Cyclotides: a natural combinatorial peptide library or a bioactive sequence player?

Abstract

In this perspective review, we focalized our attention on the application of cyclotides in drug discovery. To date, two principal approaches have been explored since now: (i) the use of cyclotides as scaffold in which bioactive peptides can be grafted to improve stability, oral bioactivity and binding to GPCRs; (ii) their application as natural peptides library. For these reasons, cyclotides probably represent today a step further in the development of new tools in drug design.     

Chemical composition and nutraceutical properties of hempseed: an ancient food with actual functional value

The seeds of hemp (Cannabis sativa L.) have been an important source of nutrition for thousands of years in Chinese and European cultures but, nowadays, in spite of the multiple clinical evidence, which highlights their huge health-promoting properties, people are still unaware about their nutritional as well as nutraceutical benefits. Indeed, the extraordinary richness of essential polyunsaturated fatty acids is odds with oxidative stability of hemp seed by-products. This is the reason for which different and innovative extractive strategies are daily employed, as well as the preservation of antioxidant hemp seed phenols and polyphenols is claimed as a key issue to pursue. Recent insights into the phenol and cannabinoid baggage of hemp seed highlight it is an inestimable source of health promoting ingredients. This review aims to provide a detailed and updated background for research on seeds of this niche crop, pointing out the chemistry and bioactivity of their phytochemicals.     

The Effects of Excipients and Particle Engineering on the Biophysical Stability and Aerosol Performance of Parathyroid Hormone (1-34) Prepared as a Dry Powder for Inhalation

Pulmonary delivery of therapeutic peptides and proteins has many advantages including high relative bioavailability, rapid systemic absorption and onset of action and a non-invasive mode of administration which improves patient compliance. In this study, we investigated the effect of spray-drying (SD) and spray freeze-drying processes on the stability and aerosol performance of parathyroid hormone (PTH) (1-34) microparticles. In this study, the stabilisation effect of trehalose (a non-reducing sugar) and Brij 97 (a non-ionic surfactant) on spray-dried PTH particles was assessed using analytical techniques including circular dichroism (CD), fluorescence spectroscopy, modulated differential scanning calorimetry and an in vitro bioactivity assay. Physical characterisation also included electron microscopy, tap density measurement and laser light diffraction. The aerosol aerodynamic performance of the formulations was assessed using the Andersen cascade impactor. Based on these studies, a formulation for spray freeze-drying was selected and the effects of the two particle engineering techniques on the biophysical stability and aerosol performance of the resulting powders was determined. CD, fluorescence spectroscopy and bioactivity data suggest that trehalose when used alone as a stabilising excipient produces a superior stabilising effect than when used in combination with a non-ionic surfactant. This highlights the utility of CD and fluorescence spectroscopy studies for the prediction of protein bioactivity post-processing. Therefore, a method and formulation suitable for the preparation of PTH as a dry powder was developed based on spray-drying PTH with trehalose as a stabiliser with the bioactivity of SD PTH containing trehalose being equivalent to that of unprocessed PTH.     

Herbal Extracts as Potential Antioxidant,Anti-Aging,Anti-Inflammatory,and Whitening Cosmeceutical Ingredients

The aim of this research was to investigate and compare the antioxidant, anti-tyrosinase, anti-aging, and anti-inflammatory activities of 16 herbal extracts for topical application in cosmetic/cosmeceutical products. Herbal plant materials were extracted by infusion in boiled water for 15 min. The total phenolic content and total flavonoid content of each extract were investigated by the Folin-Ciocalteu and aluminum chloride methods, respectively. Antioxidant activities were investigated using 2,2’-diphenyl-1-picrylhydrazyl and a ferric reducing antioxidant power assay. Anti-tyrosinase and anti-aging activities were investigated using an in vitro enzymatic-spectrophotometric method. Anti-inflammatory activities were investigated using an enzyme-linked immunosorbent assay. The findings show that the Stevia rebaudiana extract has the most significant levels of both phenols and flavonoids (p<0.05). The S. rebaudiana, Rosa damascene, and Phyllanthus emblica extracts possessed the most significant antioxidant activities (p<0.05) and a promising whitening effect with moderate anti-tyrosinase activities. Furthermore, the Echinacea purpurea extract possessed the most significant anti-collagenase (78.5±0.0 %), anti-elastase (69.0±1.4 %), and anti-hyaluronidase activity (64.2±0.3 %). The Morus alba extract possessed the most significant anti-inflammatory activity since it could inhibit the secretion of interleukin-6 and tumor necrosis factor-α (p<0.05). Therefore, these herbal extracts have promising skin benefits and have potential for use as active ingredients in cosmetic/cosmeceutical products.     

