The chemistry of cyclotides

Cyclotides are head-to-tail cyclic peptides that contain a cystine knot motif built from six conserved cysteine residues. They occur in plants of the Rubiaceae, Violaceae, Cucurbitaceae, and Fabaceae families and, aside from their natural role in host defense, have a range of interesting pharmaceutical activities, including anti-HIV activity. The variation seen in sequences of their six backbone loops has resulted in cyclotides being described as a natural combinatorial template. Their exceptional stability and resistance to enzymatic degradation has led to their use as scaffolds for peptide-based drug design. To underpin such applications, methods for the chemical synthesis of cyclotides have been developed and are described here. Cyclization using thioester chemistry has been instrumental in the synthesis of cyclotides for structure-activity studies. This approach involves a native chemical ligation reaction between an N-terminal Cys and a C-terminal thioester in the linear cyclotide precursor. Since cyclotides contain six Cys residues their syntheses can be designed around any of six linear precursors, thus providing flexibility in synthesis. The ease with which cyclotides fold, despite their topologically complex knot motif, as well as the ability to introduce combinatorial variation in the loops, makes cyclotides a promising drug-design scaffold.     

植物环蛋白研究进展*

植物环蛋白(cyclotides)是一类植物中富含二硫键、由28-37个氨基酸残基组成的大环蛋白,其分子中含有一个结构独特的环胱氨酸结(cyclic cystine knot, CCK)。由于其独特的结构和广泛的生物活性,如子宫收缩、溶血、抗肿瘤、抗微生物等活性,及其能耐常规的高温、酸解和酶解的稳定结构,可作为多肽药物设计中的模板分子进行结构修饰或活性多肽的载体,而在国际上引起广泛的关注。目前从堇菜科、茜草科和葫芦科约30种植物中发现100多个植物环蛋白,研究主要集中在澳大利亚、瑞典和美国等几个研究组,近年我们也在开展相关研究。本文主要从植物环蛋白的提取、分离、检测与结构鉴定方法,结构与性质,序列的同源性及分类,化学合成与生物合成,生物活性以及应用前景等几个方面介绍植物环蛋白的研究进展。     

Discovery, structure and biological activities of the cyclotides

The cyclotides are a family of small disulfide rich proteins that have a cyclic peptide backbone and a cystine knot formed by three conserved disulfide bonds. The combination of these two structural motifs contributes to the exceptional chemical, thermal and enzymatic stability of the cyclotides, which retain bioactivity after boiling. They were initially discovered based on native medicine or screening studies associated with some of their various activities, which include uterotonic action, anti-HIV activity, neurotensin antagonism, and cytotoxicity. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaceae families and their natural function in plants appears to be in host defense: they have potent activity against certain insect pests and they also have antimicrobial activity. There are currently around 50 published sequences of cyclotides and their rate of discovery has been increasing over recent years. Ultimately the family may comprise thousands of members. This article describes the background to the discovery of the cyclotides, their structural characterization, chemical synthesis, genetic origin, biological activities and potential applications in the pharmaceutical and agricultural industries. Their unique topological features make them interesting from a protein folding perspective. Because of their highly stable peptide framework they might make useful templates in drug design programs, and their insecticidal activity opens the possibility of applications in crop protection.     

Isolation and Characterization of Bioactive Cyclotides from Viola labridorica

Many Violaceae plants contain cyclotides, which are plant cyclopeptides distinguished by a cyclic cystine knot motif with 28–37 amino acid residues. In the current study, four new cyclotides, vila A–D ( 1 – 4 , resp.), together with a known cyclotide, varv D ( 5 ), were isolated from Viola labridorica (Violaceae). A chromatography‐based method was used to isolate the cyclotides, which were characterized using tandem mass spectrometry and 2D‐NMR spectroscopy. Several of the cyclotides showed cytotoxic activities against five cancer cell lines, i.e., U251, MDA‐MB‐231, A549, DU145, and BEL‐7402, with vila A and B ( 1 and 2 , resp.) being the most cytotoxic. The isolated cyclotides showed no antibacterial activity against Staphyloccocus aureus and Candida albicans. Homology modeling of the cyclotide structures was used to analyze structure–activity relationships.     

