Cyclotide Structure and Function: The Role of Membrane Binding and

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Cyclotide structure and function: The role of membrane binding and permeation Sónia Troeira Henriques, and David J Craik Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01212 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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Cyclotide structure and function: The role of membrane binding and permeation Sónia Troeira Henriques and David J. Craik* Institute for Molecular Bioscience, The University of Queensland, Brisbane, 4072 QLD, Australia.

Corresponding Author *E-mail: [email protected]; Phone: +61-7-3346 2019; Fax: +61-7-3346 2101

Funding source statement Research on cyclotides in our laboratory is supported by grants from the Australian Research Council (ARC; grant IDs DP150100443 and LP130100550) and the National Health and Medical Research Council (NHMRC; grant IDs APP1084965 and APP1060225). STH is the recipient of an ARC Future Fellowship (FT150100398). DJC is an ARC Australian Laureate Fellow (FL150100146).

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ABBREVIATIONS kB1, kalata B1; 3D, three-dimensional; MCoTI-I/II Momordica cochinchinensis trypsin inhibitor I/II; PE, phosphatidylethanolamine; CCK, cyclic cystine knot; AEP, asparaginyl endopeptidase; DPC, dodecylphosphocholine; tcA, tricyclon A; cyO2, cycloviolacin O2; IC50, concentration required to induce 50% of cell death; EC50, concentration required to induce antiviral cytoprotectioon to 50% of host cells; POPC, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine; SPR, surface plasmon resonance; PI, phosphatidylinositol; PEBP, PE-binding protein; MIC, minimal inhibitory concentration.


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ABSTRACT

There is a growing interest in the use of peptides as therapeutic drugs and, in particular, in the potential of cyclotides – a family of cyclic peptides with remarkable stability and amenability to sequence engineering– as scaffolds in drug design. As well as having an ultra-stable structure, many natural cyclotides have intrinsic biological activities with potential pharmaceutical or agricultural applications. Some cyclotides also have the ability cross membrane barriers and to enter into cells; in particular, cyclotides belonging to the Möbius and bracelet subfamilies have been found to harbor lipid-binding domains, which allow for the specific recognition of phosphatidylethanolamine (PE)-phospholipids in biological membranes. This lipid-selectivity is intimately correlated with the highly conserved three-dimensional structures of cyclotides, and is important for their reported biological properties and cell penetration ability. The membranebinding features of Möbius and bracelet cyclotides contrast with the lack of membrane binding of trypsin inhibitor cyclotides, which have different physicochemical properties and bioactivities from the other two subfamilies of cyclotides, but are also able to enter into cells. This article discusses the structures of cyclotides with regard to their myriad of biological activities, and describes the role of membrane binding in their functions and ability to enter inside cells.


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Cyclotides1 are a large family of plant-derived cyclic peptides whose natural function is thought to be as host defense agents against pests and pathogens, but they also have a wide range of other bioactivities with potential pharmaceutical or agricultural applications. They are characterized by a unique head-to-tail macrocyclic structure and knotted arrangement of three disulfide bonds, formed from their six conserved Cys residues, as illustrated in Figure 1. This cyclic cystine knot (CCK) topology confers cyclotides with exceptional resistance to chemical, thermal or proteolytic degradation2 and has stimulated interest in them as protein engineering frameworks.

The first cyclotide was discovered in the 1970s as a constituent of “kalata-kalata”, a traditional indigenous medicine prepared from boiled leaves of Oldenlandia affinis and ingested as a decoction by women in the Congo to accelerate childbirth.3 Chemical analysis of the herbal extract revealed that the active compound, able to resist boiling and ingestion before exerting uterotonic activity, was a 29-amino acid peptide, which was named kalata B1 (hereafter abbreviated to kB1).4,5 The structural features that confer this exceptional stability against thermal and enzymatic degradation were discovered more than 20 years later, in 1995, when the three-dimensional structure of kB1 was determined and the cyclic peptide backbone and knotted disulfide connectivity was revealed.6 Coincidentally, three other groups independently reported similarly stable macrocyclic peptides from plants in the mid-1990s, discovering them in bioassay-screening programs for anti-HIV,7 neurotensin antagonistic8 or hemolytic9 activities, but did not determine their three dimensional structures nor the unusual knotted disulfide bonding arrangement. Several botanical screening surveys in the late 1990s led to the discovery of other similar cyclic peptides that are now known to possess the distinctive CCK structure. In


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1999 the term “cyclotide” (cyclo peptide) was proposed to define this novel family of peptides, in which the CCK is the signature motif.1

Cyclotides are biosynthesized from ribosomally processed precursor proteins8,10-13 and a single plant species can express dozens to hundreds of different cyclotides, with the differences arising from sequence and size variations in their loops. (The loops are defined as the backbone segments between successive Cys residues, as shown in Figure 1). Although the discovery of cyclotides was originally guided by screening for peptide expression in plant extracts,14-17 such peptide-based screening is now often done in combination with the identification of nucleic acid sequences.18-21 To date, these screening approaches have led to the identification of over 300 cyclotide sequences in more than 60 plant species belonging to a wide range of agriculturally important plant families, including the Rubiaceae (coffee),6 Violaceae (violet),9,22 Cucurbitaceae (squash),23 Solanaceae (nightshade)24 and Fabaceae (legume) families.25

Interestingly, the occurrence of any given cyclotide in multiple plant species is uncommon, with relatively few examples of the same cyclotide being found in more than one plant species. One exception is kB1, which has been found in O. affinis, Viola odorata, V. tricolor, V. philippica, V. yedoensis, and V. baoshanensis. Furthermore, cyclotides have an uneven distribution across different plant families. For example, they have been detected in 100% of Violaceae plants screened to date, yet occur in fewer than 5% of Rubiaceae species so far investigated, and are even sparser in the other three plant families known to contain them. Genome and transcriptome sequencing efforts are likely to dramatically accelerate the pace of cyclotide discovery and promise to provide new insights into their distribution and evolution.


