Chemistry and Biology of Cyclotides: Circular Plant Peptides Outside

Review pubs.acs.org/jnp

Chemistry and Biology of Cyclotides: Circular Plant Peptides Outside the Box Robert Burman, Sunithi Gunasekera, Adam A. Strömstedt, and Ulf Göransson* Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, Box 574, SE-751 23 Uppsala, Sweden

ABSTRACT: Cyclotides stand out as the largest family of circular proteins of plant origin hitherto known, with more than 280 sequences isolated at peptide level and many more predicted from gene sequences. Their unusual stability resulting from the signature cyclic cystine knot (CCK) motif has triggered a broad interest in these molecules for potential therapeutic and agricultural applications. Since the time of the first cyclotide discovery, our laboratory in Uppsala has been engaged in cyclotide discovery as well as the development of protocols to isolate and characterize these seamless peptides. We have also developed methods to chemically synthesize cyclotides by Fmoc-SPPS, which are useful in protein grafting applications. In this review, experience in cyclotide research over two decades and the recent literature related to their structures, synthesis, and folding as well the recent proof-of-concept findings on their use as “epitope” stabilizing scaffolds are summarized.

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INTRODUCTION Cyclotides are an exceptional family of gene-encoded plant proteins: their structure comprises a continuous circular backbone because their N- and C-termini are joined by a peptide bond. That circular chain of amino acid residues typically consists of approximately 30 amino acids, including six cysteine residues that form three disulfide bonds arranged in a cyclic cystine knot (CCK) motif (Figure 1).1 During the past few years, this family of plant proteins has attracted increasing interest, as demonstrated by the number of publications covering different aspects of the field (see, for example, refs 2−10). In the current report, the status of the field is reviewed with insights into specific aspects of the development of these compounds. The history of cyclotides may be traced back more than 50 years, to the beginning of the 1960s. At that time, the late Professor Finn Sandberg of our Department surveyed medicinal and toxic plants in the Central African Republic and the Republic of the Congo. Among the recorded species was Oldenlandia af f inis, which was used to facilitate childbirth.11 Although Sandberg’s survey did not ultimately aim for the isolation of the active principles of these medicinal plants, this is to the best of our knowledge the first record of a species and its use that would prove later to open up a whole new research field. It was a Norwegian, Dr. Lorents Gran, who isolated the first cyclotides some years later. During two missions as a Red Cross physician in the Democratic Republic of the Congo, Gran observed that women in labor used a © 2014 American Chemical Society and American Society of Pharmacognosy

decoction of Oldenlandia af f inis to accelerate delivery. Triggered by the powerful uterocontractive properties of the plant, he brought the plant home to the University of Oslo for investigation. He found ultimately that peptides were responsible for the strong activity shown. The main active component was termed kalata B1 after the indigenous name of the plant, “Kalata-kalata” (the “B1” is derived from the fact that the compound was the first one isolated from the chromatographic fraction B).12 However, the complete primary structure of the peptide could not be determined at the time.13,14 It took 25 years until kalata-peptides once again came into focus. The complete structure of kalata B1 was reported in 1995, including its circular backbone and knotted arrangement of disulfide bonds as determined by NMR analysis.15,16 At that time, three different groups had published independently four similar circular peptide sequences,17−19 and a drug discovery project aimed at peptides had just been initiated in our laboratory at Uppsala. Our project was partly inspired by the story of “Kalata-kalata”, which proved that plant peptides are valid targets for finding novel types of bioactive peptides. In addition, it was clear that plant peptides had been neglected in natural products research, especially in view of the increasing awareness of peptide signaling systems in plants and the Special Issue: Special Issue in Honor of Otto Sticher Received: December 16, 2013 Published: February 14, 2014 724

dx.doi.org/10.1021/np401055j | J. Nat. Prod. 2014, 77, 724−736

Journal of Natural Products

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Figure 1. Ribbon structure of a cyclotide. The structure shows the crystal structure of varv F (PDB-file 3E4H). Note the unique features of the CCK motif: a cyclic backbone with sequence loops (1−6) and three stabilizing disulfide bonds. These disulfides are arranged in a cystine knot, constituted by two disulfides that form a ring structure together with the backbone connecting the four cysteines (I−IV; II− V) and a third disulfide that threads through the ring (III−VI). The unique cyclotide structure forces hydrophobic residues to be exposed on the surface of the protein, thereby increasing its amphipathic properties.

development of plants and plant cells as protein expression systems. A protocol for the isolation of a highly purified polypeptide fraction from plant biomass was developed.20 In particular, this protocol was used for the isolation of the first suite of cyclotides from one species, and the results confirmed them as a family of peptides.5 Hence, we reconnected to Sandberg’s discoveries in Africa some decades earlier. The peptide family has grown quickly since then, and the collective term cyclotidesafter cyclo-peptideswas suggested by one of the leaders in the field, Professor David Craik, in 1999.1 Since then, our group has focused our efforts to understand the chemistry and biology of cyclotides and to explore their possible use for biotechnological, pharmaceutical, and agricultural applications.

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LOCKING IN THE STRUCTURE Already at an early stage it was clear to us that cyclotides fall into two main subfamilies.5 In a landmark paper, Craik and coworkers then defined them as the Möbius and bracelet subfamilies.1 These subfamilies are distinguished by the presence or absence, respectively, of a cis-Pro peptide bond. With an increasing number of cyclotides identified, the two subfamilies now seem to merge, and several “hybrids”, i.e., peptides containing features from both subfamilies, have now been reported. Bracelet and Möbius cyclotides differ in size and amino acid content, with bracelets being the larger and more structurally diverse subfamily of the two; to date according to Cybase (the database of cyclic proteins),21,22 approximately two-thirds of the known cyclotides belong to the bracelet subfamily. (Cybase can be accessed at http://www.cybase.org. au.) Figure 2 displays prototypic cyclotides. Apart from the six conserved cysteines, some residues are found in all or nearly all cyclotides: the Glu residue in loop 1 and the (Asn/Asp)-Gly pair in loop 6. The former has a key structural role, further restricting flexibility and condensing the overall structure

Figure 2. (A) Surface representation of prototypic cyclotides from each subfamily: the bracelet cycloviolacin O2 (cyO2) and the Möbius kalata B1. The backbone aligned models display the distribution of hydrophobic/hydrophilic regions on the protein surface and their amphipathic structure. Hydrophobic residues (Ala, Leu, Ile, Pro, Trp, Phe, Val) are in green, cationic (Arg, Lys) in blue, and anionic (Glu) in red. (B) Representative sequences of cyclotides from the two subfamilies and a circular trypsin inhibitor. Cys residues are highlighted in gray. (C) The sequences of the bracelet and Möbius subfamilies summarized as sequence logos, generated by Weblogo.131 The overall height of the stack indicates the sequence conservation at this position, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at this position.

through a network of hydrogen bonds with loop 3;23,24 the latter residues are involved in the post-translational ring closure.25 The remaining residues are interchangeable, and although there are relatively few amino acids in a cyclotide sequence, variations are immense. The current definition of a cyclotide is strictly based on the structural motif, but this definition has become blurred lately by the discovery of a few atypical variants. Until recently, only two 725



