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.     

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。     

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.     

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.     

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.     

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.     

Squash inhibitors: from structural motifs to macrocyclic knottins

In this article, we will first introduce the squash inhibitor, a well established family of highly potent canonical serine proteinase inhibitors isolated from Cucurbitaceae. The squash inhibitors were among the first discovered proteins with the typical knottin fold shared by numerous peptides extracted from plants, animals and fungi. Knottins contain three knotted disulfide bridges, two of them arranged as a Cystine-Stabilized Beta-sheet motif. In contrast to cyclotides for which no natural linear homolog is known, most squash inhibitors are linear. However, Momordica cochinchinensis Trypsin Inhibitor-I and (MCoTI-I and -II), 34-residue squash inhibitors isolated from seeds of a common Cucurbitaceae from Vietnam, were recently shown to be macrocyclic. In these circular squash inhibitors, a short peptide linker connects residues that correspond to the N- and C-termini in homologous linear squash inhibitors. In this review we present the isolation, characterization, chemical synthesis, and activity of these macrocyclic knottins. The solution structure of MCoTI-II will be compared with topologically similar cyclotides, homologous linear squash inhibitors and other knottins, and potential applications of such scaffolds will be discussed.     

Cycloviolins A-D, anti-HIV macrocyclic peptides from Leonia cymosa

Four novel anti-HIV macrocyclic peptides containing 28-31 amino acid residues, named cycloviolins A-D, have been isolated from the hitherto unstudied tropical plant Leonia cymosa. Their primary structure, including amino acid composition and sequence, was determined by a combination of MALDI-TOF and FAB MS and by enzymatic digestion of reduced derivatives, followed by Edman degradation and mass analyses. All of the cycloviolins contain six cysteines, which are present as three intramolecular disulfide bridges. Intriguingly, cycloviolins A-D showed high degrees of sequence homology to the known cyclopsychotride A and circulins A and B from the Rubiaceae family but much less homology to the varv peptides from Viola, a member of the same family (Violaceae).     

Strategies for locating disulfide bonds in a monoclonal antibody via mass spectrometry

The location of the disulfide bonds in a recombinant monoclonal antibody was confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). A non-reduced Endoproteinase Lys-C (Endo Lys-C) digest of the antibody was analyzed directly by MALDI-TOFMS. The sample was then reduced on-plate by depositing dithiothreitol (DTT) on the sample spot and re-analyzed by MALDI-TOFMS. The disulfide bonds were assigned based on the disappearance of certain mass ions in the non-reduced digest and the appearance of product ions in the reduced digest. A rapid LC/ESI-MS protocol was also developed to determine the location of the disulfide bonds. The peptides generated from the Endo Lys-C digest of the antibody were partially separated on a high performance liquid chromatography (HPLC) column by utilizing a steep gradient and analyzed by ESI-MS. The masses of the partially resolved peptides were determined by deconvoluting the mass spectra.     

RTD-1mimic containing γPNA scaffold exhibits broad-spectrum antibacterial activities

Macrocyclic peptides with multiple disulfide cross-linkages, such as those produced by plants and those found in nonhuman primates, as components of the innate immunity, hold great promise for molecular therapy because of their broad biological activities and high chemical, thermal, and enzymatic stability. However, for some, because of their intricate spatial arrangement and elaborate interstrand cross-linkages, they are difficult to prepare de novo in large quantities and high purity, due to the nonselective nature of disulfide-bond formation. We show that the disulfide bridges of RTD-1, a member of the θ-defensin subfamily, could be replaced with noncovalent Watson-Crick hydrogen bonds without significantly affecting its biological activities. The work provides a general strategy for engineering conformationally rigid, cyclic peptides without the need for disulfide-bond reinforcement.     

A potent, versatile disulfide-reducing agent from aspartic acid

Dithiothreitol (DTT) is the standard reagent for reducing disulfide bonds between and within biological molecules. At neutral pH, however, >99% of DTT thiol groups are protonated and thus unreactive. Herein, we report on (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), a dithiol that can be synthesized from l-aspartic acid in a few high-yielding steps that are amenable to a large-scale process. DTBA has thiol pK(a) values that are ~1 unit lower than those of DTT and forms a disulfide with a similar E°' value. DTBA reduces disulfide bonds in both small molecules and proteins faster than does DTT. The amino group of DTBA enables its isolation by cation-exchange and facilitates its conjugation. These attributes indicate that DTBA is a superior reagent for reducing disulfide bonds in aqueous solution.     

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.     

Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine‐rich snakin‐1 and ‐2 antimicrobial peptides by using a combination of solid‐phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40–50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin‐2 compared to natural snakin‐2, we demonstrated that synthetic snakin‐2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin‐2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.     