Novel strategies for isolation and characterization of cyclotides: the discovery of bioactive macrocyclic plant polypeptides in the Violaceae

This review focuses on the discovery of cyclotides in the Violaceae, their isolation and their anti-cancer effects. These macrocyclic plant peptides consist of about 30 amino acids, including three conserved disulfide bonds in a cystine knotted arrangement, which renders them a remarkable stability. Their unique structure, combined with a wide array of biological activities, makes them of great interest as possible leads in drug development or as carriers of grafted peptide sequences. Here we describe the work conducted in our laboratory, which started with the overall aim of identifying peptides and small proteins of the size 10-50 amino acid residues in plants with novel chemical structures and biological profiles with a potential for drug development or for use as pharmacological tools. Thus we developed a fractionation protocol to directly address major challenges encountered when dealing with plant material, such as removal of chlorophyll, polyphenols, and low molecular compounds omnipresent in plants. Using this protocol, we then discovered a suite of cyclotides, the varv peptides, from the plant Viola arvensis (Violaceae). Following this, separation methods directly targeting cyclotides were developed, e.g. by adsorption, ion exchange chromatography and solvent-solvent partitioning, which then were used in the isolation of additional cyclotides. To structurally examine cyclotides we have also developed methods based on mass spectrometry for cyclotide sequencing and mapping of disulfide bonds. Finally, to assess structure-activity relationships, regarding their anti-cancer and cytotoxic effects that we focus upon, we have also characterized the three dimensional structure of cyclotides by homology modeling techniques.     

栝楼蛋白 2: 栝楼蛋白部分化学结构的初步测定

栝楼蛋白(Trichobitacin)是从栝楼(Trichosanthes kirilowiiMaxim, Cucurbitaceae)中新发现的核糖体失活蛋白, 分子量为27,228; pI为9.6。应用基质辅助的激光解析飞行时间质谱(MALDI-TOF-MS)和快原子轰击质谱法(FAB-MS)分别测定胰蛋白酶酶解栝楼蛋白和天花粉蛋白(Trichosanthin)的混合肽质谱, 通过比较发现了一些分子量相同的肽。由于这两种蛋白质都来源于栝楼块根, 同源性比较强, 所以这些肽序列在两种蛋白质中基本一样; 再结合蛋白N-端自动顺序仪测定栝楼蛋白N-端的结果, 确定了栝楼蛋白N-端38个氨基酸的顺序, 栝楼蛋白经胰蛋白酶酶解后所得肽段用HPLC分离纯化, 再用蛋白质自动顺序仪, DABITC/PITC双偶合手工法和质谱法共确定了栝楼蛋白N-端, C-端等100多个氨基酸残基的序列。     

Comparative Study of the Chemical Compositions and Antioxidant Activities of Fresh Juices from Romanian Cucurbitaceae Varieties

Cucurbitaceae is a family of health-promoting plants due to their compounds with beneficial effects. The aim of this study was to analyze, for the first time, the chemical composition, the antioxidant activity and the metal chelating properties of fruit juices obtained from four different species of the Cucurbitaceae family cultivated in Romania, namely Momordica charantia, Cucumis metuliferus, Benincasa hispida and Trichosanthes cucumerina. The samples of juice were analyzed by high-performance liquid chromatography (HPLC) and all the four species displayed high levels of the two triterpenes, oleanolic and ursolic acids, and also in phenolic compounds, including catechin, (−)-epicatechin and gallic acid. The juices demonstrated significant antioxidant activity against the free radical 2,2-diphenyl-1-picrylhydrazyl (ranging from 20 to 95%,), a good iron binding ability (ranging from 7.45 ± 0.28% to 86.95 ± 0.97%) and also promising antioxidant potential against the ABTS radical (ranging from 4.97 to 32.60 μETx/mL juice). Our findings raise interesting questions for further research on Cucurbitaceae fruit juices and, consequently, their very good antioxidant potential suggests these fruits should be further explored for their protective effect against oxidative damage. This is the first time the chemical composition and antioxidant activities of fruit juices from these four Romanian Cucurbitaceae varieties have been investigated.     