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Scheme 1. Schematic Overview of the Procedures To Detect Novel Cyclotides by Chemical Meansa

To first evaluate the cyclotide content of a plant, an aqueous extract is made and analyzed using liquid chromatography−mass spectrometry (LCMS). After isolation of pure cyclotides by HPLC, the Cys residues should be reduced and alkylated so that the sample can be digested enzymatically, yielding linear products that can be sequenced further by MS-MS. The sequence determination can be elusive, but in combination with amino acid analysis full sequence coverage is usually obtained. To obtain a complete 3-D structure, NMR spectroscopy or X-ray crystallography can be used. a

only a minority of species in this family and the distribution is concentrated within a few tribes.40,41 The Violaceae family includes 22 genera and approximately 930 species; these are predominantly tropical, growing as perennial herbs, shrubs, and trees or treelets.42,43 The Violaceae has so far proved to be the richest source of cyclotides, with these compounds having been found in all species investigated. Remarkably, these two primary cyclotide-bearing plant families are not closely related to one another. For this reason, it is likely that cyclotides may be found more widely in the plant kingdom. Outside the Violaceae, Rubiaceae, Solanaceae, and Fabaceae, cyclotide-like genes,44,45 sequences, and linear peptides46 have been identified in Poaceae, but no circular cyclotides have been detected among these plants. A chemical screen has also shown that members of the family Apocynaceae (closely related to Rubiaceae) contain small proteins with six Cys residues, which possibly could indicate the presence of cyclotides.40 Developing Methods for Cyclotide Detection and Isolation. The early methods for extracting and fractionating cyclotides from plant biomass were time- and resourceconsuming and developed to isolate polypeptides in general.20 Over time, knowledge has grown regarding the cyclotides, and new isolation methods have been developed incrementally, with much improved yields and more simplified procedures.7,47 For example, in our laboratory, the isolation procedure before final purification with reversed-phase HPLC has been trimmed down to include only three steps (extraction with 60% methanol in water, liquid−liquid extraction with dichloromethane, and reversed-phase solid-phase extraction, RP-SPE) instead of five more laborious steps in the original protocol.20 The current approach for detecting and characterizing novel cyclotides is illustrated in Scheme 1. To evaluate if a plant possibly contains cyclotides, a fast small-scale extraction using only milligrams of plant material is advised before going to a large extraction or for screening purposes.48 The extract directly or after RP-SPE can then be analyzed using an LC-MS system. Long retention times coupled with masses ranging from 2800 to 3500 strongly suggest further studies.40,48

linear variants of cyclotides had been isolated from cyclotideexpressing plants that clearly have the same genetic origin as “true” cyclotides, but they lack the amino acid residues that are needed for cyclization.26,27 At present, several more of such linear cyclotide-like sequences have been found, and these are now referred to as “acyclotides” or “uncyclotides”.28,29 Furthermore, among the many trypsin-inhibitory cystine knotted proteins that have been isolated from the cucurbit (Cucurbitaceae) plant family, a few have a circular backbone and, thus, fulfill the criteria for inclusion in the cyclotide family.30,31 However, they have more sequence similarity with their linear counterparts (i.e., linear cucurbit trypsin inhibitors) than with cyclotides in general, and they miss some key residues that are found in the common cyclotides, e.g., the structurally important Glu residue. As such, they are sometimes simply referred to as “cyclic knottins”.32 It is interesting to note that the disulfide connectivity was the subject of a long debate, as a consequence of the inherent difficulties of assigning disulfides in molecules with closely packed cysteines. The cystine knot structure of kalata B1 was first deduced by NMR spectroscopy,15,23,33 but has also been questioned by results obtained using this same technique.34 Nevertheless, today the cystine knot is undisputed and has been verified chemically by partial reduction of disulfides combined with stepwise alkylation35 and by X-ray crystallography.36

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CYCLOTIDES ARE ALL AROUND US Since the discovery of the first cyclotide in Oldenlandia af f inis, about 280 different cyclotides have been isolated from more than 40 plant species. Patterns in occurrence are beginning to be unraveled, but the vast majority of cyclotides have been discovered in the violet (Violaceae) and coffee (Rubiaceae) families. Recently, cyclotides have also been found in Clitoria ternatea of the pea (Fabaceae) family37,38 and Petunia x hybrida of the potato (Solanaceae) family.28,39 The distribution of cyclotides within the Fabaceae and Solanaceae at large is yet unknown. Although the Rubiaceae is a large family of plants, with 600 genera and over 13 000 species, cyclotides have been found in 726



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have been obtained (see, for example, ref 4 for a short outline of cyclotide structure determination by NMR and visit Cybase21,22 for updated lists of available cyclotide structures). It should be noted that NMR structures have also been derived for synthetic peptide fragments representative of certain segments within cyclotide precursors (Oak1 and Vok1),55 linear cyclotides,26 cyclotide folding intermediates,56,57 and a number of cyclotide derivates and mutants.24,56,58−60 Owing to the compact and highly structured fold, cyclotides display “textbook“ peptide NMR spectra with well-dispersed signals.23,47 Hence, solution NMR spectroscopy is the preferred choice for the determination of cyclotide structures. In comparison, only one structure has been obtained by X-ray crystallography. The cyclotide varv F was crystallized successfully (after extensive and long screenings for the crystallization conditions), and the structure was determined at a resolution of 1.8 Å. The X-ray structure confirmed the cystine knot, the cis-Pro bond characteristic for Möbius cyclotides, and the intricate network of hydrogen bonds that stabilizes the structure.36 NMR spectroscopy has also been used to study cyclotides because it allows their stability and flexibility to be monitored at different temperatures and pHs or in the presence of denaturants61 and to study cyclotide structure in membranemimicking environments.62,63 The latter is of significant interest given that their biological functions appear to involve membrane interactions. The tertiary structure during membrane interactions is maintained,62−64 and quaternary structures have been suggested to play a role in cyclotide activity.65,66 It appears clear that oligomeric structures are formed in solution,67,68 but it is doubtful if structurally ordered oligomers have a functional role.