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Cyclotides were originally classified into two major subfamilies, referred to as the bracelet and Möbius subfamilies, distinguished by either the absence or presence of Pro residue preceded by a cis-peptide bond in loop 5, respectively.1,3 The presence of the cis-Pro induces a 180º twist in the peptide backbone of members of the Möbius subfamily, conceptually resulting in a molecular Möbius strip. A single plant species typically expresses a cocktail of cyclotides belonging to both subfamilies (split roughly 2:1 between the bracelet and Möbius members), suggesting a common biosynthetic origin and a degree of correlation between the distribution of the two subfamilies. Some sample cyclotide sequences from these two subfamilies are given in Table 1. A third independent subfamily, the trypsin inhibitor cyclotides (see Table 1), has quite different sequences to the bracelet and Möbius subfamilies but possesses the characteristic CCK motif, so is classified in the cyclotide family.26,27 A few dozen trypsin inhibitor cyclotides have been identified to date, and all are found in seeds of Cucurbitaceae plants of the Momordica genus.18,23,28 Trypsin inhibitor cyclotides are also referred to as cyclic knottins29 due to their high sequence homology with (acyclic) trypsin inhibitors known as knottins.

Given the naturally occurring sequence diversity in the backbone loops of cyclotides, the CCK scaffold has been proposed as a natural combinatorial template.30 This sequence diversity is highlighted by the ‘diversity wheel’ in Figure 1B for Möbius cyclotides, which shows the sequence variations that have so far been found to occur in the various loops. A more comprehensive listing of sequences is available in CyBase, www.cybase.org.au,31 an online database dedicated to cyclotides and other naturally occurring cyclic peptides.


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The fact that cyclotides can be chemically synthesized and are very tolerant to substitutions in their loop regions, makes them useful as ‘grafting’ templates in protein engineering and drug design applications.32-34 In addition to being stable and versatile scaffolds, some cyclotides have the much sought after ability (from a drug design perspective) to penetrate cells.35,36 Recent studies in which cyclotides were engineered to stabilize linear bioactive sequences and used as a delivery system to cross cell membranes and target intracellular pharmaceutical targets33,37 have enhanced their potential as a new generation of therapeutic peptide scaffolds. In this article we focus on the topic of the membrane permeation of cyclotides, with particular reference to pharmaceutical activities.

Cyclotide structures The first full characterization of a cyclotide was reported in 1995, when the amino acid sequence and three-dimensional (3D) structure of the Möbius cyclotide kB1 was defined.6 The knotted arrangement of disulfide bonds determined by solution state NMR was subsequently seen again in the structure of cycloviolacin O1 (a bracelet cyclotide) and reported in a 1999 paper,1 which formally recognized the protein family and defined the term ‘cyclotide.’ Figure 1A shows the structure of kB1 and illustrates the CCK motif and the six backbone loops between the six conserved Cys residues. Loops 1 and 4 form part of the cystine knot motif, whereas loops 2, 3, 5 and 6 can be regarded as protruding from the molecular core and are typically the subject of protein engineering or ‘grafting’ applications.


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These first NMR structures of cyclotides were used for modeling the structures of newly discovered cyclotides, including vodo M and vodo N.38 The rigidity of cyclotide structures makes them amenable to such modeling studies, and is also the reason for their exceptional stability.2 Interestingly, synthetic acyclic analogs of cyclotides have a similar 3D structure to the native cyclic peptides,39 suggesting that the cystine knot motif, rather than the cyclic backbone, is the major factor determining the tertiary fold. Although it appears not to be critical for defining the 3D structure, the cyclic backbone has been shown to be crucial for the maintenance of biological activity; for example, the anti-HIV activity of kB1 is lost upon linearization of the backbone.40

It was not until 2009 that the first crystal structure of a cyclotide was reported,41 some 14 years after the NMR solution structure determination of kB1. The crystal structure confirmed the knotted disulfide arrangement previously determined from NMR studies and showed that the membrane-bound structure was similar to the structure of the cyclotide in solution.41 Overall, NMR has proven to be the dominant technique for the determination of cyclotide structures.42 NMR has also been useful in defining the structures of folding intermediates of cyclotides, which are of interest in deciphering how the cystine knot forms. For example, a two-disulfide analogue of kB1 that mimics a transient folding intermediate was found to have a similar structure to the native cyclotide43 based on the similarity of αH NMR chemical shifts. Similar studies were later done for the trypsin inhibitor cyclotide MCoTI-II, where it proved possible to directly determine the structure of a similar two-disulfide folding intermediate since the intermediate was long lived.44


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The finding that cyclotide 3D structures are relatively invariant has important implications for membrane binding studies, in that cyclotides can be regarded as rigid bodies for the modeling of membrane-binding interactions. For example, using dodecylphosphocholine (DPC) micelles as a membrane mimic, Shenkarev et al.45 were able to determine the orientation of kB1 bound to a micelle surface. These studies demonstrated that the surface characteristics of cyclotides are likely to have important roles in defining their intermolecular interactions and hence biological activities.

