Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V. hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.     

Natural and grafted cyclotides in cancer therapy: An insight

Cyclotides is a rapidly growing class of plant‐derived cyclic peptides exhibiting several bioactivities with potential applications in the agricultural and pharmaceutical sectors. Both natural and grafted cyclotides have shown promise in cancer therapy. Approximately 70 natural cyclotides belonging to three plant families (Fabaceae, Rubiaceae, and Violaceae) have shown cytotoxicity against several cancer cell lines. Cyclotides exhibit considerable stability against thermal and enzymatic proteolysis, owing to their unique structure with knotted topology and head to tail cyclization. Further, their small size, high stability, oral bioavailability, and tolerance to amino acid substitution in structural loops make them an ideal platform for designing peptide‐based drugs for cancer. Thus, cyclotides provide ideal scaffolds for bioactive epitope grafting and facilitating drug delivery in cancer treatment. Many anticancer linear peptides have been grafted in cysteine knotted cyclic framework of cyclotide for enhancing their cell permeability across cellular membranes, thereby improving their delivery and pharmacokinetics. The present review comprehensively discusses the distribution, toxicity, and anticancer bioactivity of natural cyclotides. Further, it systematically elaborates on the role and action of epitopes' into grafted cyclotides in targeting cancer. The review also encompasses related patents landscape study and future challenges in peptide‐based cancer therapy.     

Plant cyclotides: an unusual class of defense compounds

Plant cyclotides are unusual peptides with low molecular masses and a three-dimensional structure characterized by the presence of a cyclic fold. Synthetic peptides can adopt this circular conformation, but it is not a common feature for most members of other peptide groups. Cyclotides present a wide range of functions, such as the ability to induce stronger contractions during childbirth and anti-tumor activity. Additionally, some cyclotides present anti-viral, insecticidal or proteinase inhibitory activity. In this paper, we describe the structural and functional characteristics of plant cyclotides, their most conserved features and the development of these peptides for human health and biotechnological applications.     

15N cyclotides by whole plant labeling

Cyclotides are 28-37 amino acid peptides incorporating three disulfide bonds and a cyclic backbone. Their cyclic and knotted topology renders them immune to denaturation by heat or organic solvents and highly resistant to proteolysis. They have a range of interesting and potentially useful pharmaceutical properties and have been proposed as scaffolds within which peptides with drug activities can be stabilized for delivery. Some members of the family also have agricultural applications deriving from their potent insecticidal activity. Labeling peptides with the NMR-active and stable 15N isotope facilitates a range of studies by NMR, including structural and dynamics studies and their use as tracers. However, owing to their head-to-tail cyclized peptide backbone labeled cyclotides are not amenable to conventional recombinant labeling strategies. We have developed an approach to overcome this limitation by growing the cyclotide-bearing plant Oldenlandia affinis on nitrogen-free agar media supplemented with 15N salts and obtaining complete labeling at no detriment to plant biomass. We purified the insecticidal cyclotides kalata B1 and kalata B2 as examples and provide heteronuclear single quantum coherence (HSQC) NMR spectra for each. This method of labeling cyclotides involves only a fraction of the cost of uniform labeling by solid-phase peptide synthesis.     

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.     

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.     

Isolation and characterization of cytotoxic cyclotides from Viola philippica

Cyclotides are a large family of plant peptides characterized by a macrocyclic backbone and knotted arrangement of three disulfide bonds. This unique structure renders cyclotides exceptionally stable to thermal, chemical and enzymatic treatments. They exhibit a variety of bioactivities, including uterotonic, anti-HIV, cytotoxic and hemolytic activity and it is these properties that make cyclotides an interesting peptide scaffold for drug design. In this study, eight new cyclotides (Viphi A-H), along with eight known cyclotides, were isolated from Viola philippica, a plant from the Violaceae family. In addition, Viba 17 and Mram 8 were isolated for the first time as peptides. The sequences of these cyclotides were elucidated primarily by using a strategy involving reduction, enzymatic digestion and tandem mass spectroscopy sequencing. Several of the cyclotides showed cytotoxic activities against the cancer cell lines MM96L, HeLa and BGC-823. The novel cyclotides reported here: (1) enhance the known sequence variation observed for cyclotides; (2) extend the number of species known to contain cyclotides; (3) provide interesting structure-activity relationships that delineate residues important for cytotoxic activity. In addition, this study provides insights into the potential active ingredients of traditional Chinese medicines.     

Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.     

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.     

Quantitative analysis of backbone-cyclised peptides in plants

Progress in understanding the biosynthetic pathway of the cyclotides has been hampered as this unique family of cyclic plant peptides are notoriously difficult to analyse by standard proteomic approaches such as gel electrophoresis. We have developed a simple, rapid and robust strategy for the quantification of cyclotides in crude plant extracts using MALDI-TOF MS making use of generic peptides similar in mass to the analyte as internal standards for calibration. Linearity (r(2)>0.99) over two orders of magnitude (down to femtomole levels) was achieved in plant extracts, allowing quantitative analysis of transgenic and endogenous peptide expression.     

Phosphatidylethanolamine Binding Is a Conserved Feature of Cyclotide-Membrane Interactions

Cyclotides are bioactive cyclic peptides isolated from plants that are characterized by a topologically complex structure and exceptional resistance to enzymatic or thermal degradation. With their sequence diversity, ultra-stable core structural motif, and range of bioactivities, cyclotides are regarded as a combinatorial peptide template with potential applications in drug design. The mode of action of cyclotides remains elusive, but all reported biological activities are consistent with a mechanism involving membrane interactions. In this study, a diverse set of cyclotides from the two major subfamilies, Möbius and bracelet, and an all-d mirror image form, were examined to determine their mode of action. Their lipid selectivity and membrane affinity were determined, as were their toxicities against a range of targets (red blood cells, bacteria, and HIV particles). Although they had different membrane-binding affinities, all of the tested cyclotides targeted membranes through binding to phospholipids containing phosphatidylethanolamine headgroups. Furthermore, the biological potency of the tested cyclotides broadly correlated with their ability to target and disrupt cell membranes. The finding that a broad range of cyclotides target a specific lipid suggests their categorization as a new lipid-binding protein family. Knowledge of their membrane specificity has the potential to assist in the design of novel drugs based on the cyclotide framework, perhaps allowing the targeting of peptide drugs to specific cell types.     

Backbone cyclization of a recombinant cystine-knot peptide by engineered Sortase A

Cyclotides belong to the family of cyclic cystine-knot peptides and have shown promise as scaffolds for protein engineering and pharmacological modulation of cellular protein activity. Cyclotides are characterized by a cystine-knotted topology and a head-to-tail cyclic polypeptide backbone. While they are primarily produced in plants, cyclotides have also been obtained by chemical synthesis. However, there is still a need for methods to generate cyclotides in high yields to near homogeneity. Here, we report a biomimetic approach which utilizes an engineered version of the enzyme Sortase A to catalyze amide backbone cyclization of the recombinant cyclotide MCoTI-II, thereby allowing the efficient production of active homogenous species in high yields. Our results provide proof of concept for using engineered Sortase A to produce cyclic MCoTI-II and should be generally applicable to generating other cyclic cystine-knot peptides.     

Mutagenesis of bracelet cyclotide hyen D reveals functionally and structurally critical residues for membrane binding and cytotoxicity