Using this approach, up to 50 different cyclotides have been detected from a single plant species.49 Individual peptides can usually be isolated using preparative RP-HPLC, but the process is often challenging because of the complex mix of highly homologous cyclotides. Several ways have been used to solve this separation problem, including using tetraethylenepentamine and formic acid instead of trifluoroacetic acid-based gradients.48,49 Furthermore, charge50 and aromaticity5 have been used to separate cyclotides into groups before final purification. In some cases, the choice of starting material is crucial for the success of the purification. For example, kalata B1 and varv A, differing in only one amino acid (Thr and Ser, respectively), are coexpressed in Viola odorata21 and are practically impossible to separate from one another. However, varv A is expressed without any interfering kalata B1 in Viola tricolor, and the best source of kalata B1 is still the first plant from which it was discovered, Oldenlandia af f inis. Characterization of Primary to Quaternary Structures. The CCK motif is intrinsically problematic for peptide sequencing, as conventional methods require a single linear peptide chain for sequencing. Hence, the disulfide bonds must be broken and the backbone must be made linear to determine the primary structure of cyclotides. This is achieved by using a reducing agent [e.g., dithiothreitol or tris(2-carboxyethyl)phosphine] in combination with enzymatic digestion. In most cases, the free thiols are protected (e.g., by iodoacetamide, Nethylmaleimide, or bromoethylamine) to inhibit reoxidation of cysteines.35,51 The reduction and alkylation of cysteines has also been exploited as an indication of the presence of three disulfide bonds.40 Conveniently, the conserved Glu residue in loop 1 can be exploited for enzymatic cleavage (using endoproteinase GluC) to yield a single linear chain for most cyclotides. Following cleavage, the first indication of a cyclic backbone is given by a mass increase of 18 Da following cleavage, i.e., the uptake of water. This procedure can be used successfully to monitor cyclotide content in mixtures and more or less crude plant extracts. The first cyclotide sequences originated from Edman sequencing. Today, MS-MS has become the main tool.51−54 However, despite the current development in MS technology, it is still less than straightforward to determine the full sequence from one single 30 amino acid long fragment. Shorter, more readily interpretable, peptides may be obtained using other enzymes (e.g., trypsin or chymotrypsin), alone or in combination. Either method needs to be confirmed by independent data, such as quantitative amino acid analysis or genetic information. There is an inherent problem in distinguishing between isobaric residues (with the same mass), e.g., Leu and Ile, using MS-MS. Use of chymotrypsin can sometimes solve the problem since it cleaves sequences after Leu and not Ile, but if Leu is followed by a Pro, cleavage is impossible and hence the results are difficult to interpret. It may then be hard to solve the complete sequence without support from Edman sequencing or sequence information from DNA/ RNA analyses. Currently, new developments in nucleic acid sequencing are proving advantageous also in cyclotide research. Sequencing total RNA has now become among the first steps before entering into any peptide discovery project, and we foresee that this will significantly change the way we work and enhance the number of cyclotides that will be discovered. As mentioned above, the first three-dimensional structure of a cyclotide (kalata B1) was determined in the mid-1990s,15 but since then specific details on the structures of many cyclotides

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ACTIVITY OF CYCLOTIDES Kalata B1 and the few cyclotides reported in the early 1990s all resulted from bioassay-guided isolation procedures. These early reports included the hemolytic violapeptide I,19 the neurotensin-binding inhibitor cyclopsychotride A,17 and circulins A and B, with anti-HIV properties.18 Subsequently, cyclotides have shown activity in the low micromolar range against a wide range of organisms such as insect larvae,25 snails,69 nematode parasites in sheep,70 hookworms,71 and larvae of barnacles.50 During the early years of cyclotide research, Tam and coworkers reported that synthetic cyclotides from both subfamilies displayed activity against both Gram-positive and Gram-negative bacteria as well as against Candida spp.72 Although all cyclotides in this study were active against Staphylococcus aureus, the spectrum of cyclotide antimicrobial activity has been shown to be more restricted in our hands.73 For example, the bracelet cycloviolacin O2 showed no activity on Staphylococcus, but was active against several Gram-negative bacteria at the low micromolar range, and the Möbius cyclotides tested showed little antibacterial potency. However, both cycloviolacin O2 and Möbius cyclotides (kalata B1 and B2) have shown growth-impairing qualities on heterogeneous soil bacteria samples as well as on green algae and plant roots.74 This broad activity against organism types that are associated with infection and grazing of plants, and resource competition supports the hypothesis that they are components of the plant defense system. Today, one of the main research areas in our laboratory is to evaluate the antimicrobial effects of cyclotides. Our group has for a long time used a human-cell-based cytotoxicity assay for bioactivity screening of cyclotides. The 727



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Figure 3. Proposed lytic mechanism of the cyclotide cyO2 on a PE/PG membrane. (1) Cyclotide adsorption driven by hydrophobic patch (green), cationic residues (blue), and PE affinity, results in demixing of PE and cyclotide (by PE affinity and negative curvature contribution). (2) Depletion of PE lipids from the membrane by micellization results in membrane thinning and increased positive curvature stress. (3) When a higher local cyclotide concentration threshold is reached, this forces membrane perforations to occur, resulting in leakage.

Several models of membrane-disrupting mechanisms have been suggested for lytic peptides, including specific pore formations and more general curvature stress generated perforations. A peptide multimer formation with a defined pore diameter has been claimed as the membrane-disrupting mechanism of kalata B1 based on stepwise current increase in a patch clamp experiment.66 However, due to lack of supporting data, combined with a report showing that kalata B1 does not readily self-associate,67 conclusive evidence for such a specific pore formation has yet to be produced. The membrane composition is of special importance for the lytic activity of cyclotides. Some selectivity in cytotoxicity against leukemic lymphocytes rather than normal lymphocytes has been shown for cycloviolacin O2.75 Tumor cell membranes have a higher negative charge density with 3−7 times more phosphatidylserine, which is to a large extent located on the outer leaflet of the membrane.82 In addition, a decrease in cholesterol content can be expected to some degree for many tumor cell lines;83 both characteristics are likely consequences of rapid cell division. Curiously, it has been reported that cholesterol content significantly increases lytic potency for equivalent bulk concentrations of kalata B1 on model membranes.66 This is unprecedented in the field of membrane lytic peptides and also contrary to our experience with the same peptide.84 It should be noted that the area of the target membrane affects the amount of peptide needed to reach the adsorption threshold required for lysis. Hence, the peptide-tolipid ratio dictates the activity in membrane leakage assays of molecules with high adsorption isotherms such as cyclotides and not the total concentration. Therefore, it is difficult to compare selectivity of lytic potential between different cell types and microbes with certainty, and even between model membrane systems, if care is not taken to ensure equivalent target areas.85 Cyclotides belong to a small group of peptides having affinity for phosphatidylethanolamine (PE) membrane lipids, which is a conserved feature among both subfamilies of cyclotides.86 Although this affinity has been known for some time for Möbius cyclotides,87,88 the importance of this affinity for their lytic activity and its structural key elements was revealed only recently.84,88 The bracelet cyclotide cycloviolacin O2 was shown to preferentially disrupt PE-containing membranes rather than those with phosphatidylcholine (PC), with this activity dependent on the conserved Glu residue.84 The underlying mechanism for this selective lytic activity could be ascribed to a higher adsorption on PE-containing membranes due to structural affinity,88 and a subsequent specific extraction of PE lipids. This results in thinning of the membrane, which in turn makes it permeable and sensitive to peptide-induced