Rapid sequencing and disulfide mapping of peptides containing disulfide bonds by using 1,5-diaminonaphthalene as a reductive matrix

MS/MS is indispensable for the amino acid sequencing of peptides. However, its use is limited for peptides containing disulfide bonds. We have applied the reducing properties of 1,5-diaminonaphthalene (1,5-DAN) as a MALDI matrix to amino acid sequencing and disulfide bond mapping of human urotensin II possessing one disulfide bond, and human guanylin possessing two disulfide bonds. 1,5-DAN was used in the same manner as the usual MALDI matrices without any pre-treatment of the peptide, and MS/MS was performed using a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (MALDI QIT TOFMS). The results demonstrated that MS/MS of the molecular ions reduced by 1,5-DAN provided a series of significant b-/y-product ions. All 11 amino acid residues of urotensin II were identified using 1,5-DAN, while only 5 out of 11 residues were identified using 2,5-dihydroxybenzoic acid (DHB); similarly 11 out of 15 amino acid residues of guanylin were identified using 1,5-DAN, while only three were identified using DHB. In addition, comparison of the theoretical and measured values of the mass differences between corresponding MS/MS product ions using 1,5-DAN and DHB narrowed down the possible disulfide bond arrangement candidates. Consequently, 1,5-DAN as a reductive matrix facilitates rapid amino acid sequencing and disulfide mapping for peptides containing disulfide bonds.     

Synthesis of Disulfide Surrogate Peptides Incorporating Large-Span Surrogate Bridges Through a Native-Chemical-Ligation-Assisted Diaminodiacid Strategy

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide-containing peptides. However, peptides incorporating large-span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)-assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible-to-obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X-ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.     

Fragmentation of peptides with intra-chain disulfide bonds in triple quadrupole mass spectrometry and its quantitative application to biological samples

A growing number of peptides are being used today in bioanalytical laboratories. Because of this, there is an increasing interest in the development of highly sensitive, specific and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays for the quantitative analysis of peptides in biological samples. Among the mass spectrometers previously used for peptide quantification, triple quadrupole mass spectrometers are generally not considered the instrument of choice. With this instrumentation, collision cascades or multiple fragmentations tend to generate multiple peaks that have weak intensities. This leads to a loss in detection sensitivity. However, in cases where immonium product ions were formed in abundance, it was found that peptide quantification succeeded. A common feature of these peptides is their intra-loop structure. To elucidate the usefulness of this feature in fragmentation, several peptide analytes with intra-chain disulfide bonds were investigated in this study, including a newly synthesized analog having a single amino acid substitution. The results presented here indicate that abrupt bond cleavage from the intra-loop structure of peptides could be one of the premises for intense immonium ion generation. In contrast, any preferential cleavage of peptide bonds (e.g., proline effect) that gives rise to a linearized sequence would break the intactness of the loop and prevent it from completely dissociating. In addition, the utilization of immonium product ions in LC/MS/MS was demonstrated for the determination of peptides with intra-chain disulfide bonds in biological fluids.     

Synthesis of Disulfide Surrogate Peptides Incorporating Large‐Span Surrogate Bridges Through a Native‐Chemical‐Ligation‐Assisted Diaminodiacid Strategy

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide‐containing peptides. However, peptides incorporating large‐span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)‐assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible‐to‐obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X‐ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.     

Photoredox-Mediated Hydrogen Isotope Exchange Reactions of Amino-Acids,Peptides, and Peptide-Derived Drugs

Hydrogen isotopically labelled compounds are essential diagnostic tools in drug research and development, as they provide vital information about the biological metabolism of drug candidates and their metabolites. Herein we report a photoredox-initiated hydrogen atom transfer (HAT) protocol which efficiently and selectively introduces deuterium or tritium at C(sp³)−H bonds, utilizing heavy water (D₂O or T₂O) as the hydrogen isotope source, and a guanidine base. This protocol has been successfully applied to the incorporation of deuterium in several amino acids (lysine, glycine and proline) and small peptides. Finally, the method has been applied to tritium, because tritium-labelled peptides are essential for application in biological experiments, such as ligand-binding assays, or absorption, distribution, metabolism, and excretion (ADME) studies.     

Context-dependence of the contribution of disulfide bonds to beta-hairpin stability

Incorporation of disulfide bonds to stabilize protein and peptide structures is not always a successful strategy. To advance current knowledge on the contribution of disulfide bonds to beta-hairpin stability, a previously reported beta-hairpin-forming peptide was taken as a template to design a series of Cys-containing peptides. The conformational behavior of these peptides in their oxidized, disulfide-cyclized peptides, and reduced, linear peptides, was investigated on the basis of NMR parameters: NOEs, and 1H and 13C chemical shifts. We found that the effect of disulfide bonds on beta-hairpin stability depends on its location within the beta-hairpin structure, being very small or even destabilizing when connecting two hydrogen-bonded facing residues. When the disulfide bond is linking non-hydrogen-bonded facing residues, we estimated that its contribution to the free-energy change of beta-hairpin folding is approximately -1.0 kcal mol(-1). This value is larger than those reported for most beta-hairpin-stabilizing cross-strand side-chain-side-chain interactions, except for some aromatic-aromatic interactions, in particular the Trp-Trp one, and the cation-pi interaction between Trp and the non-natural methylated Arg/Lys. As disulfide bonds are frequently used to stabilize peptide conformations, our conclusions can be useful for peptide, peptidomimetic, and protein design, and may even extend to other chemical cross-links.