The surfaces of cyclotides are somewhat unusual due to the constraints imposed by the CCK motif occupying the protein core. Unlike most proteins, where hydrophobic residues are buried in the protein core, Möbius and bracelet cyclotides have their hydrophobic residues solvent exposed. These cyclotides thus typically elute late in reversed-phase HPLC separations (~45% 60% acetonitrile), a phenomenon that has been used to guide cyclotide isolation and discovery, and accounts in part for their membrane binding properties. On the other hand, members of the trypsin inhibitor subfamily display a more positively-charged surface and have fewer hydrophobic residues (see Table 1), and therefore elute earlier. Their more hydrophilic surface correlates with a lack of membrane binding for this subfamily.

Biological functions: Möbius and bracelet cyclotides Pesticidal activities and potential agricultural applications


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The main biological functions of natural cyclotides appear to be in plant defense, based on observations of their pesticidal activities.46 In particular, insecticidal activity was first noted in 2001, when Helicoverpa armigera caterpillars – a major cotton and corn crop pest – were fed with an artificial diet containing kB1 at the same concentration as is naturally expressed in plant leaf (~0.8 µmol/g)21. Larvae fed with a kB1-laced diet showed markedly impaired growth and development, as well as a higher mortality rate, than larvae fed on a control diet lacking cyclotides (Figure 2A).21 Electron microscopy showed that kB1 induces lysis of the larval midgut cells, which suggested a mechanism dependent on an ability to disrupt membranes (Figure 2B & C).47 Insecticidal activity against H. armigera larvae was later reported for other cyclotides, including kB2 from O. affinis48 and Cter M from Clitoria ternatea.13 Cyclotides are also toxic to other larval insects, including Diatrea saccharalis49 and Drosophila melanogaster.50 Cyclotides have activity against a range of non-insect pests, including the Golden Apple snail, Pomacea canaliculata,51 a major pest of rice in South East Asia. They are also active against gastrointestinal nematodes such as Trichostrongylus colubriformis, the livestock parasite Haemonchus contortus,52,53 and the human parasite Necator americanus.54 Given the range of pesticidal activities of cyclotides demonstrated to date, it is reasonable to postulate that cyclotides might also be toxic to other, as yet untested, pests and thus have a significant untapped agricultural potential. At this stage, no cyclotide-based product has reached the market as a pesticidal agent, but this situation is likely to change soon. One cyclotide-bearing product, SeroX, was approved in 2016 in Australia for external application to cotton for insect control. In our opinion there seems to be exciting potential for the use of cyclotides in agricultural applications as pesticides, and potentially also for the control of livestock pests.


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Cyclotides have sometimes been referred to as plant antimicrobial peptides;55-57 however, few studies have addressed their activity against plant-pathogenic bacteria or fungi. In one such study semi-purified cyclotide fractions from V. odorata were tested against the plant pathogenic bacteria Ralstonia solanacearum and Xanthomonas oryzae in a radial diffusion assay.58 The authors of that study concluded that the cyclotide fractions were active against these two Gramnegative bacteria; however, the methods used to confirm the presence of cyclotides, the purity and the number of cyclotides in these semi-pure fractions was unclear. In another study,59 the Möbius cyclotides kB1 and kB2, and the bracelet cyclotide cycloviolacin O2 (cyO2) were tested against a mixed culture of soil bacteria. In that study the minimal inhibitory concentration (MIC, i.e., the lowest concentration of a compound that prevents visible growth of a bacterium) was higher than 50 µM for the tested cyclotides, so the activity is very weak. Overall, unequivocal evidence supporting antimicrobial activity of cyclotides in host defense is yet to be provided, and in our opinion suggestions that cyclotides are plant host defense antimicrobial peptides should be tendered carefully.

Bioactivities with potential pharmaceutical applications In addition to their putative host defense properties, other activities that have been reported for native cyclotides belonging to the bracelet and Möbius subfamilies include antimicrobial activity against human pathogens, anti-HIV activity and anti-cancer properties. With regard to antimicrobial activity, a study published in 1999,60 reported that cyclotides have promising activity against human pathogenic bacteria and fungi, and suggested that cyclotides might be an alternative to conventional antibiotics. In that study, synthetically produced kB1 and circulin A


