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.     

Solution structure of the cyclotide palicourein: implications for the development of a pharmaceutical framework

The cyclotides are a family of disulfide-rich proteins from plants. They have the characteristic structural features of a circular protein backbone and a knotted arrangement of disulfide bonds. Structural and biochemical studies of the cyclotides suggest that their unique physiological stability can be loaned to bioactive peptide fragments for pharmaceutical and agricultural development. In particular, the cyclotides incorporate a number of solvent-exposed loops that are potentially suitable for epitope grafting applications. Here, we determine the structure of the largest known cyclotide, palicourein, which has an atypical size and composition within one of the surface-exposed loops. The structural data show that an increase in size of a palicourein loop does not perturb the core fold, to which the thermodynamic and chemical stability has been attributed. The cyclotide core fold, thus, can in principle be used as a framework for the development of useful pharmaceutical and agricultural bioactivities.     

Discovery and structures of the cyclotides: novel macrocyclic peptides from plants

Summary Circular disulfide-rich polypeptides were unknown a decade ago but over recent years a large family of such molecules has been discovered, which we now refer to as the cyclotides. They are typically about 30 amino acids in size, contain an N- to C-cyclised backbone and incorporate three disulfide bonds arranged in a cystine knot motif. In this motif, an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments is penetrated by the third disulfide bond. The combination of this knotted and strongly braced structure with a circular backhone renders the cyclotides impervious to enzymatic breakdown and makes them exceptionally stable. This article describes the discovery of the cyclotides in plants from the Rubiaceae and Violaceae families, their chemical synthesis, folding, structural characterisation, and biosynthetic origin. The cyclotides have a diverse range of biological applications, ranging from uterotonic action, to anti-HIV and neurotensin antagonism. Certain plants from which they are derived have a history of uses in native medicine, with activity being observed after oral ingestion of a tea made from the plants. This suggests the possibility that the cyclotides may be orally bioavailable. They therefore have a range of potential applications as a stable peptide framework.     

Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry

Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis, whereof ten (vigno 1–10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides.     

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 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.     

Factors Influencing the Stability of Cyclotides: Proteins with a Circular Backbone and Cystine Knot Motif

Cyclotides are a large family of mini-proteins that have the distinguishing features of a head-to-tail cyclised backbone and a cystine knot formed by six conserved cysteine residues. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaceae families. The unique structural features of the cyclotides make them extremely resistant to chemical, thermal and proteolytic degradation. In this article we review recent studies from our laboratory that dissect the role of the individual structural elements in defining the stability of cyclotides. The resistance of cyclotides to chemical and proteolytic degradation is in large part due to the cystine knot, whereas the thermal stability is a composite of several features including the cystine knot, the cyclic backbone and the hydrogen bonding network. A range of biological activities of cyclotides is critically dependent on the presence of the cyclic backbone.Australian Peptide Conference Issue.     

From nature to creation: Going around in circles,the art of peptide cyclization

Cyclic peptides and cyclotides are becoming common identities within the present efforts seen in peptide engineering – as we seek approaches to achieve potent biological activity, pharmacological selectivity, structurally stability and oral bioavailability. Yet this unique family of peptides has faced uncommon hurdles in their discovery, synthesis and bioengineering, retaining to characteristics that truly deviate these from their linear counterparts. In this mini-review we take a board spectrum approach to introduce this novel family of biomolecules and the troubles that they face in their sequence and disulfide connectivity assignment, together highlighting the present combined strategies involved in cyclic peptide/cyclotide synthesis and modification. These efforts have circumvented otherwise impossible hurdles in their manipulation and production that are only now advancing cyclic peptides/cyclotides as research probes and future pharmaceutical templates.     