High concentrations of sodium chloride facilitate the use of immobilized chymotrypsin for separating virgin forms of specific trypsin inhibitors.

It has been shown that specific trypsin inhibitors exhibit also antichymotrypsin activity in the presence of high NaCl concentrations. Taking advantage of this phenomenon a simple procedure of separation of the virgin forms of trypsin inhibitors from squash seeds and porcine pancreas (Kazal) was elaborated. In a typical experiment the inhibitor sample was loaded onto immobilized chymotrypsin equilibrated with 5 M NaCl at pH 8. After washing out unadsorbed material the virgin forms of inhibitors could be eluted either with water, buffer pH 8.0 or 0.02 M citrate buffer pH 2.6 containing no NaCl.     

A liquid chromatography-electrospray ionization-mass spectrometry method for quantification of cyclotides in plants avoiding sorption during sample preparation

Cyclotides are plant-produced, bioactive, cyclic mini-proteins with interesting pharmaceutical and agricultural applications. A reverse phase liquid chromatography electrospray ionization mass spectrometry (RP-LC-ESI-MS) method for analysis of cyclotides in plant materials with a minimum of sample pre-treatment is presented. Three exemplary cyclotides (kalata B1, kalata B2 and cycloviolacin O2) were used as reference substances for the method development. Linearity (r(2)>0.99) was achieved in the concentration range 0.05-10 mg/L and the limit of detection was 1.7-4.0 μg/L. The present study is the first to demonstrate that cyclotides dissolved in water sorb to glass vials, but the addition of 15% of acetonitrile or 40 mg/L of bovine serum albumin is sufficient to keep the cyclotides in solution. Cyclotides were extracted from candied violets, violet tea, and the plants Oldenlandia affinis and Viola odorata using 70% methanol containing 0.1% formic acid (v/v). The plant content was determined to be 23.5-14,200 μg/g (dry weight). The highest content of cyclotide was found in wild Danish V. odorata, and it is the highest content of cyclotide in a plant reported hitherto. Candied violets contained 0.00-8.66 μg/g (dry weight), while no cyclotides were detected in commercial violet tea.     

Enabling Efficient Folding and High-Resolution Crystallographic Analysis of Bracelet Cyclotides

Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, hampering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.     

Cytotoxic potency of small macrocyclic knot proteins: structure-activity and mechanistic studies of native and chemically modified cyclotides

The cyclotides are a family of circular and knotted proteins of natural origin with extreme enzymatic and thermal stability. They have a wide range of biological activities that make them promising tools for pharmaceutical and crop-protection applications. The cyclotides are divided into two subfamilies depending on the presence (M?bius) or absence (bracelet) of a cis-Pro peptide bond. In the current work we report a series of experiments to give further insight into the structure-activity relationship of cyclotides in general, and the differences between subfamilies and the role of their hydrophobic surface in particular. Selective chemical modifications of Glu, Arg, Lys and Trp residues was tested for cytotoxic activity: derivatives in which the Trp residue was modified showed low effect, demonstrating the existence of a connection between hydrophobicity and activity. However, over the full set of cyclotides examined, there was no strong correlation between the cytotoxic activity and their hydrophobicity. Instead, it seems more like that the distribution of charged and hydrophobic residues determines the ultimate degree of potency. Furthermore, we found that while the Glu residue is very important in maintaining the activity of the bracelet cyclotide cycloviolacin O2, it is much less important in the M?bius cyclotides. Despite these differences between cyclotide subfamilies, a systematic test of mixtures of cyclotides revealed that they act in an additive way.     