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were reported to have MICs of 0.26 µM, and 0.19 µM, respectively, in in vitro assays against Staphylococcus aureus, but this was in media with no salt; at physiological salt concentrations the tested cyclotides were inactive, even at concentrations as high as 500 µM.60 Although the antimicrobial activity of kB1 against S. aureus has been tested by other groups,61-63 the original results have yet to be replicated. In a study reported in 2011, cyclotides from Clitoria ternatea (butterfly pea) were tested against some Gram negative bacteria in an agar radial diffusion assay and two were reported to have antimicrobial activity. Specifically, cliotide T1 had a MIC of 1.1 µM against Escherichia coli, 2.7 µM against Klebesiela pneumonia and 4.7 µM against Pseudomonas aeruginosa, and cliotide T4 showed MICs of 1.0 µM against E. coli, 1.9 µM against K. pneumonia and 1.5 µM against P. aeruginosa.12 More recently, cliotides T7-T21 from the same plant were tested against E. coli and some were shown to be active, with MICs between 0.5 and 3.1 µM in an agar radial diffusion assay. All the peptides were less potent (MIC values varied between 2.5 and >20 µM) when tested in a microbroth dilution assay using 100 mM of salt.64 Based on AFM micrographs, the authors suggested that the tested cyclotides possess bactericidal activity and act by disrupting the bacterial outer membrane.64 We have tested the antimicrobial activity of a range of isolated cyclotides from several plants,63,65,66 as well as cyclotide extracts from C. ternatea (unpublished results), against Gram negative and Gram positive bacteria using a microtitre broth method. Of all the cyclotides tested so far, only cyO2 was observed to have significant antimicrobial activity and then only against E. coli,66 in agreement with the findings from another group.62 Furthermore, we found that at the reported in vitro MIC against E.coli (MIC = 25 µM and MIC50 = 6.8 ± 0.4 µM) cyO2was toxic to human red blood cells- the concentration required to induce 50% of cell death was 5 ± 0.1 µM.66 Therefore, in our opinion cyO2 cannot be considered to be a promising antimicrobial candidate


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for treatments against human-pathogenic bacteria. Nevertheless, other native or engineered cyclotides that have not yet been tested, might have useful antimicrobial properties. The HIV-cytoprotective effect of cyclotides was first reported for two bracelet cyclotides, circulin A and circulin B, during a National Cancer Institute natural products screening program.7 In that study both circulin A and B were found to confer a dose-dependent cytoprotective effect against HIV, with antiviral cytoprotective concentrations (EC50) ranging from 40 to 260 nM, depending on the virus strain and host cell, and to act by decreasing the level of infectious virions by a mechanism independent of HIV-reverse transcriptase activity.7 Since that first report, other native cyclotides belonging to both the bracelet and Möbius subfamilies have also been found to possess anti-HIV activities,40,67-70 with comparisons of the efficacies of the tested cyclotides suggesting that their surface hydrophobicity correlates with anti-HIV potency.69,70 NMR studies of cyclotide binding to DPC micelles41,45 have shown that the hydrophobic patch of cyclotides binds to the hydrophobic region of the micelles, suggesting that the latter region is relevant for interaction with membranes. Given this finding, a mechanism of interaction dependent on membrane binding and the ability to inhibit the binding and/or fusion of HIV has been proposed.71 This hypothesis was evaluated in studies of kB1 and its mutants,63 as well as for kB2 and tcA,66 with the results supporting a mechanism dependent on binding to HIV particles and inactivating them by disrupting their enveloping membrane in a dose-dependent manner. Potential anticancer applications of cyclotides were first described in 2002, when two Möbius family members, varv A and varv F, and the bracelet cyclotide cyO2, were reported to be toxic to a panel of immortalized and primary tumor cells in in vitro assays in a dose-dependent manner, with IC50s ranging from 0.11 to 7.49 µM, depending on the specific peptide and cell line.72


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Cytotoxicity against other cancer cell lines was later reported for other Möbius and bracelet cyclotides, suggesting that native cyclotides might be promising anticancer agents.73-79 However, despite the promise of the in vitro results, when cyO2 was tested in mice no significant antitumor effects were observed, suggesting that is does not have anti-cancer potential in vivo.80 In fact, cyclotides also have toxicity against non-cancer cells, as demonstrated through their hemolytic activity with IC50s ranging from 5 to > 64 µM,66,69,70,81 but there is limited quantitative information available on their selectivity for some cell types over others. In a recent study we aimed to quantify any selectivity towards cancer cells,82 but found that toxicity against cancer and non-cancer cells was similar. That study also found that cyclotides exert their anticancer activity by killing cells though cell membrane disruption and that toxicity against both cancerous and healthy cells correlated with the ability of individual cyclotides to bind to and disrupt cell membranes.

Membrane binding properties of Möbius and bracelet cyclotides Kalata B1: Many activities, one mechanism? Biophysical studies using model membranes have provided evidence demonstrating the ability of cyclotides to bind to phospholipid bilayers and have revealed that kB1 has selectivity for certain lipids.63 Specifically, the membrane-binding affinity (Figure 3A) and efficiency of kB1 for inducing vesicle leakage (Figure 3B) correlates with the phosphatidylethanolamine (PE) content in model membrane systems, as demonstrated using surface plasmon resonance (SPR), isothermal calorimetry and fluorescence methodologies.63,83,84 A specific interaction between kB1 and PE is further supported by studies conducted with PIP strip membranes,35 in which the