Cyclotides have a wide range of bioactivities relevant for agricultural and pharmaceutical applications. This large family of naturally occurring macrocyclic peptides is divided into three subfamilies, with the bracelet subfamily being the largest and comprising the most potent cyclotides reported to date. However, attempts to harness the natural bioactivities of bracelet cyclotides and engineer-optimized analogs have been hindered by a lack of understanding of the structural and functional role of their constituent residues, which has been challenging because bracelet cyclotides are difficult to produce synthetically. We recently established a facile strategy to make the I11L mutant of cyclotide hyen D that is as active as the parent peptide, enabling the subsequent production of a series of variants. In the current study, we report an alanine mutagenesis structure-activity study of [I11L] hyen D to probe the role of individual residues on peptide folding using analytical chromatography, on molecular function using surface plasmon resonance, and on therapeutic potential using cytotoxicity assays. We found that Glu-6 and Thr-15 are critical for maintaining the structure of bracelet cyclotides and that hydrophobic residues in loops 2 and 3 are essential for membrane binding and cytotoxic activity, findings that are distinct from the structural and functional characteristics determined for other cyclotide subfamilies. In conclusion, this is the first report of a mutagenesis scan conducted on a bracelet cyclotide, offering insights into their function and supporting future efforts to engineer bracelet cyclotides for biotechnological applications.     

Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.     

Sequencing cyclic peptides by multistage mass spectrometry

Some of the most effective antibiotics (e.g. Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of MS-based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage MS, and show its advantages over single-stage MS. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria.     

Computational Design of Hypothetical New Peptides Based on a Cyclotide Scaffold as HIV gp120 Inhibitor

Cyclotides are a family of triple disulfide cyclic peptides with exceptional resistance to thermal/chemical denaturation and enzymatic degradation. Several cyclotides have been shown to possess anti-HIV activity, including kalata B1 (KB1). However, the use of cyclotides as anti-HIV therapies remains limited due to the high toxicity in normal cells. Therefore, grafting anti-HIV epitopes onto a cyclotide might be a promising approach for reducing toxicity and simultaneously improving anti-HIV activity. Viral envelope glycoprotein gp120 is required for entry of HIV into CD4+ T cells. However, due to a high degree of variability and physical shielding, the design of drugs targeting gp120 remains challenging. We created a computational protocol in which molecular modeling techniques were combined with a genetic algorithm (GA) to automate the design of new cyclotides with improved binding to HIV gp120. We found that the group of modified cyclotides has better binding scores (23.1%) compared to the KB1. By using molecular dynamic (MD) simulation as a post filter for the final candidates, we identified two novel cyclotides, GA763 and GA190, which exhibited better interaction energies (36.6% and 22.8%, respectively) when binding to gp120 compared to KB1. This computational design represents an alternative tool for modifying peptides, including cyclotides and other stable peptides, as therapeutic agents before the synthesis process.     

Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule.     

Microwave-assisted Boc-solid phase peptide synthesis of cyclic cysteine-rich peptides.

In this study we describe the first protocols for the synthesis of cystine-rich peptides in the presence of microwave radiation with Boc-solid phase peptide synthesis (SPPS). This method is exemplified for macrocyclic peptides known as cyclotides, which comprise approximately 30 amino acids and incorporate a cystine knot arrangement of their three disulfide bonds. However, the method is broadly applicable for a wide range of peptides using Boc-SPPS, especially for SPPS of large peptides via native chemical ligation. Microwave radiation produces peptides in high yield and with high purity, and we were able to reduce the time for the assembly of approximately 30 mer peptide chains to an overnight reaction in the automated microwave-assisted synthesis.     

Chemical synthesis and biosynthesis of the cyclotide family of circular proteins

Cyclotides are a recently discovered class of proteins that have a characteristic head-to-tail cyclized backbone stabilized by a knotted arrangement of three disulfide bonds. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. Cyclotides have a range of bio-activities, including uterotonic, anti-HIV, anti-bacterial and cytotoxic activity but their insecticidal properties suggest that their natural physiological role is in plant defense. They are genetically encoded as linear precursors and subsequently processed to produce mature cyclic peptides but the mechanism by which this occurs remains unknown. Currently most cyclotides are obtained via direct extraction from plants in the Rubiaceae and Violaceae families. To facilitate the screening of cyclotides for structure-activity studies and to exploit them in drug design or agricultural applications a convenient route for the synthesis of cyclotides is vital. In this review the current chemical, recombinant and biosynthetic routes to the production of cyclotides are discussed.