Biological activities and potential cosmeceutical applications of bioactive components from brown seaweeds: a review

Seaweeds are the primary producers of all aquatic ecosystems. Chemical constituents isolated from diverse classes of seaweeds exert a wide range of nutritional, functional and biological activities. Unique metabolites of seaweeds possess specific biological properties that make them potential ingredients of many industrial applications such as functional foods, pharmaceuticals and cosmeceuticals. Cosmeceuticals of natural origin are becoming more popular than synthetic cosmetics. Hence, the investigation of new seaweeds derived functional components, a different source of natural products, has proven to be a promising area of cosmeceutical studies. Brown seaweeds also produce a range of active components including unique secondary metabolites such as phlorotannins and many of which have specific biological activities that give possibilities for their economic utilization. Brown seaweeds derived active compounds have been shown various functional properties including, antioxidant, antiwrinkling, whitening, antiinflammatory and antiallergy. It is well-known that these kind of biological effects are closely associated with cosmeceutical preparations. This communication reviews the current knowledge on brown seaweeds derived metabolites with various biological activities and the potential use as cosmeceutical ingredients. It is hoped that the reviewed literature on multifunctional properties of brown seaweeds will improve access to the seaweed based natural products specially the ability to incorporate these functional properties in cosmeceutical applications.     

Hydrolysis of milk-derived bioactive peptides by cell-associated extracellular peptidases of Streptococcus thermophilus

The trend to confer new functional properties to fermented dairy products by supplementation with bioactive peptides is growing in order to encounter the challenge of health-promoting foods. But these functional ingredients have not to be hydrolysed by proteases of bacteria used in the manufacture of these products. One of the two yoghurt bacteria, Streptococcus thermophilus, has long been considered as weakly proteolytic since its only cell wall-associated subtilisin-like protease, called PrtS, is not always present. Nevertheless, a recent study pointed out a possible peptidase activity in certain strains. In this present study, the stability of milk-derived bioactive peptides, e.g. the anxiolytic peptide, α_s1-CN-(f91-97), in the presence of two different S. thermophilus strains with PrtS⁺ or PrtS^? phenotype was studied. Both strains appeared to be capable of hydrolysing the α_s1-CN-(f91-97) and other bioactive peptides by recurrent removal of N-terminal residues. The hydrolysis was neither due to intracellular peptidases nor to HtrA protease. Results obtained showed that the observed activity originates from the presence at the surface of both strains of an extracellular aminopeptidase activity. Moreover, a cell wall-associated X-prolyl dipeptidyl peptidase activity was also highlighted when β-casomorphin-7 was used as substrate. All of these findings suggest that, in order to use fermented milks as vector of bioactive peptides, the stability of these bioactive peptides in this kind of products implies to carefully characterize the potential action of the surface proteolytic enzymes of S. thermophilus.     

Role of a FMRFamide‐like family of neuropeptides in the pharyngeal nervous system of Caenorhabditis elegans