compounds varv A, varv F, and cycloviolacin O2 showed potent activity in a panel of 10 different human tumor cell lines at low correlations with the activity patterns varying from anticancer drugs in clinical use today,75 suggesting a mode of action different from that of known anticancer drugs. Additionally, a comparison was made between the cytotoxic activity of varv A and cycloviolacin O2 against tumor cells from patients and normal lymphocytes from healthy human subjects. In this study, the cyclotides showed an almost 10-fold selective toxicity against leukemia cells.75 Subsequently, it was shown that the cytotoxic effect on tumor cell lines can be distinguished morphologically within a few minutes.76 Such a rapid process is indicative of necrosis, while apoptosis requires activation of several intracellular cascades as well as gene expression resulting in longer onset periods. Both the rapid kinetics and the broad spectrum of the toxic effect produced by many cyclotides point toward membrane disruption as responsible for activity.76 Cyclotides Interact with Cellular Membranes. Most biological activities assigned to cyclotides concern toxicity on a broad range of organisms, including mammalian tumor cells,75 nematodes,77 insect epithelial cells,78 bacteria,72,73 fungi,72 and enveloped viruses.18 One thing these organisms have in common is their phospholipid membrane: the loss of membrane integrity inevitably leads to death of whatever life form is encased by it. In order for cyclotides to exert their membrane-disrupting activity, they must have a high affinity for the target membrane. The pronounced amphiphilic and frequently cationic (in the bracelet subfamily) nature of cyclotides is shared by most antimicrobial peptides. These qualities confer the hydrophobic and electrostatic forces that drive adsorption of cyclotides toward the lipid core and anionic surface of the membranes.79 Other characteristics that promote membrane adsorption include tryptophan affinity for the glyceryl/carbonyl region of the phospholipids,80 which anchor peptides at the membrane interfaces, and membrane-induced formation of intramolecular hydrogen bonds that are stabilized by the hydrophobic environment of the membrane.79,81 Although cyclotides have the possibility of benefiting from both these characteristics, for example, the semiconserved tryptophan next to the cis-Pro among the Möbius cyclotides, their exceptionally rigid structure cannot be expected to undergo much further hydrogen bonding as a consequence of membrane adsorption, with the possible exception of the loops of the hydrophobic patch in certain circumstances. However, the same rigid structure not only improves enzymatic stability but also forces hydrophobic residues to the surface of the molecule, thereby improving their amphiphilic properties. From these aspects, cyclotides appear as ideal antimicrobial peptide structures. 728



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Figure 4. Biosynthesis and structure of cyclotides. Cyclotides are synthesized as precursor proteins, with a conserved endoplasmic reticulum (ER) signal region, a pro-region, an N-terminal repeat (NTR) signal, the mature cyclotide sequence, and a short C-terminal tail. The NTR and cyclotide region can be repeated up to three times in different precursors, encoding different or identical cyclotides. The arrow below the ER signal indicates a highly conserved region that has been used as a target for a degenerative primer encoding for the sequence AAFALPA.

leakage. To our knowledge, cyclotides are the first peptides that have been demonstrated to display PE-selective lytic activity and to mediate selective extraction of phospholipids from a membrane.84 Cyclotide affinity for PE-containing membranes could be linked subsequently with activity of biological membranes on erythrocytes, bacteria, and enveloped viruses.86 Selectivity between the human membrane model PC/ cholesterol (60:40) and E. coli model membranes [PE/ phosphatidylglycine (PG)/diphosphatidylglycine (dPG), 67:23:10] was 100-fold in lytic activity for cycloviolacin O2, predominately due to PE selectivity, but also by a combination of electrostatic attraction to the anionic phospholipids and the lack of cholesterol of the bacterial membrane.84 The results indicated that when a cyclotide binds to the membrane, it associates with PE lipids, promoted by both specific affinity and by favorable packing parameters associated with peptides that expand the lipid headgroup region. The high local concentration of cyclotides and the lipid demixing that follows result in membrane thinning and curvature stress, which ultimately results in perforations, possibly by toroidal pores or fissures lined by cyclotides (Figure 3). Structure−Activity Relationships. Naturally occurring cyclotides vary in size between 28 and 37 amino acid residues and have a net charge between −2 and +3. Cyclotides with a positive net charge have been found generally to be more membrane active than those that are neutral or negatively charged.49,75,89,90 This may be seen as a natural observation since biomembranes normally carry a net negative charge of various densities. The importance of this electrostatic attraction is seen clearly for the cationic residues in the bracelet family, as exemplified by experiments where successive charge neutralization on cycloviolacin O2 may be correlated with declines in tumor cell cytotoxicity.91 Furthermore, Ala mutated variants of Glu in kalata B1 have been shown to be inactive in hemolytic and insecticidal assays.65 Structural studies reveal that the carboxylic acid of Glu is oriented toward the interior of the peptide and is responsible for maintaining structure rather than peptide surface electrostatics.24 In the bracelet family, the Glu side chain forms hydrogen bonds with the backbone structure of loop 3, a hydrophobic sequence forming a helical structure that otherwise would be distorted. As such, the Glu is important for membrane adsorption and possibly also for aggregation and specific interactions, but via loop 3. The interactions of the Glu are present also in the Möbius subfamily. However, in this case loop 3 is hydrophilic and not directly involved with membrane adsorption. Substitution of any residue in the highly conserved three residues of loop 1 (including the conserved Glu) with Ala

abolished the lytic activity of kalata B1, as did mutations of hydrophobic residues in loop 5.66 Most cyclotides carry an aromatic residue (usually tryptophan): bracelets in loop 2 and Möbius in loop 5. In terms of membrane adsorption this confers a membrane interface anchoring residue, which is reasonable since both these loops are hydrophobic and would be expected to constitute the patch directly associated with the membrane on adsorption of the respective subfamily. When summarizing the activities of native cyclotides, the conclusion is that in order to correlate the structures of cyclotides with their potency and to understand their mechanisms of action, it is necessary to consider their structure as a whole rather than focusing specifically on single residues. So far, no systematic study has been performed to monitor these whole-molecule SARs (or QSARs), but a tool for developing this is being investigated in our laboratory. So will the cell membrane and phosphatidylethanolamine prove to be the final molecular target of cyclotides? Although membrane disruption may explain most biological and pharmacological activities, the current model does not explain all effects of cyclotides. For example, the seemingly specific neurotensin-binding of cyclopsychotride A may be explained by cyclotide binding to the lipids present in the receptor/ membrane preparation. However, it is difficult, if not impossible, to explain the reversible paralysis of barnacle larvae in the antifouling assay.50 Indeed, our group was able recently to show the first functional indication of a secondary target other than the membrane/membrane disruption, namely, DNA-damaging effect at a concentration 100 times lower than the lytic concentration.92 This is intriguing because at the middle concentration (i.e., 10 times lower than the lytic concentration) peptides showed neither lytic nor DNAdamaging effects. In addition, it has been shown that kalata B1 and MCoTI-I/II do cross the cell membrane, at subtoxic concentrations.93−95 More recently, cyclotide activity came full circle when Gruber and co-workers demonstrated that a cyclotide (kalata B7) indeed does bind to the oxytocic receptor.96 As such, even though there is now a clear hypothesis for how cyclotides affect the membrane, we are only at the beginning of understanding the whole spectrum of cyclotide function and activity.