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binding of kB1 to immobilized phospholipids in a nitrocellulose membrane revealed that, even in a non-bilayer environment, kB1 only binds to phospholipids with PE-headgroups (Figure 3C). Comparisons between kB1 and various analogues have provided evidence for a common mechanism by which kB1 exerts its diverse activities, and have highlighted the importance of molecular topology and topography in these activities. For instance, with regard to topology, backbone linearized forms of kB1 do not display the anti-HIV,40 hemolytic39 or insecticidal properties of the cyclic parent molecule, demonstrating a key role for the cyclic backbone. With regard to topography, the role of individual residues has been probed using point mutagenesis. For example, an Ala-scan revealed that specific point mutations render kB1 inactive in both insecticidal and hemolytic assays,50 whereas a Lys-scan showed that the insertion of a Lys residue at certain positions increased toxicity against red blood cells and nematodes.85 The residues that are critical for the various bioactivities of kB1 are surface exposed and colocalize on one face of the molecule,50,85 termed the ‘bioactive face’, which is composed of mainly hydrophilic residues (Figure 4A). Single mutations at any position in this face with Lys85 or Ala50 (e.g., E7A, E7K or R28A) render the peptide inactive. A separate ‘hydrophobic face’ (Figure 4A) is not affected by Ala substitutions, but Lys substitutions cause a loss of activity (e.g., W23K, V25K). Even very conservative mutations in some residues (e.g., G6A, W23Y, E7D, T16S) can significantly decrease the bioactivity of kB1 in a wide range of assays.50,52,82,86,87 On the other hand, mutations at other residues either do not change potency, or in some cases improve the potency of kB1 (e.g. kB1[T20K] has improved anthelmintic properties).85 The region formed by residues that can be mutated to increase the potency of kB1 is referred to as the ‘amendable face’ and is highlighted in Figure 4A.


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A correlation between membrane-binding affinity and bioactivity was clearly established when kB1 and a range of mutants were compared with respect to their affinity for lipid membranes.63 Specifically, ‘inactive’ kB1 mutants are unable to bind to model membranes, even when PEphospholipids were part of their composition. Conversely, mutants that are more potent than kB1 have a higher affinity for model membranes that include PE-phospholipids (Figure 4B). Furthermore, studies conducted with D-kB1, a mirror image of kB152,88 (Figure 4C) revealed that the enantiomer was active in all the activities tested (i.e. insecticidal, anti-HIV, hemolytic, anticancer, anthelmintic), albeit slightly less active than L-kB1, as shown in Figure 4D. The slightly lower bioactivity of

D-kB1

correlated with slower binding and reduced affinity for model

membranes containing PE-phospholipids (Figure 4E).88 The lower membrane-binding affinity of D-kB1

compared to L-KB1, suggests that the chiral environment present in the phospholipid

bilayers affects the activity of kB1 and provides further support for a mechanism dependent on membrane interactions. NMR studies conducted on kB1 suggest that the bioactive face is involved in specific binding to PE-phospholipids,63 whereas the hydrophobic face is important for insertion into the membrane (Figure 5).45 We proposed that the geometry of the bioactive patch facilitates interactions between the carboxylate group of Glu in loop 1 of kB1 and the ammonium group of PE, whereas the side chain of the Arg in loop 6 of kB1 can interact with the PE phosphate group. The hydrophobic side chains, and in particular the side chain of Trp23, are likely to anchor the peptide in the membrane through extra hydrophobic and H-bond interactions.63 Neutron diffraction studies on PE-containing membranes have further confirmed the ability of kB1 to insert deeply into lipid bilayers.84


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The importance of the bioactive face in the activity of kB1 and its ability to target PEphospholipids was further illustrated by studies on kB1[E7D], a kB1 analogue with a conservative mutation (Glu to Asp) at the center of the bioactive face. Although kB1[E7D] has the same charge and overall structure as kB1, it is strikingly inactive against red blood cells and unable to bind to PE-containing membranes, as shown by SPR and NMR spectroscopy.63 The importance of the hydrophobic patch has been shown using analogues in which Trp23 was replaced by other aromatic residues. For instance, kB1[W23Y] is unable to bind to lipid bilayers and is non-toxic. In fact, just replacing the Trp with 5-hydroxytryptophan (5-HT), i.e., kB1[W23(5-HT)], leads to much slower membrane association and lower toxicity than native kB1.82 Despite the evidence that targeting PE-phospholipids and insertion into the lipid bilayer are required for the activity of kB1, a recent modeling study proposed an alternative mechanism of action, not involving deep insertion into lipid bilayers.89 In that study the interaction of kB1 was modelled using a lipid bilayer lacking PE-phospholipids, and the findings suggested the hydrophilic patch is not involved in membrane binding. In our opinion, the lack of penetration and the involvement of the bioactive patch in the binding mode are likely to result from the absence of PE-phospholipids, and support the notion that the presence of PE-phospholipids is important for efficient insertion into the membrane. After insertion into a membrane kB1 induces permeabilization, as has been demonstrated by studies on both model membranes and cells. Specifically, electrophysiology studies87 suggest that kB1 induces ion conduction through lipid bilayers by a mechanism that probably involves membrane disruption.87 The formation of local disturbances in the lipid bilayer is consistent with microscopy studies conducted using giant unilamellar vesicles composed of a mixture of 1-