The nervous system of C. elegans has a remarkable abundance of flp genes encoding FMRFamide‐like (FLP) neuropeptides. To provide insight into the physiological relevance of this neuropeptide diversity, we have tested more than 30 FLPs (encoded by 23 flps) for bioactivity on C. elegans pharynx. Eleven flp genes encode peptides that inhibit pharyngeal activity, while eight flp genes encode peptides that are excitatory. Three potent peptides (inhibitory, FLP‐13A, APEASPFIRFamide; excitatory, FLP‐17A, KSAFVRFamide; excitatory, FLP‐17B, KSQYIRFamide) are encoded by flp genes, which, according to reporter gene constructs, are expressed in pharyngeal motoneurons. Thus, they may act through receptors localized on the pharyngeal muscle. The two other potent peptides, FLP‐8 (excitatory AF1, KNEFIRFamide,) and FLP‐11A (inhibitory, AMRNALVRFamide), appear to be expressed in extrapharyngeal neurons and are therefore likely to act either indirectly or as neurohormones. Intriguingly, a single neuron can express peptides that have potent but opposing biological activity in the pharynx. Only five flp genes encode neuropeptides that have no observable effect on the pharynx, but none of these have shown reporter gene expression in the pharyngeal nervous system. To examine the roles of multiple peptides produced from single precursors, a comparison was made between the bioactivity of different neuropeptides for five flp genes (flp‐3, flp‐13, flp‐14, flp‐17, and flp‐18). For all but one gene (flp‐14), the effects of peptides encoded by the same gene were similar. Overall, this study demonstrates the impressive neurochemical complexity of the simple circuit that regulates feeding in the nematode, C. elegans. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Purification of Gekko Small Peptide Fraction and Its Effect of Inducing Apoptosis of EC 9706 Esophageal Cancer Cells by Inhibiting PI3K/Akt/GLUT1 Signaling Pathway

This study aimed to isolate and purify a cytotoxic extraction from Gekko japonicus, identify its components and determine its cytotoxic activity in vitro. We isolated and identified the most potent cytotoxic Gekko small peptide LH-20-15. The identification and analysis of peptide sequences of LH-20-15 were performed by de novo peptide sequencing, and two new peptides were found. LH-20-15 significantly inhibited the proliferation of human esophageal squamous carcinoma EC 9706 cells in a dose-dependent manner. Furthermore, LH-20-15 induced apoptosis in esophageal cancer cells by activating the mitochondrial apoptotic pathway. Further research showed that LH-20-15 inhibited the PI3 K/Akt/GLUT1 signaling pathway. In conclusion, LH-20-15 from Gekko japonicus is a peptide mixture and may inhibit EC 9706 cell proliferation and induce apoptosis by activating the mitochondrial apoptotic pathway. It also regulates glucose metabolism by targeting the PI3 K/Akt/GLUT1 signaling pathway. These small peptides could be new sources of natural cytotoxic ingredients against esophageal cancer with potential drug values.     

Biotechnological production of sphingoid bases and their applications

Sphingolipids are not only essential components of biological membranes but also play numerous other vital functions in living cells. Moreover, they are major constituents of the outermost layer of human epidermis which acts as permeability barrier of the skin. Therefore, they have a high potential to be used in a wide variety of application fields such as antibacterial and antifungal agents, active pharmaceutical ingredients of therapeutics as well as active ingredients in cosmeceutical or nutraceutical formulations. However, their chemical synthesis is a complex and cost-intensive process. As the yeast Wickerhamomyces ciferrii has been found to be a natural producer of acetylated sphingoid bases, it provides a promising alternative for their biotechnological synthesis. In the last years, this yeast has been established by classical strain improvements as well as modern genetic engineering for the industrial production of phytosphingosine. Moreover, routes for the synthesis of sphinganine and sphingosine have been implemented. This mini-review summarizes the current knowledge about biosynthesis of sphingoid bases, genetic engineering of W. ciferrii for their biotechnological production, as well as their applications in cosmetic formulations.     

The Peptaibiotics Database – A Comprehensive Online Resource

In this work, we present the ‘Peptaibiotics Database’ (PDB), a comprehensive online resource, which intends to cover all Aib‐containing non‐ribosomal fungal peptides currently described in scientific literature. This database shall extend and update the recently published ‘Comprehensive Peptaibiotics Database’ and currently consists of 1,297 peptaibiotic sequences. In a literature survey, a total of 235 peptaibiotic sequences published between January 2013 and June 2014 have been compiled, and added to the list of 1,062 peptides in the recently published ‘Comprehensive Peptaibiotics Database’. The presented database is intended as a public resource freely accessible to the scientific community at peptaibiotics‐database.boku.ac.at. The search options of the previously published repository and the presentation of sequence motif searches have been extended significantly. All of the available search options can be combined to create complex database queries. As a public repository, the presented database enables the easy upload of new peptaibiotic sequences or the correction of existing informations. In addition, an administrative interface for maintenance of the content of the database has been implemented, and the design of the database can be easily extended to store additional information to accommodate future needs of the ‘peptaibiomics community’.     