Genotoxicity and cellular uptake of cyclotides: evidence for multiple modes of action

Cyclotides are a family of ultra-stable, head-to-tail cyclic mini-proteins from plants, with each member comprising about 30 amino acid residues. Their stability derives from the unique structural topology where the cyclic backbone and two disulfide bonds make up an embedded ring, which is knotted by a third disulfide bond. The cyclotides find potential applications in the pharmaceutical industry as stable peptide-based scaffolds for unstable drugs, and also as medicinal agents, due to the wide range of their inherent pharmacological activities. However, there is a lack of fundamental toxicological studies on this type of compound. The current study determined the possible DNA-damaging effects of three cyclotides, i.e., cycloviolacin O2, vaby D, and kalata B1, in human lymphoma cells by use of the alkaline version of the comet assay. The three cyclotides induced massive DNA fragmentation at lethal concentrations. At a sub-lethal concentration, cycloviolacin O2 and vaby D gave a bell-shaped dose-response curve for their DNA-damaging effect. Kalata B1 caused no significant DNA damage at sub-cytotoxic concentrations. Single-cell micro-autoradiography was carried out on tritium-labeled cycloviolacin O2 in order to understand the mechanism behind the dose-response curve. The results revealed that the peptide is taken up into the cell, both at cytotoxic and at low concentrations. Most biological effects of the cyclotides have been taken to follow from the disruption of cell membranes, but even if the intracellular mechanisms and targets still remain unknown, the current study has unequivocally demonstrated that cyclotides also must have other dose-dependent modes of action.     

Optimization of cyclotide extraction parameters

Cyclotides are gene-encoded plant mini-proteins that contain a unique circular and cystine knotted amide backbone. Because of that ultra stable scaffold and the ability to harness a wide variety of sequences and biological activities within the scaffold, cyclotides find interesting potential applications for drug discovery and in agriculture. However, some fundamental knowledge is still missing to exploit these plant compounds, including finding the optimal process of their extraction from plant material. In the current work, the extraction parameters solvent type, time of extraction, number of re-macerations and the plant material to solvent ratio have been compared using the sweet violet (Viola odorata L.) as a model plant. That species is a well-characterized and rich source of cyclotides that contains prototypic cyclotides with different chemical and physical properties. We found that hydroalcoholic solutions of medium polarity give good yield of the cyclotide cocktail. In conclusion, single maceration with 50% MeOH for 6 h at a plant material to solvent ratio of 0.5:10 (g/mL) represents an optimum extraction method.     

The cyclotide family of circular miniproteins: nature's combinatorial peptide template

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The structural features that contribute to their remarkable stability are described in this review.     

Analysis of the Structural Stability Among Cyclotide Members Through Cystine Knot Fold that Underpins Its Potential Use as a Drug Scaffold

Recent emergence of plant derived peptide cyclotides, characterized with a cyclized head-to-tail backbone and three disulfide bonds forming cyclic cystine knot, has advanced the field of biopharmaceutics to next level. This conserved structural feature of cyclotides holds responsible for its outstanding resistance towards thermal, chemical and enzymatic degradation. Besides, the cyclotides are preferred widely in current research to develop them as potent peptide therapeutics, where the improvement of structural stability is a demanding task in pharmaceutical firm. Hence, in this work, the structural stability of six cyclotides of kalata family (kalata B1, kalata B2, kalata B5, kalata B7, kalata B8 and kalata B12) was investigated. Among all, maximum number of intra-molecular interactions was observed only in kalata B1 (kB1). In addition, geometrical observables using conformational sampling of six kalata cyclotides also revealed that kB1 exhibited statistically significant structural stability in terms of contours of root mean square fluctuation, gyration radius, ovality and surface area (polar and non-polar). Furthermore, the distance of disulfide bridges (S–S within 2.2 Å) also confirmed that kB1 achieved maximum strength in terms of structural stability and accomplished remarkable functionality in terms of ovality as compared to other five kalata cyclotides. Accordingly, kB1 could be demonstrated as a stable template for the advancement of peptide therapeutics.     

Discovery and structures of the cyclotides: novel macrocyclic peptides from plants

Circular disulfide-rich polypeptides were unknown a decade agobut over recent years a large family of such molecules hasbeen discovered, which we now refer to as the cyclotides. They are typically about 30 amino acids in size, contain an N- to C-cyclised backbone and incorporate three disulfide bondsarranged in a cystine knot motif. In this motif, an embeddedring in the structure formed by two disulfide bonds and theirconnecting backbone segments is penetrated by the thirddisulfide bond. The combination of this knotted and stronglybraced structure with a circular backbone renders thecyclotides impervious to enzymatic breakdown and makes themexceptionally stable. This article describes the discovery ofthe cyclotides in plants from the Rubiaceae and Violaceaefamilies, their chemical synthesis, folding, structuralcharacterisation, and biosynthetic origin. The cyclotides havea diverse range of biological applications, ranging fromuterotonic action, to anti-HIV and neurotensin antagonism.Certain plants from which they are derived have a history ofuses in native medicine, with activity being observed afteroral ingestion of a tea made from the plants. This suggeststhe possibility that the cyclotides may be orallybioavailable. They therefore have a range of potentialapplications as a stable peptide framework.     