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CYCLOTIDE PRODUCTION: IN PLANTS AND LABORATORIES Cyclotides comprise one of the few classes of natural macrocyclic gene products discovered to date.97 Analysis of cyclotide precursor sequences obtained from cDNA have 729



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cyclotide chemistry in the laboratory, solid-phase peptide synthesis (SPPS) is still unrivaled. Solid-Phase Peptide Synthesis. Intriguingly, cyclotides are amenable to chemical synthesis despite their knotted and circular nature. Deriving a mature cyclotide from the building block amino acids involves the synthesis of the linear cyclotide precursor, cyclization of its N- and C-termini, and formation of the native disulfides to achieve the correct cystine knot topology. Currently, the linear cyclotide precursors are synthesized chemically by standard solid-phase peptide synthesis based on either tert-butoxycarbonyl (Boc) or 9H-fluoren9-ylmethoxycarbonyl (Fmoc) chemistry. For a long time, native as well as grafted cyclotides have been synthesized predominantly by Boc-SPPS.113−115 However, in recent years there has been a greater inclination to develop Fmoc-SPPS-based protocols that do not require handling of potentially hazardous chemicals (e.g., TFA for deprotection and the use of HF for final cleavage as in Boc-SPPS). Once the linear peptide is obtained, the next step is to ligate its N- and C-termini to obtain the cyclized peptide. Most commonly, this is achieved using a C-terminal thioester and an N-terminal Cys via native chemical ligation,116 which remains the most robust synthesis approach to date.113,114,117−119 Peptide thioesters can be assembled readily on resin using Boc-SPPS, because the thioester group withstands the acidic deprotection step used. On the contrary, it is challenging to synthesize thioester peptides by Fmoc-SPPS, because thioesters are intrinsically unstable also to the weak base that is used for deprotection (e.g., piperidine). The simplest way to overcome this problem is by generating/activating the C-terminal thioester group once the peptide exits the Fmoc synthesis route, but peptides attached to the resin via a C-terminal sulfonamide linker can be activated to generate a C-terminal thioester. This approach has been used in cyclotide synthesis.120 Similarly, a C-terminally attached thioester group can be generated in solution on a protected peptide that is cleaved off from the resin.118 In both cases, cyclization via native chemical ligation is ultimately achieved as the requirements of C-terminal thioester and a N-terminal cysteine are met. Very recently, our group has adapted the N-acylureamediated approach for native chemical ligation121 for microwave-assisted SPPS for cyclotide syntheses.122 The use of microwave irradiation enhances efficiency and purity of the synthetic peptides. With this method, a linear peptide is assembled on a C-terminal diaminobenzoic acid linker, which upon activation and acylation leads to an N-acylurea/Nbenzymidazolinone (Nbz) moiety. In thiol-rich environments, the Nbz-peptide undergoes in situ thioesterification and native chemical ligation to give a cyclized peptide by native chemical ligation. The manual cyclotide synthesis protocols have also been improved with the assistance of microwaves and automation to accelerate the speed of peptide synthesis during the amino acid coupling and thioesterification steps.115,122 Presumably, thioesterification of the linear peptide leads to a vigorous “thia-zip” reaction in which the cyclic peptide backbone is zipped from the C- to the N-terminal in a series of thiol and thiolactone exchanges between the carbonyl group of the C-terminal thioester and the intertwining Cys side chains. Ultimately, the head-to-tail cyclized peptide is derived by a spontaneous S,N acyl-migration from the terminal Cys side chain to the peptide backbone. The characteristic feature of this thioester-mediated strategy is that cyclization of the peptide chain occurs prior to

shown that the genes encoding them consist of an endoplasmic reticulum (ER) signal domain, a pro-region, and one to three mature cyclotide domains, each preceded by an N-terminal repeat (NTR) sequence.25,48,98,99 Figure 4 shows the cleavage points of a schematic cyclotide after a Lys/Gly/Asn residue in the NTR sequence and the Asn or Asp in the cyclotide domain. Details of the processing of the precursors, involving oxidative folding, excision of the mature cyclotide sequence, and head-totail cyclization, are not fully understood nor is the order of the events concerned. However, an asparaginyl-endoproteinase (AEP) has been suggested to be involved in the cleavage of the C-terminal tail and simultaneous cyclization of the cyclotide domain, at least for the prototypic cyclotide kalata B1.100−102 Additionally, a protein-disulfide isomerase seems to play a major role in the oxidative folding of cyclotides through reshuffling (isomerization) of disulfide bonds.103,104 Current insights into cyclotide biosynthesis have been reviewed recently,6 but some of the points that need to be addressed are highlighted here. None of the functional studies in planta have used plants that express cyclotides; only the common model plants (Nicotiana benthamiana, N. tabacum, and/or Arabidopsis thaliana) have been used. Indeed, expression of the cyclotide precursor protein in these plants results in a cyclic, correctly folded peptide, but in low yields. Accumulating evidence suggests that cyclotide biosynthesis shares common features with both of the smaller circular trypsin inhibitory peptides from sunflower (e.g., SFTI-1) and the cyclic cystine knotted trypsin inhibitory peptides from the family Cucurbitaceae (e.g., McoTI-I/II). This involves cyclization by hijacking existing AEPs,31,105 but conclusive evidence is still lacking for plant species expressing cyclotides. As such, until definite proof emerges showing that AEP activity is the common denominator for the biosynthesis of cyclotides, our opinion is that one should not limit the search to AEPs only. This is of particular importance in light of the recent discovery of the first specific peptide cyclase,106 which has been found in Saponaria vaccaria (Caryophyllaceae). Although the substrate in this case is a shorter precursor and the products are cyclic peptides of only five to eight amino acid residues, this study demonstrates clearly that one should not rule out the occurrence of specific enzymes also for the biosynthesis of cyclotides. A more detailed understanding of the biosynthesis is of fundamental scientific interest, but it may also pave the road toward more efficient systems for the screening and production of cyclotides.107 Camarero and co-workers have produced successfully folded (cucurbit cyclotide) MCoTI-II, using Escherichia coli,108 and intein fusion protein, which generates cyclic and folded kalata B1 in a one-pot reaction after purification of the fusion protein.109 Very recently, peptide ligands specific for the VEGF-A binding site on neuropilin-1 were identified by screening a ∼6 × 109-membered E. coli display library of disulfide-rich peptides derived from the cyclotide kalata B1.110 However, these peptides were neither cyclic nor folded, but the study shows proof of concept of a technique to find cyclotide-like binders that then can be developed further. Still, there is no example of the production of a non-natural cyclotide in planta. Cell suspension cultures of Oldenlandia af f inis have been established, expressing yields of up to 0.7 mg of kalata B1 per liter of medium,111 but plant cell cultures have yet to be proven as a viable alternative for the production of bioactive cyclotide mutants.112 To access 730