Noncholecystokinin peptides in human serum which cause gallbladder contraction

In fasting human serum, cholecystokinin (CCK) is not the principal substance which causes $\text{in}$$\text{vitro}$ rabbit gallbldder contraction. Removal of CCK by affinity chromatography from fasting sera from 8 healthy adults reduced bioactivity only by 18 ± 4% (SEM). Unlike CCK, the bioactivity of serum was enhanced by 30 to 57% rather than destroyed by pronase and chymotrypsin respectively and was not inhibited by dibutyryl cGMP. Reduction of serum bioactivity by carboxypeptidase Y indicated that the bioactive substances in serum are peptides. On Sephadex G-50, bioactive substances eluted in positions different from any known form of CCK. Thus, the principal substances in fasting human serum causing $\text{in}$$\text{vitro}$ gallbladder contraction are not CCK but are most likely small peptides which act at receptors different from the receptors for CCK.     

石斛属植物抗肿瘤活性成分及其机制研究进展

石斛属(Dendrobium)是兰科植物中的第二大属,很多石斛属植物是传统的名贵药用植物,具有良好的抗肿瘤作用。近年来石斛属植物抗肿瘤研究取得了显著进展,该文对石斛属植物的抗肿瘤主要活性成分、提取方法以及抗肿瘤机制等方面进行了归纳。石斛属植物抗肿瘤主要活性成分有多糖、生物碱、菲类、联苄类、芴酮类化合物等,抗肿瘤作用机制主要有增强机体免疫力、抑制癌细胞增殖、促进癌细胞凋亡、调控或阻滞癌细胞周期、抗氧化和清除自由基、改变信号通路传导等。在此基础上,进一步提出加强对石斛属植物抗癌方面的深入研究,挖掘更多的石斛属药用资源及其特征成分,深入解析它们的抗肿瘤作用机制,建立全面的评价体系,为开发石斛属植物抗癌药物提供理论基础,为合理、有效地利用石斛属资源提供科学依据。     

Novel anti-inflammatory peptides as cosmeceutical peptides

Ultraviolet (UV) radiation induced inflammation plays an important role in the aging of human skin. Prostaglandin (PG) E₂ is the primary mediator of UVB induced photoinflammation. We screened an internal library for dipeptides that inhibited UVB induced PGE₂ synthesis but showed no cytotoxicity toward human keratinocytes. We identified three highly active inhibitory sequences, LE (Leu + Glu), MW (Met + Trp) and MY (Met + Tyr). To evaluate their efficacy in human skin, 24 sites of abdomen skin were irradiated with a 308 nm excimer laser (300 mJ/cm²), after which 2% LE, MW, MY or a control were applied to the irradiated sites for 24 h. The erythema index (EI) was measured before and 24 h after treatment. The results showed that LE and MW significantly decreased UVB induced erythema (p = 0.041 and p = 0.036, respectively), but ME did not. Overall, LE and MW are candidate cosmeceutical peptides that can protect skin from UVB induced photoinflammation.     

Comprehensive N-Methyl Scanning of a Potent Peptide Inhibitor of Malaria Invasion into Erythrocytes Leads to Pharmacokinetic Optimization of the Molecule

Apical membrane antigen-1 is a protein found in the merozoite stage of the malaria parasite Plasmodium falciparum and has been shown to be critical in the invasion of host erythrocytes. Using a random peptide library displayed on the surface of phage, a 20-residue peptide, R1 (VFAEFLPLFSKFGSRMHILK), was identified which specifically recognized and bound to P. falciparum AMA-1. Moreover, the peptide was found to competitively inhibit parasite invasion of red blood cells (RBC). In this study we report the synthesis and properties of the R1 peptide modified by comprehensive N-methyl scanning along the backbone of the peptide. The native R1 and eighteen R1 analogues containing single N-methyl substitutions were synthesized by manual solid-phase Fmoc peptide chemistry. The set of N-methylated peptides was assessed for relative binding affinity with Pf AMA-1 and RBC invasion inhibitory ability. N-methylation in positions 1, 8, and 14 in the R1 peptide produced analogues exhibiting stronger binding characteristics to Pf AMA-1. A tri N-methylated peptide was more than 10 times more active in the inhibition of RBC invasion assay. This peptide also exhibited enhanced serum stability which may be partially responsible for the increase in binding and bioactivity. We have therefore shown that modifications to a biologically active peptide can dramatically enhance activity and potentially provide a lead to a peptide therapeutic molecule with optimized pharmacokinetic properties.     