The role of conserved Glu residue on cyclotide stability and activity: a structural and functional study of kalata B12, a naturally occurring Glu to Asp mutant

Cyclotides are a family of plant defense proteins with a unique cyclic backbone and cystine knot. Their remarkable stability under harsh thermal, enzymatic, and chemical conditions, combined with their range of bioactivities, including anti-HIV activity, underpins their potential as protein drug scaffolds. The vast majority of cyclotides possess a conserved glutamate residue in loop 1 of the sequence that is involved in a structurally important network of hydrogen bonds to an adjacent loop (loop 3). A single native cyclotide sequence, kalata B12, has been discovered that has an aspartic acid in this otherwise conserved position. Previous studies have determined that methylation of the glutamate or substitution with alanine abolishes the membrane disrupting activity that is characteristic of the family. To further understand the role of this conserved structural feature, we studied the folding, structure, stability, and activity of the natural aspartic acid variant kalata B12 and compared it to the prototypical cyclotide kalata B1, along with its glutamate to alanine or aspartate mutants. We show that the overall fold of kalata B12 is similar to the structure of other cyclotides, confirming that the cyclotide framework is robust and tolerant to substitution, although the structure appears to be more flexible than other cyclotides. Modification of the glutamate in kalata B1 or replacing the aspartate in kalata B12 with a glutamate reduces the efficiency of oxidative folding relative to the native peptides. The bioactivity of all modified glutamate cyclotides is abolished, suggesting an important functional role of this conserved residue. Overall, this study shows that the presence of a glutamic acid in loop 1 of the cyclotides improves stability and is essential for the membrane disrupting activity of cyclotides.     

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.     

Processing of a 22 kDa precursor protein to produce the circular protein tricyclon A

Cyclotides are a family of plant proteins that have the unusual combination of head-to-tail backbone cyclization and a cystine knot motif. They are exceptionally stable and show resistance to most chemical, physical, and enzymatic treatments. The structure of tricyclon A, a previously unreported cyclotide, is described here. In this structure, a loop that is disordered in other cyclotides forms a beta sheet that protrudes from the globular core. This study indicates that the cyclotide fold is amenable to the introduction of a range of structural elements without affecting the cystine knot core of the protein, which is essential for the stability of the cyclotides. Tricyclon A does not possess a hydrophobic patch, typical of other cyclotides, and has minimal hemolytic activity, making it suitable for pharmaceutical applications. The 22 kDa precursor protein of tricyclon A was identified and provides clues to the processing of these fascinating miniproteins.     

NMR as a tool for elucidating the structures of circular and knotted proteins

Cyclotides are a recently discovered family of mini-proteins that have a head-to-tail cyclised backbone stabilized by a knotted arrangement of three disulfide bonds. They have a wide range of biological activities, including uterotonic, anti-bacterial, anti-HIV, and anti-tumour activity but their insecticidal activities suggest that their natural function is in plant defense. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. This stability and resistance to proteolysis makes them a potentially valuable protein engineering tool at the interface of chemistry and biology: they have the structure of proteins but the stability and biophysical properties of organic molecules. In this review the role of NMR in defining the structures of cyclotides is described.     

Cyclotides,a versatile ultrastable micro-protein scaffold for biotechnological applications

Cyclotides are fascinating microproteins (≈30–40 residues long) with a unique head-to-tail cyclized backbone, stabilized by three disulfide bonds forming a cystine knot. This unique topology makes them exceptionally stable to chemical, thermal and biological degradation compared to other peptides of similar size. Cyclotides have been also found to be highly tolerant to sequence variability, aside from the conserved residues forming the cystine knot, able to cross cellular membranes and modulate intracellular protein–protein interactions both in vitro and in vivo. These properties make them ideal scaffolds for many biotechnological applications. This article provides and overview of the properties of cyclotides and their applications as molecular imaging agents and peptide-based therapeutics.     

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.     

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.