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oxidation of the Cys residues. In fact, cyclization of the two termini of the cyclotide precursor appears to have a dramatic effect on folding, by enhancing the propensity for the correct folding.117 The cyclized peptide then attains its native disulfide connectivity under basic (∼pH 8) folding conditions. However, obtaining the correct native cystine knot topology of cyclotides is not always straightforward. This problem can be tackled already at the peptide synthesis stage by selectively protecting side chains of the Cys residues using chemically distinct protecting groups.32,72,114 This allows the disulfides in the peptide to be formed in two consecutive steps, with disulfides CysI−CysIV and CysIII−CysVI formed first and CysII−CysV formed later, thus limiting the number of misfolded disulfide isomers. A synthetic approach that has only rarely been used is Cys oxidization prior to cyclization of the peptide backbone, as exemplified in the early kalata B1 synthesis attempts.117 In this instance, folding of the peptide chain into the native cystine knot topology preorganizes the N- and C-termini in close proximity. However, the use of this strategy has been limited because specific achiral residues are required at the termini of the linear chain to prevent racemization and also due to the possibility of side reactions at the Lys and Asp residues during cyclization.117 The different syntheses strategies are outlined in Figure 5. No general sequence-dependent synthesis problems have been reported for cyclotides, with the exception being the cyclic cucurbit trypsin inhibitor MCoTI-II that contains an Asp-Gly motif.118,120 This motif is prone to aspartimide formation, which results from a ring-closure between the β-carboxy side chain of Asp and the nitrogen of the α-carboxamide and is dependent on the total exposure time of the growing peptide chain to the piperidine used in Fmoc-SPPS and by extensive folding times. However, MCoTI-II has been successfully synthesized by suppressing aspartimide formation, by the introduction of the Asp-Gly dipeptide with the Gly backbone nitrogen protected by 2-hydroxy-4-methoxybenzyl or 2,4dimethoxybenzyl protecting groups.118 A chemoenzymatic biomimetic strategy has also been utilized in the synthesis of MCoTI-I and MCoTI-II analogues.120 Taking advantage of the linear cyclotide backbone acid bearing P1 residue at the C-terminus, which is a viable substrate for protease-mediated ligation, the synthesis of the cyclic backbone without the need for a C-terminal thioester has been achieved. In this approach, the open chain MCoTI analogues were first refolded to the cystine knot topology and subsequently cyclized using polymer-supported proteases. The method has, however, not been used widely, but it is not unlikely that AEPs or even specific cyclization enzymes might find use in future cyclotide syntheses. Tying the Knot. Folding refers to the oxidative formation of the native cystine knot and establishment of the native threedimensional molecular topology. This appears to be highly dependent on the inherent propensity of each cyclotide to adopt the cystine knot. Understanding cyclotide folding is of fundamental interest as well as important from an applications perspective, as cyclotides are potentially valuable scaffolds in drug design. Insights have been obtained into the in vitro cyclotide folding pathways by folding experiments, although the mechanisms of in vivo cyclotide synthesis and folding are only beginning to be unraveled.100,101 A single cyclotide precursor with six Cys residues can adopt potentially 75 different disulfide con-

Figure 5. Schematic illustration of different cyclotide synthesis strategies. (A) Thioester-mediated Boc-SPPS strategy. (B) Boc-SPPS without thioesterification. (C) Boc-SPPS using selective disulfide protection. (D) Thioester-mediated Fmoc-SPPS folding.

formations (15 one-disulfide, 45 two-disulfide, and 15 threedisulfide species), and it is fascinating that a cyclotide precursor navigates its way through the correct folding pathway into the final native fold under optimized folding conditions. In general, it appears that folding of Möbius and MCoTI cyclic knottins are easier when compared to bracelet cyclotides. Both Möbius and MCoTI fold conveniently into the native conformation in buffers maintained at pH ∼8. The yield may in some cases be improved by adding cosolvents (e.g., 2propanol) to increase hydrophobicity in the buffers.123 For kalata B1, it appears that 2-propanol is needed to stabilize the distinct hydrophobic patch of residues that becomes exposed to the surface upon folding. Importantly, in the absence of 2propanol, kalata B1 folds at a slower rate, leaving the flexible disulfide intermediates susceptible to deamidation at the sensitive Asn-Gly.123 2-Propanol is not required for MCoTIII folding, presumably because the native form has more hydrophilic amino acids exposed at its surface. The rate and yield are heavily influenced by the redox potential of the buffer, which may be controlled by the addition of glutathione (reduced and oxidized). Redox potential is also influenced by “external” factors, e.g., the amount of air (oxygen) dissolved in the solution or in contact with the solution. As such, it is good practice to degas folding buffers, seal reaction tubes with argon or nitrogen, and use freshly prepared solutions of glutathione. 731



Review

Figure 6. Overview of oxidative folding pathways of cyclotides. Fully reduced cyclotides are folded via the main folding intermediates (containing two or three disulfide bonds, 2SS or 3SS) to the native structure. Kalata B1, a representative member of the Möbius subfamily, has a native-like main folding intermediate [2SS(IIa)], which is not a direct precursor to the native form, during its oxidative folding. A twodisulfide intermediate (2SS-IIb), present during kalata B1 unfolding, has been suggested as the possible direct precursor during kalata B1 oxidative refolding. Cycloviolacin O2, representing the bracelet subfamily, appears to fold via 3SS intermediates with non-native disulfide bonds. The cyclic knottin MCoTI-II also has a native-like 2SS folding intermediate, but this is in fact the direct precursor to the native form.

reduced form rather than to the native form, although it has two native disulfide bonds (CysI−CysIV and CysIII−CysVI). Bracelet cyclotides appear more problematic with respect to their in vitro folding. They do not simply fold under the optimized conditions for Möbius or MCoTI and generally give a range of misfolded products during their folding. However, they have been successfully folded, by selective Cys protection during synthesis, as previously mentioned. Furthermore, our group has established optimized in vitro folding conditions for the prototypical bracelet cycloviolacin O2, which included the presence of detergents and folding at lower temperatures.123 That cyclotide, which is the only bracelet cyclotide that has been studied in any detail, shows a major misfolded intermediate with three non-native disulfides. The native form requires a major conformational change, but also breaking of the disulfide bonds to form the native form. It is alleged that both detergents and cosolvents act as stabilizers for the native cyclotide conformation by interacting with the hydrophobic residues that are exposed to the surface upon folding. Despite the conserved cystine knot, why are the folding pathways of these proteins so different? For Möbius cyclotides and the cucurbit cyclotides (McoTI-I/II), there are differences in size and sequence of individual loops. Mainly the size of the cystine knot, which is larger in MCoTI-II, appears to be responsible for the differences in the folding pathways. Despite similar structures of the backbones and overall sizes of the molecules, the degree and the location of the hydrophobic and charged resides are different in Möbius and bracelet cyclotides. Mainly residues in loops 5 and 6 form the hydrophobic patch in kalata B1, but the corresponding loops are more hydrophilic and charged in the bracelet cyclotide cycloviolacin O2.24 Furthermore, a large hydrophobic patch involving loop 2 and an α-helix in loop 3 is present in cycloviolacin O2. Interestingly, the corresponding part is hydrophilic in kalata B1.23 As a result of these structural differences, it is suggested that cycloviolacin O2 requires not only a hydrophobic cosolvent but also detergents to stabilize the more amphipathic character of its native form. Thus, specific structural characteristics of individual cyclotides appear to dictate their folding behavior. Although some details can start to be revealed and some attempts to systematic studies have been done,123,124 it still cannot be predicted if and how cyclotides will fold or not.