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palmitoyl-2-oleoyl-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-glycero-3phosphoethanolamine (POPC/POPE) in which dyes of different sizes (10 and 70 kDa) were shown to leak from the vesicles concomitantly, before the vesicles burst.66 Toxicity that is dependent on the ability to bind to and permeabilize cell membranes was recently confirmed in a flow cytometry study using HeLa cells,82 where the concentration of kB1 required to induce cell membrane permeability and toxicity was the same. Extrapolations to other Möbius and bracelet cyclotides All Möbius and bracelet cyclotides tested so far share common biological activities and physicochemical properties. Like kB1, all Möbius and bracelet cyclotides have a hydrophobic face and a bioactive face containing a Glu residue in loop 1 and a Lys or Arg residue in loop 6, as exemplified by Möbius kB2 and bracelet cyO2 in Figure 6A; these features suggest that Möbius and bracelet cyclotides might all possess a common ability to bind to and disrupt cell membranes. This suggestion is supported by the observation that mutations in either the Glu or Lys/Arg decrease the activity of Möbius varv A90 and bracelet cyO2.90,91 Furthermore, studies conducted with DPC micelles using NMR spectroscopy show that, similar to what was observed with kB1,45 the hydrophobic faces in kB2,92 kB7,93 cyO292 and varv F41 are responsible for micelle binding. In a 2012 study, a strong correlation between the ability to bind to PEphospholipids, membrane-binding affinity and bioactivity was shown for native cyclotides from the Möbius and bracelet subfamilies.66 In that study all the tested cyclotides selectively bound to and permeabilized membranes that contained PE-phospholipids (Figure 6B & C) and the trend in their ability to bind and disrupt model membranes (Figure 6C & D) correlated with their potency in hemolytic, anti-HIV and cytotoxicity assays66,82 (Figure 6E & F) as exemplified with Möbius cyclotides kB1 and kB2 and the bracelet cyclotides cyO2 and tcA. In summary, the ability to


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selectively bind to and disrupt membranes containing PE-phospholipids has now been confirmed for a range of native Möbius and bracelet cyclotides.65,94 Thus, the mode-of-action proposed for kB1 appears to be generalizable to other Möbius and bracelet cyclotides. That all Möbius and bracelet cyclotides tested so far have selectivity for PE-phospholipids suggests that cyclotides can be classified as a ‘lipid-binding domain’ family of peptides.66 Cyclotides and classic lipid-binding domain families95,96 share some features: a pocket for the recognition of a specific lipid headgroup, in which electrostatic interactions are involved; in some cases, the requirement of extra hydrophobic interactions; and the ability to insert into membranes. Lipid-binding domains are common in proteins involved in membrane trafficking and signal transduction;96 for example, members of the plant PE-binding protein (PEBP) family, are involved in modulating plant architecture and flowering, and can regulate the transition from the vegetative to the reproductive phase.97 Interestingly, two highly homologous members of the PEBP family have opposite activities in Arabidopsis thaliana: one represses flowering whereas the other activates it. These functions can be interchanged by swapping a single amino acid.98 Although cyclotides do not resemble PEBP members, which are typically ~25kDa in size, the fact that residues important for insertion into the membrane and recognition of the PE-headgroup are conserved in Möbius and bracelet subfamilies and that a single plant species can produce dozens of them, leads us to speculate that this lipid specificity might be associated with other more complex roles for cyclotides, independent of their insecticidal properties. Such speculation does not extend to trypsin inhibitor cyclotides, which do not bind to membranes. Trypsin inhibitor cyclotides: maintain a CCK motif but do not bind membranes


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The two first identified trypsin inhibitor cyclotides, MCoTI-I and MCoTI-II, were discovered in the seeds of the tropical vine Momordica cochinchinensis and, as their names suggest, are highly potent trypsin inhibitors.23 Recently, additional cyclic and acyclic trypsin inhibitors were identified in seeds of other Momordica plants at either the nucleotide and/or peptide level.18,28,99,100 Trypsin inhibitors and other proteinase inhibitors are commonly found in plant seeds and it is thought that they have a defense role against insects through inhibition of digestive enzymes,101-103 or in protecting seeds from degradation in the digestive tracts of animals that play a role in seed dispersal via ingestion of fruits. As noted earlier in this article, trypsin inhibitor cyclotides have different physicochemical properties from Möbius or bracelet cyclotides. Specifically, they lack the bioactive and hydrophobic faces and appear not to bind to membranes, which is consistent with their relative lack of toxic properties compared to Möbius and bracelet cyclotides.35 Their lack of membrane binding also is beneficial in their presumed function of protecting seeds in the digestive tracts of dispersing animals- toxicity to such animals would presumably be unfavorable to excretion of the seeds. Furthermore, the lack of toxicity has advantages for their applications as pharmaceuticals or animal health products, and trypsin inhibitor cyclotides have been engineered to inhibit a range proteases involved in human diseases, including matriptase,104,105 a serine protease overexpressed in a range of human epithelial tumors and an attractive cancer target, and 3C protease,106 a protease essential for the replication of the foot-and-mouth-disease virus. The promising results obtained in these early protease inhibitory studies and the tolerance to sequence variation in the active site loop (loop 1) suggests that trypsin inhibitor cyclotides might be reengineered to selectively inhibit other proteases with applications in medicine or agriculture.106 More broadly, all three subfamilies of cyclotides are tolerant to sequence


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substitutions in many of their loops, thus facilitating their application as grafting frameworks whose targets extend well beyond just proteases, as described in the following section.