Structural and initial biological analysis of synthetic arylomycin A2

The growing threat of untreatable bacterial infections has refocused efforts to identify new antibiotics, especially those acting by novel mechanisms. While the inhibition of pathogen proteases has proven to be a successful strategy for drug development, such inhibitors are often limited by toxicity due to their promiscuous inhibition of homologous and mechanistically related human enzymes. Unlike many protease inhibitors, inhibitors of the essential type I bacterial signal peptidase (SPase) may be more specific and thus less toxic due to the enzyme's unique structure and catalytic mechanism. Recently, the arylomycins and related lipoglycopeptide natural products were isolated and shown to inhibit SPase. The core structure of the arylomycins and lipoglycopeptides consists of a biaryl-linked, N-methylated peptide macrocycle attached to a lipopeptide tail, and in the case of the lipoglycopeptides, a deoxymannose moiety. Herein, we report the first total synthesis of a member of this group of antibiotics, arylomycin A2. The synthesis relies on Suzuki-Miyaura-mediated biaryl coupling, which model studies suggested would be more efficient than a lactamization-based route. Biological studies demonstrate that these compounds are promising antibiotics, especially against Gram-positive pathogens, with activity against S. epidermidis that equals that of the currently prescribed antibiotics. Structural and biological studies suggest that both N-methylation and lipidation may contribute to antibiotic activity, whereas glycosylation appears to be generally less critical. Thus, these studies help identify the determinants of the biological activity of arylomycin A2 and should aid in the design of analogs to further explore and develop this novel class of antibiotic.     

Sunflower trypsin inhibitor-1

SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar K(i) value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors.     

A new pentacyclic cucurbitane glucoside and a new triterpene from the fruits of Gymnopetalum integrifolium

A new pentacyclic cucurbitane glucoside, named aoibaclyin (1) and a new triterpene (2) have been isolated from the EtOH extract of the fruits of Gymnopetalum integrifolium Kurz (Cucurbitaceae), together with three known compounds, bryoamaride (3), 25-O-acetylbryoamaride (4) and beta-sitosterol 3-O-beta-D-glucopyranoside (5). The structures of these compounds were elucidated by spectroscopic analyses.     

Mass Spectrometric Analysis of Cyclotides from Clitoria ternatea: Xxx-Pro Bond Fragmentation as Convenient Diagnostic of Pro Residue Positioning

Cyclotides, a class of macrocyclic plant peptides, characterized by a cyclic backbone and three inter-locking disulfide bonds, may be divided into two major structural subfamilies, Möbius and Bracelet, based on the presence or absence of a specific proline residue. The present study describes the suite of cyclotides obtained from Clitoria ternatea, characterized by LC−MS and MS/MS techniques. Notable variations in product ion distributions were observed in cyclotides belonging to different structural subfamilies based on the number and positions of proline residues. For instance, Cter M which is an abundant Möbius cyclotide in this plant containing three proline residues, displayed distinct b- and y- ion characteristics in the MS/MS spectra compared to Cliotide T1, another commonly identified cyclotide but belonging to the Bracelet subfamily having two proline residues. The distinct fragmentation pattern of prototypical cyclotides of each structural subfamily, determined by Xxx-Pro bond fragmentation, was used to rapidly identify and sequence a novel cyclotide ctr pep 30 from this plant.     

Anti-HIV cyclotides

The cyclotides are a recently discovered, structurally unique family of bioactive plant peptides. Their discovery spawned a series of structural analyses, synthetic efforts, and studies to define the biosynthesis and biological properties of these novel peptide metabolites. Cyclotides have a head-to-tail cyclized amino acid backbone and a conserved cystine knot motif that provides an extremely stable structural framework. They all share a common global fold and are highly resistant to denaturation and to cleavage by proteolytic enzymes. However, these macrocyclic peptides are quite permissive to amino acid substitutions or additions in several peripheral loop regions, since changes in these loops do not alter the core cyclotide structure. These features make cyclotides attractive templates for incorporating desired amino acid sequences and then delivering these peptide sequences in a well defined, highly stable framework. Cyclotides likely function in a defensive role in the source plants since they exhibit a broad spectrum of antimicrobial activity and are detrimental to the growth and survival of herbivorous insects. Cyclotides are gene-encoded polypeptides that are cleaved from larger precursor proteins and then cyclized. This review summarizes research done on a subset of cyclotides that were discovered due to their HIV inhibitory properties. It details the isolation and characterization of these compounds and describes this work in the context of our current state of knowledge of the entire cyclotide family.     