intermediate, des(CysI-CysIV) or 2SS(IIa), is a kinetic trap present during the Möbius oxidative folding.56 This intermediate is not the direct precursor to the native form and does not readily refold into the native conformation, unless with the assistance of glutathione, which facilitates disulfide reshuffling. In MCoTI-II oxidative folding, a similar major two-disulfide intermediate with a native-like structure is present, which, unlike kalata B1, easily converts to the native form and hence the direct precursor to the native form. Another two-disulfide intermediate, des(CysII-CysV) or 2SS(IIb), is present during kalata B1 unfolding. This intermediate has been suggested as the possible direct precursor during oxidative refolding. Structural analysis indicates the disulfide CysII−CysV is the most surface-exposed disulfide in kalata B1 and the easiest bond to be broken, indicating that 2SS(IIb) could be the most likely candidate to be the direct precursor to the native form. It is interesting to note that this intermediate does not have a native-like structure, as it elutes on RP-HPLC very close to the

A CIRCLE OUTSIDE THE BOX So can the fundamental knowledge of these cyclic peptides be translated into applied sciences? For drug design, as well as in the field of natural product chemistry, peptides and proteins are sometimes considered as rather fragile biomolecules that are easily degraded by chemical and biotic factors. Currently, this view is changing. Clearly, the circular backbone and the cystine knot confer an extreme structural stability that lacks parallels in other protein families. The main natural role of this structure appears to be in plant defense, most notably against insect pests, but cyclotides do have the potential to be exploited in many ways in both agricultural and pharmaceutical applications (Figure 7). With the rising number of hypervariable cyclotide sequences and supporting evidence for their ultrastable scaffold, cyclotides have been implicated as ideal molecular templates for the design of pharmaceutical and agricultural agents. As a first line of evidence for the flexibility of the cyclotide template, Daly et al. designed acylic permutants of kalata B1, to prove that the cyclotide backbone could be opened in four of the backbone

In addition to these obvious differences in the folding buffers, there are also fundamental differences in the major folding intermediates between different cyclotide subfamilies, as depicted in Figure 6. A major native-like two-disulfide

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Figure 7. Overview of potential pharmaceutical and agricultural applications of cyclotides. In agricultural applications, cyclotide gene sequences could be expressed in crop plants to enhance resistance to pests, while in pharmaceutical applications biologically active peptide epitopes could be grafted into the CCK framework of natural cyclotides. A link between pharmaceutical and agricultural applications is indicated with an arrow; pharmaceutically modified cyclotides might, in the future, be produced in plants or plant cell cultures, via transformation with genes encoding modified cyclotides. Some of these applications have been claimed in patents.132

loops, without disrupting the overall native fold.59 Soon it became evident that specific residues in the backbone loops of cyclotides belonging to distinct subfamilies could be translocated without altering the native cyclotide fold, and more recently complete backbone loops across subfamilies were exchanged.60,124 Along similar lines, each noncysteine residue in kalata B1 could be exchanged for an alanine and the native-like cyclotide conformation could be maintained in the resulting alanine mutants.65 Interestingly, the undesirable hemolytic activity of the parent cyclotide scaffold could be removed with these single alanine mutations, suggesting that cyclotides hold great promise in terms of playing the role of a molecular scaffold with potential therapeutic value. The term “epitope grafting” was coined to describe the grafting of a completely novel/foreign bioactive epitope onto the cyclotide backbone loops, and once confined within a cyclic backbone, the linear epitopes may be harbored from exoproteases.125 In an ideal situation, the receptor binding conformation of the epitope will still be maintained on the cyclotide scaffold and the epitope will be sufficiently flexible to interact with the putative receptors, but not too flexible to be penetrated by endoproteases. Thus, a finite balance between flexibility and rigidity of an epitope is desirable to ensure that the epitope is not locked into an inactive conformation. In the first proof-of-concept study, epitopes that antagonize the VEGF-A receptor were incorporated into the Möbius kalata B1. These mutants target the therapeutic area of antiangiogenesis, which has relevance in anticancer treatment.125 It was possible to design an engineered cyclotide that is stable in serum, with activity in the low micromolar region. With information on the ability of cyclotides to enter cells,93−95 it is speculated that engineered cyclotides may be valuable in a therapeutic context against both extracellular and intracellular targets. In the past few years, a number of other studies have emerged that followed this concept of cyclotide grafting: MCoTI-II has been engineered into human leukocyte elastase and human mast cell tryptase beta-based inhibitors;126,127 a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity has been designed based on the knottin cyclotide MCoTI-I;128 and kalata B1

cyclotide has been engineered into melanocortin agonists.129 Recently, Tam and co-workers showed that grafting exploiting the cyclotide scaffold also confers other advantages than mere stability: kalata B1 grafted with an epitope antagonistic against the bradykinin receptor was found to be active orally in an inflammatory pain assay.10 To the best of our knowledge, these published reports represent only a fraction of all grafting efforts undertaken by different research groups over the years. The challenges with grafting lie not in the “synthesis component”, but rather in the “folding phase” when a native-like engineered cyclotide conformation is desired. (Notably, most grafting studies have been done using McoTIs and kalata B1 as scaffolds because of their relatively easy folding.) The challenges of folding also increase when a completely foreign epitope is used, which often is very different from the original sequence. Thus, many grafting attempts end up with misfolded or non-natively folded cyclotide isomers, depending on the nature of the epitopes and the tolerance of the cyclotide framework. However, is it necessary to limit the search for bioactive substances to correctly (natively) folded peptides? The answer appears to be no. For example, the recent development of a cyclotide-based bacterial display system led to the discovery of potent neuropilin antagonists, but even the most active peptide resulted in non-native cyclotide conformations.110 In fact, cyclization appears to assist the stability of the engineered analogues, as they were resistant to endopeptidases and had improved binding to neuropilin compared to control linear peptides. Most of the current grafting studies have only reached the point of biochemically characterizing the grafted cyclotides and determining their serum stability in vitro. However, to progress in the drug design pipeline, pharmacokinetic and pharmacodynamic studies of cyclotides are essential. Currently, studies of these aspects are lagging behind. How stable are cyclotides in blood? How fast do the kidneys excrete them? And is it possible that cyclotides, native or engineered, are immunogenic? These are the critical questions that need to be addressed if cyclotidebased therapeutic agents are to be realized. Notably, cyclotides and other naturally occurring cyclic peptides have also inspired the use of the concept of cyclization 733