The CCK framework of cyclotides: A drug design scaffold with cell-penetrating properties Figure 7 illustrates the principle of how the amenability of cyclotides to loop grafting has inspired applications in drug design in which the CCK scaffold is used to stabilize bioactive peptide sequences.71,107,108 Although the intrinsic toxicity of some Möbius and bracelet cyclotides would be a concern in pharmaceutical applications, in practice this is easily overcome, as single mutations can render Möbius and bracelet cyclotides non-toxic. Currently, there are around 25 examples which demonstrate that the CCK scaffold can be redesigned to target proteins with specific functions.34 Recent examples include engineered cyclotides with immunoregulatory potential for the treatment of multiple sclerosis;109 grafted angiogenic epitopes with potential cardiovascular or wound healing applications;32 a melanocortin agonist with the potential to treat obesity;110 and a substrate-based inhibitor against BCR-ABL kinase, a target implicated in chronic myeloid leukemia.111 These examples include engineered cyclotides targeting either extracellular or intracellular proteins, thus highlighting the versatility of the CCK framework.

Intracellular targets are particularly challenging, and cell-penetrating peptides are regarded as exciting vectors to carry bioactive sequences inside cells to reach such targets. MCoTI-II was the first cyclotide shown to internalize into cells36 and more recently MCoTI-I112 and kB135,113 were also shown to have cell penetrating properties. The amenability of cyclotides to loop replacements, together with their high stability and cell-penetrating properties, make them a very


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attractive scaffold to stabilize and deliver peptides across cell membranes and bind to specific intracellular targets. This capability was demonstrated in a seminal study by Camarero and coworkers33 whereby MCoTI-I was engineered with a 12-amino acid sequence (PMI) to modulate the p53 tumor suppressor pathway. PMI is known to block the interaction of the cellular guardian p53 with the oncoprotein Hdm2, and the grafted MCoTI-PMI was shown to acquire this ability while retaining the characteristic stability of cyclotides. The activity of MCoTI-PMI was confirmed in vivo in a mouse xenograft model, demonstrating the ability of cyclotides to functionally inhibit intracellular targets.33 The internalization mechanism used to cross membranes is dependent on the cyclotide subfamily (Figure 8). Studies with kB1 suggest that it enters cells via direct membrane translocation and endocytosis, and both pathways are initiated by binding to PE-phospholipids.113 The importance of the cell membrane was revealed when the internalization rate of membrane-inactive kB1 analogues (i.e., kB1[E7K], kB1[T16K] and kB1[V25K]) was found to be significantly lower than that of the membrane-active kB1[T20K]. Although kB1 enters endocytic compartments, its internalization is not blocked by compounds that inhibit specific endocytic pathways, suggesting that its internalization probably occurs through multi-endocytic mechanisms.113 Internalization of kB1 via direct membrane translocation is consistent with its ability to translocate through pure model membranes, and by an overall increase, both at 4ºC and 37ºC, in the uptake of kB1[T20K], but not of the membrane inactive kB1[T16K], when cells are treated with Myr-PI, an endocytic inhibitor that improves the exposition of PE-phospholipids. Biophysical studies suggest that the translocation of kB1 through membranes might involve the formation of a nonbilayer intermediate and a peptide-PE-phospholipid complex.113 The 3D structure of kB1 is clearly important, as when the overall fold of a cyclotide is compromised (e.g., by linearization


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or disulfide bond reduction) its membrane-binding properties and bioactivities are abolished.63,114 Quantitative

analysis

using

liquid

chromatography-multiple

reaction

monitoring

has

demonstrated that a large portion (~50%) of kB1 enters into cells and is in its oxidized form, confirming that the kB1 structure resists the reducing intracellular environment.113 MCoTI-I and MCoTI-II are internalized by energy-dependent processes (i.e. endocytic routes) and are unable to cross cell membranes by direct permeation.35,36,112 Their internalization by a mechanism dependent on endocytic routes is consistent with internalization being inhibited by endocytic inhibitors or when cells are cooled to 4ºC.35,112 In addition, co-localization of MCoTI-I with lysosomes and with late endosomes was observed after 1 h incubation, suggesting that MCoTI-I traffics through an endosomal pathway.112 Although unable to bind to any lipid membranes tested, including negatively-charged membranes, nor to bind to negative proteoglycans, mutagenic studies suggest that the patch of positively-charged residues on the surface of MCoTI-II is important for its uptake.35,115 Direct binding of MCoTI-II with phosphatidylinositol (PI)-phosphates, including strong binding to PI(4,5)P2, has been observed in vitro. PI(4,5)P2 is the most abundant PI-phosphate in the plasma membrane and is known to be involved in endocytic processes, helping in the recruitment of proteins to the cell membrane and in their subsequent internalization. Therefore, it has been hypothesized that electrostatic interactions, between the positive residues in MCoTI-II and the negatively-charged PI(4,5)P2 in the plasma membrane, might assist the uptake of MCoTI-II by activating endocytosis.35 Although MCoTI-I or MCoTI-II are favorable scaffolds in drug design due to their low toxicity and ability to enter inside cells, their internalization efficiency is low compared to typical linear CPPs115,116 and this led to the modification of MCoTI-II to improve its intracellular uptake. One reengineered MCoTI-II, MCo-CTP, includes a highly-positively charged segment in loop 2,


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while maintaining loop 6, the preferred loop for grafting, untouched. MCo-CTP is non-toxic, retains the high resistance to protease degradation and has an internalization rate comparable to that of TAT, the gold standard CPP.116 We would anticipate that ongoing efforts to further improve internalization efficiency will lead to even better examples of cyclic cell penetrating peptides.