Prevention of biofilm formation on titanium surfaces modified with conjugated molecules comprised of antimicrobial and titanium-binding peptides

Specific binding of antimicrobial peptides to titanium (Ti) surfaces may serve to prevent biofilm formation, leading to a reduction in peri-implantitis. This study evaluated the binding behavior of conjugated molecules consisting of antimicrobial and hexapeptidic Ti-binding peptides (minTBP-1) using the quartz crystal microbalance (QCM-D) technique, and investigated the effect of modification of Ti surfaces with these peptides on the bioactivity of Porphyromonas gingivalis. Four kinds of peptide were prepared: histatin 5 (DSHAKRHHGYKRKFHEKHHSHRGY), minTBP-1 + histatin 5 (RKLPDAPDSHAKRHHGYKRKFHEKHHSHRGY), lactoferricin (FQWQRNMRKVR), and minTBP-1 + lactoferricin (RKLPDAPGGFQWQRNMRKVR). The QCM-D analysis demonstrated that significantly larger increases in peptide adsorption were observed in the conjugated peptides than in antimicrobial peptides alone. In addition, ATP activity in P. gingivalis in peptide-modified specimens significantly decreased compared to that in the Ti control. These results indicate that surface modification with conjugated molecules consisting of antimicrobial and Ti-binding peptides is a promising method for reduction of biofilm formation on Ti surfaces.     

Yeast cells for encapsulation of bioactive compounds in food products: A review

Nowadays bioactive compounds have gained great attention in food and drug industries owing to their health aspects as well as antimicrobial and antioxidant attributes. Nevertheless, their bioavailability, bioactivity, and stability can be affected in different conditions and during storage. In addition, some bioactive compounds have undesirable flavor that restrict their application especially at high dosage in food products. Therefore, food industry needs to find novel techniques to overcome these problems. Microencapsulation is a technique, which can fulfill the mentioned requirements. Also, there are many wall materials for use in encapsulation procedure such as proteins, carbohydrates, lipids, and various kinds of polymers. The utilization of food-grade and safe carriers have attracted great interest for encapsulation of food ingredients. Yeast cells are known as a novel carrier for microencapsulation of bioactive compounds with benefits such as controlled release, protection of core substances without a significant effect on sensory properties of food products. Saccharomyces cerevisiae was abundantly used as a suitable carrier for food ingredients. Whole cells as well as cell particles like cell wall and plasma membrane can act as a wall material in encapsulation process. Compared to other wall materials, yeast cells are biodegradable, have better protection for bioactive compounds and the process of microencapsulation by them is relatively simple. The encapsulation efficiency can be improved by applying some pretreatments of yeast cells. In this article, the potential application of yeast cells as an encapsulating material for encapsulation of bioactive compounds is reviewed.     

Characterization of New Milk-derived Inhibitors of Angiotensin Converting Enzyme In Vitro and In Vivo

Inhibition of angiotensin converting enzyme (ACE) has been observed with a variety of different peptides, and peptide fragments with inhibitory capabilities have been identified within many different proteins, including milk proteins. The purpose of this study therefore was to identify new short peptides with inhibitory properties from the primary structure of milk proteins and to characterize them in vitro and in vivo, since no milk derived ACE inhibitors have previously been evaluated for their ability to inhibit ACE in vivo. In vitro, 8 of 9 dipeptides were found to be competitive inhibitors of ACE. The IC₅₀ was significantly lower when an angiotensin I-like substrate was used, than when a bradykinin-like substrate was used. Using three different in vivo models for ACE inhibition, a very moderate effect was observed for three of the new peptides, but only for up to 6 or 12 minutes. Nothing was observed with two reference compounds that are reported to be hypotensive ACE-inhibitors derived from milk proteins. This raises the question whether the mechanism of hypotensive action is straightforward inhibition of ACE in vivo.     