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(5) Göransson, U.; Luijendijk, T.; Johansson, S.; Bohlin, L.; Claeson, P. J. Nat. Prod. 1999, 62, 283−286. (6) Craik, D. J.; Malik, U. Curr. Opin. Chem. Biol. 2013, 17, 546−554. (7) Göransson, U.; Svangård, E.; Claeson, P.; Bohlin, L. Curr. Protein Pept. Sci. 2004, 5, 317−329. (8) Craik, D. J.; Swedberg, J. E.; Mylne, J. S.; Cemazar, M. Expert Opin. Drug Discovery 2012, 7, 179−194. (9) Ji, Y.; Majumder, S.; Millard, M.; Borra, R.; Bi, T.; Elnagar, A. Y.; Neamati, N.; Shekhtman, A.; Camarero, J. A. J. Am. Chem. Soc. 2013, 135, 11623−11633. (10) Wong, C. T.; Rowlands, D. K.; Wong, C. H.; Lo, T. W.; Nguyen, G. K.; Li, H. Y.; Tam, J. P. Angew. Chem., Int. Ed. 2012, 51, 5620−5624. (11) Sandberg, F. In Cahièrs de la Maboké; Buffon: Paris, 1965; Vol. III, Fascicule 1, p 27. (12) Gran, L. Lloydia 1973, 36, 174−178. (13) Gran, L. Acta Pharmacol. Toxicol. 1973, 33, 400−408. (14) Sletten, K.; Gran, L. Medd. Nor. Farm. Selsk. 1973, 35, 69−82. (15) Saether, O.; Craik, D. J.; Campbell, I. D.; Sletten, K.; Juul, J.; Norman, D. G. Biochemistry 1995, 34, 4147−4158. (16) Pallaghy, P. K.; Nielsen, K. J.; Craik, D. J.; Norton, R. S. Protein Sci. 1994, 3, 1833−1839. (17) Witherup, K. M.; Bogusky, M. J.; Anderson, P. S.; Ramjit, H.; Ransom, R. W.; Wood, T.; Sardana, M. J. Nat. Prod. 1994, 57, 1619− 1625. (18) Gustafson, K. R.; Sowder, R. C.; Henderson, L. E.; Parsons, I. C.; Kashman, Y.; Cardellina, J. H.; McMahon, J. B.; Buckheit, L. K.; Pannell, L. K.; Boyd, M. R. J. Am. Chem. Soc. 1994, 116, 9337−9338. (19) Schöpke, T.; Hasan, A. M. I.; Kraft, R.; Otto, A.; Hiller, K. Sci. Pharm. 1993, 61, 145−153. (20) Claeson, P.; Göransson, U.; Johansson, S.; Luijendijk, T.; Bohlin, L. J. Nat. Prod. 1998, 61, 77−81. (21) Wang, C. K.; Kaas, Q.; Chiche, L.; Craik, D. J. Nucleic Acids Res. 2008, 36, D206−D210. (22) Mulvenna, J. P.; Wang, C.; Craik, D. J. Nucleic Acids Res. 2006, 34, D192−D194. (23) Rosengren, K. J.; Daly, N. L.; Plan, M. R.; Waine, C.; Craik, D. J. J. Biol. Chem. 2003, 278, 8606−8616. (24) Göransson, U.; Herrmann, A.; Burman, R.; Haugaard-Jönsson, L. M.; Rosengren, K. J. ChemBioChem 2009, 10, 2354−2360. (25) Jennings, C.; West, J.; Waine, C.; Craik, D.; Anderson, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10614−10619. (26) Ireland, D. C.; Colgrave, M. L.; Nguyencong, P.; Daly, N. L.; Craik, D. J. J. Mol. Biol. 2006, 357, 1522−1535. (27) Gerlach, S. L.; Burman, R.; Bohlin, L.; Mondal, D.; Göransson, U. J. Nat. Prod. 2010, 73, 1207−1213. (28) Poth, A. G.; Mylne, J. S.; Grassl, J.; Lyons, R. E.; Millar, A. H.; Colgrave, M. L.; Craik, D. J. J. Biol. Chem. 2012, 287, 27033−27046. (29) Nguyen, G. K.; Lim, W. H.; Nguyen, P. Q.; Tam, J. P. J. Biol. Chem. 2012, 287, 17598−17607. (30) Hernandez, J. F.; Gagnon, J.; Chiche, L.; Nguyen, T. M.; Andrieu, J. P.; Heitz, A.; Trinh Hong, T.; Pham, T. T.; Le Nguyen, D. Biochemistry 2000, 39, 5722−5730. (31) Mylne, J. S.; Chan, L. Y.; Chanson, A. H.; Daly, N. L.; Schaefer, H.; Bailey, T. L.; Nguyencong, P.; Cascales, L.; Craik, D. J. Plant Cell 2012, 24, 2765−2778. (32) Chiche, L.; Heitz, A.; Gelly, J. C.; Gracy, J.; Chau, P. T.; Ha, P. T.; Hernandez, J. F.; Le-Nguyen, D. Curr. Protein Pept. Sci. 2004, 5, 341−349. (33) Nair, S. S.; Romanuka, J.; Billeter, M.; Skjeldal, L.; Emmett, M. R.; Nilsson, C. L.; Marshall, A. G. Biochim. Biophys. Acta 2006, 1764, 1568−1576. (34) Skjeldal, L.; Gran, L.; Sletten, K.; Volkman, B. F. Arch. Biochem. Biophys. 2002, 399, 142−148. (35) Göransson, U.; Craik, D. J. J. Biol. Chem. 2003, 278, 48188− 48196. (36) Wang, C. K.; Hu, S. H.; Martin, J. L.; Sjögren, T.; Hajdu, J.; Bohlin, L.; Claeson, P.; Göransson, U.; Rosengren, K. J.; Tang, J.; Tan, N. H.; Craik, D. J. J. Biol. Chem. 2009, 284, 10672−10683.

to linear peptides. One example is the recent study of a natural α-conotoxin, Vc1.1, with exciting potential for the treatment of neuropathic pain (by inhibiting the HVA Ca2+ channel currents via activation of GABAB receptors).130 By engineering a cyclic Vc1.1 analogue, it was possible to increase the stability in gastric fluid, simulated intestinal fluid, and human serum. Whether cyclotides will be further developed and commercially used remains to be seen. The patents on cyclotides mainly concern aspects of cyclotide activity or inventions regarding cyclotide genes. Using this knowledge, developments toward an agricultural application have been made; for example, a cyclotide gene has been transferred to crop plants in an attempt to improve natural defenses against pests.101 The successful start, i.e., production of transgenic plants, is promising, but the studies so far have relied on the insertion of only a single gene. Coexpression with auxiliary proteins such as folding and circularization enzymes from cyclotide-bearing plants has the potential to increase the yields. Another potential application is to directly use the pesticide effects of cyclotides directly to inhibit the growth of bacteria, algae, and fungi. Cyclotides have already been shown to have a potent, nontoxic, and reversible effect against fouling barnacles.50 However, the importance of the fundamental aspects of science should not be underestimated, nor should it be forgotten that the best source of new knowledge likely is curiosity alone. Without curiosity as the driving force, cyclotides may still be awaiting discovery. In fact, it is also the fundamental features of the cyclotides that attract the most interest: namely, the cyclic cystine knot and its inherent stability and rigidity. It is these features that define this peptide circle as a molecule outside the box.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +46 184715031. Fax: +46 18509101. E-mail: ulf. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Work by the authors has been supported by the Swedish Research Council (621-2007-5167) and the Swedish Foundation for Strategic Research (F06-0058). We thank current and former members of the group and collaborators. U.G. thanks Dr. P. Claeson and Prof. L. Bohlin for their guidance during the first year of cyclotide research in Uppsala, Prof. G. Samuelsson for his inspiring early work in the field of plant peptides, and the late Prof. F. Sandberg for his enthusiastic support.

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DEDICATION Dedicated to Prof. Dr. Otto Sticher, of ETH-Zürich, Zürich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry.

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REFERENCES

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