PDA) lipids were shown to exhibit striking color changes upon interactions with antimicrobial membrane peptides. The color changes in the system occur because of the structural perturbation of the lipids following their interactions with antimicrobial peptides. The assay was also sensitive to the antibacterial properties of structurally and functionally related peptide analogs.     

Structural diversity of marine cyclic peptides and their molecular mechanisms for anticancer,antibacterial, antifungal,and other clinical applications

Many cyclic peptides and analogues derived from marine sources are known to possess biological properties, including anticancer, antitumor, antibacterial, antifungal, antiparasitic, anti-inflammation, anti-proliferative, anti-hypertensive, cytotoxic, and antibiotic properties. These compounds demonstrate different activities and modes of action according to their structure such as cyclic oligopeptide, cyclic lipopeptide, cyclic glycopeptide and cyclic depsipeptide. The recent advances in application of the above-mentioned cyclic peptides were reported in dolastatins, soblidotin, didemnin B, aplidine, salinosporamide A, kahalalide F and bryostatin 1 and they are currently in clinical trials. These cyclic peptides are possible novel drugs discovered and developed from marine origin. Literature data concerning the potential properties of marine cyclic peptides were reviewed here, and the structural diversity and biological activities of marine cyclic peptides are discussed in relation to the molecular mechanisms of these marine cyclic peptides.     

Milk derived bioactive peptides and their impact on human health – A review

Milk-derived bioactive peptides have been identified as potential ingredients of health-promoting functional foods. These bioactive peptides are targeted at diet-related chronic diseases especially the non-communicable diseases viz., obesity, cardiovascular diseases and diabetes. Peptides derived from the milk of cow, goat, sheep, buffalo and camel exert multifunctional properties, including anti-microbial, immune modulatory, anti-oxidant, inhibitory effect on enzymes, anti-thrombotic, and antagonistic activities against various toxic agents. Majority of those regulate immunological, gastrointestinal, hormonal and neurological responses, thereby playing a vital role in the prevention of cancer, osteoporosis, hypertension and other disorders as discussed in this review. For the commercial production of such novel bioactive peptides large scale technologies based on membrane separation and ion exchange chromatography methods have been developed. Separation and identification of those peptides and their pharmacodynamic parameters are necessary to transfer their potent functional properties into food applications. The present review summarizes the preliminary classes of bioactive milk-derived peptides along with their physiological functions, general characteristics and potential applications in health-care.     

Antifungal peptides: Origin, activity, and therapeutic potential

Antifungal peptides have been identified in a wide range of life forms which include plants, mammals, and microorganisms. Their structures are as varied as their antifungal properties. Semisynthetic and fully synthetic analogs have been developed from a few of these natural peptides that are superior to the parent compound. A few of these peptides hold promise in combating fungal infections and have entered clinical trials.     

Biological activity and enzymic degradation of substance P analogs: implications for studies of the substance P receptor.

Partial sequences of Substance P, either free or blocked at their amino terminal, have been examined for their stability towards inactivation by homogenate or particulate fractions of rat brain and for their relative potencies as smooth muscle contractors. The C-terminal hexapeptide in both the free and blocked forms displays activity comparable to that of the longer C-terminal peptides as well as to that of the native undecapeptide. The blocked peptides, however, are much more stable than their corresponding free peptides. Among the free peptides Substance P is degraded slower than the free hexa- and hepta-peptides, suggesting that the N-terminal tetrapeptide part may play a role in stabilizing the molecule. Blocked hepta- and octapeptide analogs, carrying probe properties, may be useful for studies of the Substance P receptor.     

Antibacterial mechanisms of GN-2 derived peptides and peptoids against Escherichia coli

Escherichia coli is the main etiological agent of urinary trait infections, able to form biofilms in indwelling devices, resulting in chronic infections which are refractory to antibiotics treatment. In this study, we investigated the antimicrobial and anti-biofilm properties exerted against E. coli ATCC 25922, by a set of peptoids and peptides modeled upon the peptide GN-2, previously reported as a valid antimicrobial agent. The putative antimicrobials were designed to evaluate the effect of cationicity, hydrophobicity and their partitioning on the overall properties against planktonic cells and biofilms as well as on LPS binding, permeabilization of Gram-negative bacteria membranes and hemolysis. The data demonstrated that peptides are stronger antimicrobials than the analogue peptoids which in return have superior anti-biofilm properties. In this study, we present evidence that peptides antimicrobial activity correlates with enhanced LPS binding and hydrophobicity but is not affected by partitioning. The data demonstrated that the enhanced anti-biofilm properties of the peptoids are associated with decreased hydrophobicity and increased penetration of the inner membrane, compared to that of their peptide counterpart, suggesting that the characteristic flexibility of peptoids or their lack of H-bonding donors in their backbone, would play a role in their ability to penetrate bacterial membranes.     

抗菌肽基因工程研究及其表达策略

抗菌肽广泛存在于多种生物体内,具有广谱抗菌、调节免疫、抑制肿瘤等多种生物学功能,作用机制独特,是目前基因工程研究的热点之一。本文综述了抗菌肽的一般性质及其国内外基因工程研究进展,探讨了在抗菌肽转基因研究中采用的表达策略及理论依据。     

Aqueous solubility and membrane interactions of hydrophobic peptides with peptoid tags

Lysine tagging of hydrophobic peptides of parent sequence KKAAALAAAAALAAWAALAAAKKKK-NH(2) has been shown to facilitate their synthesis and purification through water solubilization, yet not impact on the intrinsic properties of the hydrophobic core sequence with respect to its insertion into membranes in an alpha-helical conformation. However, due to their positively charged character, such peptides often become bound to phospholipid head groups in membrane surfaces, which inhibits their transbilayer insertion and/or prevents their transport across cellular bilayers. We sought to develop more neutral peptides of membrane-permeable character by replacing most Lys residues with uncharged peptoid [N-(R)glycyl] residues, which might similarly confer water solubility while retaining membrane-interactive properties of the hydrophobic core. Several "peptoid-tagged" derivatives of the parent peptide were prepared with varying peptoid content, with five of the six Lys residues replaced with peptoids Nala and/or Nval. Conformations of these peptides measured by circular dichroism spectroscopy demonstrated that these water-soluble peptides retain the alpha-helix structure in micelles (lysophosphatidylcholine and sodium dodecyl sulfate) notwithstanding the known helix-breaking capacity of the peptoid tags. Blue shifts in Trp fluorescence spectra and quenching experiments with acrylamide confirmed that peptoid-tagged peptides insert spontaneously into micellar membranes. Results suggest that upon introduction of uncharged tags, the interaction between the membrane and the peptides is dominated by the hydrophobicity of the peptide core rather than the electrostatic interactions between the Lys and the head groups of the lipids. The overall findings indicate that peptoid residues are effective surrogates for Lys as uncharged water-solubilizing tags and, as such, provide a potentially valuable feature of design of membrane-interactive peptides.