Inhibition of degradation and aggregation of recombinant human consensus interferon-α mutant expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with complex medium in bioreactor

The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins. However, limitations such as protein degradation and aggregation became obvious when secreting heterologous protein-recombinant human consensus interferon-α mutant. Here, we investigate the effect of induction temperature on the yield and stability of interferon mutant expressed by P. patoris with buffered complex medium. The best results in terms of interferon mutant bioactivity and specific bioactivity were obtained when the microorganism was induced at 15°C, which were 2.91 × 10⁸ ± 0.3 × 10⁸ and 2.26 × 10⁸± 0.23 × 10⁸ IU mg⁻¹, respectively. At the same time, the cells grew fast owing to high AOX1-specific activity, and interferon mutant expression level reached 1.23 g l⁻¹, which was almost 30 times higher than that in the flask. Also, the proteolytic degradation of interferon mutant was inhibited completely because of lower protease bioactivity probably due to a reduced cell death rate at lower temperatures as well as protection of yeast extract and peptone in complex medium. In addition, interferon mutant aggregation was repressed significantly by the addition of Tween-80, and a specific bioactivity of 7.35 × 10⁸ ± 0.56 × 10⁸ IU mg⁻¹ was obtained. These results should be applicable to other low-stability recombinant proteins expressed in P. pastoris.     

Colostrum and bioactive,colostral peptides differentially modulate the innate immune response of intestinal epithelial cells

Characterization and identification of peptides with bioactivity from food have received considerable interest recently since such bioactive components must be adequately documented if they are part of functional food claims. We have characterized peptides from colostrum or those generated by a simulated gastrointestinal digest (GI) and tested them for bioactivity using murine intestinal (mIC_c12) cells and compared with bioactivity of intact colostrum. The peptides were recovered in the permeate after dialysis. The presence of peptides in the permeate was confirmed by C₁₈ RP‐HPLC, determination of free amino termini and MALDI MS. The bioactivity of the intact colostrum and colostral peptides in the permeate was tested using mIC_c12 cells stimulated in the absence or presence of different bacterial ligands that mediate cellular activation through stimulation of Toll‐like receptors (TLR). Whereas intact colostrum generally reduced TLR‐mediated signaling, the isolated peptides seemed to either stimulate or reduce the immune response depending on the bacterial ligand used for stimulation. Interestingly, the most potent bioactive peptides originated from nondigested colostrum, which had only been subject to endogenous protease activity. Identified peptides in the nondigested colostrum originated exclusively from the casein fraction of colostrum as shown by MALDI MS/MS identification. Thus, multiple components with different bioactivities towards the innate immune response appear in bovine colostrum. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Probing the influence of sequence-dependent interactions upon α-helix stability in alanine-based linear peptides

The observation that short, linear alanine-based polypeptides form stable α-helices in aqueous solution has allowed the development of well-defined experimental systems with which to study the influence of amino acid sequence upon the stability of secondary structure. We have performed detailed conformational searches upon six alanine-based peptides in order to rationalize the observed variation in the α-helical stability in terms of side-chain-backbone and side-chain-side-chain interactions. Although a simple, gas-phase, potential model was used to obtain the conformational energies for these peptides, good agreement was obtained with experiment regarding their relative α-helical stabilities. Our calculations clearly indicate that valine, isoleucine, and phenylalanine residues should destabilize the α-helical conformation when included within alanine-based peptides because of energetically unfavorable side-chain-backbone interactions, which tend to result in the formation of regions of 3₁₀-helix. In the case of valine, the destabilization most probably arises from entropic effects as the isopropyl side chain can assume more orientations in the 3₁₀-helical form of the peptide. A detailed examination of very short-range interactions in these peptides has also indicated that an interaction, involving fewer than five consecutive residues, whose stabilizing effect reinforces that of the (i, i + 4) hydrogen bond may be the basis of the requirement for increased nucleation (σ) and propagation parameters (s) required by Zimm–Bragg theory to predict the α-helical content for compounds in this class of short peptides. Our calculations complement recent work using modified Zimm–Bragg and Lifson–Roig theories of the helix–coil transition, and are consistent with molecular dynamics simulations upon linear peptides in aqueous solution. © 1993 John Wiley & Sons, Inc.