The growth inhibition of Streptococcus mutans by 5'-nucleotidase inhibitors from Areca catechu L

New 5'-nucleotidase inhibitors named NF-86I, NF-86II were recently isolated from the seeds of Areca catechu L. NF-86I and NF86II showed inhibitory effects on the growth of Streptococcus mutans MT8148(c) and Streptococcus mutans MT6715(g), respectively. In addition, these inhibitors could inhibit insoluble glucan formation from sucrose. NF-86I and NF-86II were found to be polyphenolic substances. Some polyphenols such as tannic acid bind non-specifically to proteins (tannic activity). The 5'-nucleotidase inhibitors that we isolated did not show any such activity. However, the growth inhibitory activity and the inhibitory effect on water-insoluble glucan production were equal to tannic acid. It is therefore considered that these inhibitors bind specifically to the bacterial cell surface. Our findings suggest that the 5'-nucleotidase inhibitors NF-86I and NF-86II may be useful anti-plaque preventing agents.     

Ultrafiltration‐LC–MS combined with semi‐preparative HPLC for the simultaneous screening and isolation of lactate dehydrogenase inhibitors from Belamcanda chinensis

Stroke represents the fourth leading cause of death in the USA and the second leading cause of death worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke and natural products are considered a promising source of novel lactate dehydrogenase inhibitors. In this study, we used PC12 cells to determine the protective effect of extracts from the herb Belamcanda chinensis following toxic challenge. Using ultrafiltration high‐performance liquid chromatography coupled with photo‐diode array detection and electrospray ionization mass spectrometry, we screened and identified isoflavonoids from Belamcanda chinensis extracts. Semi‐preparative high‐performance liquid chromatography was then applied to separate and isolate the active constituents. Using these methods, we identified six major compounds in Belamcanda chinensis as lactate dehydrogenase inhibitors: tectoridin, iristectorin A, iridin, tectorigenin, irigenin, and irisflorentin, which were then isolated to >92% purity. This is the first report that Belamcanda chinensis extracts contain potent lactate dehydrogenase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from Belamcanda chinensis guided by ultrafiltration high‐performance liquid chromatography coupled with photo‐diode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.     

Isolation of chemical constituents from Enicosanthum cupulare (King) Airy-Shaw

Six chemical constituents were isolated from Enicosanthum cupulare (King) Airy-Shaw. The structures of the isolated compounds were determined using 1D, 2D-NMR and were found to be identical to those previously reported for these compounds. Three aporphine alkaloids, O-methylmoschatoline, liriodenine and oxostephanine were isolated from this plant for the first time. In addition, we examined the effects of these compounds on melanogenesis in murine B16 melanoma cells; O-methylmoschatoline increased melanin content in these cells.     

Peptide quantification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry: investigations of the cyclotide kalata B1 in biological fluids

A rapid method has been developed for the quantification of the prototypic cyclotide kalata B1 in water and plasma utilizing matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. The unusual structure of the cyclotides means that they do not ionise as readily as linear peptides and as a result of their low ionisation efficiency, traditional LC/MS analyses were not able to reach the levels of detection required for the quantification of cyclotides in plasma for pharmacokinetic studies. MALDI-TOF-MS analysis showed linearity (R2 > 0.99) in the concentration range 0.05-10 microg/mL with a limit of detection of 0.05 microg/mL (9 fmol) in plasma. This paper highlights the applicability of MALDI-TOF mass spectrometry for the rapid and sensitive quantification of peptides in biological samples without the need for extensive extraction procedures.