Summary and outlook The biological activities and the ability to enter into cells of cyclotides belonging to the Möbius and bracelet subfamilies are dependent on their structure and membrane-binding properties. In particular, Möbius and bracelet cyclotides are highly hydrophobic, have conserved residues that form a bioactive face at the molecular surface and their overall 3D fold is important for their ability to target PE-phospholipids in the cell membrane and exert their functions. Small mutations in the overall structure and in the hydrophobic or bioactive patches render these cyclotides unable to exert their biological functions or enter into cells. On the other hand, cyclotides belonging to the trypsin inhibitor subfamily are more hydrophilic, have several positively-charged surface exposed residues, have low affinity for lipid bilayers, and are less toxic than Möbius or bracelet cyclotides. Although having a weaker ability to enter cells than linear CPPs or other cyclotides, MCoTI-II has been successfully engineered to improve its cell penetrating ability without compromising its stability. Overall, the cyclotide family can thus be regarded as a set of stable scaffolds that can be tailored to inhibit intracellular or extracellular targets with high selectivity and potency, and can be considered as complementary to smallmolecule drugs or biologics.


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In our opinion, one of the most exciting applications of cyclotides relates to their potential to target intracellular protein-protein interactions involved in cancer pathways. Traditional thinking considers intracellular protein-protein interactions to be “undrugabble” as the interface is too large to be targeted by small-molecule drugs, and larger biologics have low cell penetration and hence cannot effectively modulate intracellular targets. Two reports from independent groups have now shown that engineered cyclotides are able to target intracellular cancer pathways,33,37 confirming their potential as anticancer drug leads.

The value of cyclotides, and of other CPPs, as drug leads and delivery systems could be greatly enhanced by improving their internalization efficiency, and their ability to target a specific cell type (e.g. cancer cells) or to direct them to a required location inside cells. To achieve these goals, we are currently investigating the distribution of cyclotides inside cells (e.g. cytosol, membrane bound, or in a specific organelle) and developing strategies that might engender them with the ability to target a specific cell type or reduce their entrapment within endosomes to increase their concentration in the cytosol, where target proteins normally reside. We also believe that the ultimate uptake of cyclotides by the pharmaceutical industry will be facilitated by improvements in their synthesis yields, using either chemical or biological approaches. Ongoing research into their structures, distribution and biosynthesis will continue to be valuable in underpinning the development of new synthetic or biosynthetic strategies.

ACKNOWLEDGMENTS We thank the many staff, students and collaborators who have worked with us on cyclotides and whose names are listed amongst the references.


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(17) Plan, M. R. R., Göransson, U., Clark, R. J., Daly, N. L., Colgrave, M. L., and Craik, D. J. (2007) The cyclotide fingerprint in oldenlandia affinis: elucidation of chemically modified, linear and novel macrocyclic peptides. Chembiochem 8, 1001–1011. (18) Mylne, J. S., Chan, L. Y., Chanson, A. H., Daly, N. L., Schaefer, H., Bailey, T. L., Nguyencong, P., Cascales, L., and Craik, D. J. (2012) Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis. Plant Cell 24, 2765–2778. (19) Koehbach, J., Attah, A. F., Berger, A., Hellinger, R., Kutchan, T. M., Carpenter, E. J., Rolf, M., Sonibare, M. A., Moody, J. O., Wong, G. K.-S., Dessein, S., Greger, H., and Gruber, C. W. (2013) Cyclotide discovery in Gentianales revisited-identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants. Biopolymers 100, 438–452. (20) Trabi, M., Mylne, J. S., Sando, L., and Craik, D. J. (2009) Circular proteins from Melicytus (Violaceae) refine the conserved protein and gene architecture of cyclotides. Org. Biomol. Chem. 7, 2378–2388. (21) Jennings, C., West, J., Waine, C., Craik, D., and Anderson, M. (2001) Biosynthesis and insecticidal properties of plant cyclotides: the cyclic knotted proteins fromOldenlandia affinis. Proc. Natl. Acad. Sci. U.S.A. 98, 10614–10619. (22) Claeson, P., Göransson, U., Johansson, S., Luijendijk, T., and Bohlin, L. (1998) Fractionation Protocol for the Isolation of Polypeptides from Plant Biomass. J. Nat. Prod. 61, 77–81. (23) Hernandez, J. F., Gagnon, J., Chiche, L., Nguyen, T. M., Andrieu, J. P., Heitz, A., Trinh Hong, T., Pham, T. T., and Le-Nguyen, D. (2000) Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure. Biochemistry 39, 5722–5730. (24) Poth, A. G., Mylne, J. S., Grassl, J., Lyons, R. E., Millar, A. H., Colgrave, M. L., and Craik, D. J. (2012) Cyclotides associate with leaf vasculature and are the products of a novel precursor in petunia (solanaceae). J. Biol. Chem. 287, 27033–27046. (25) Poth, A. G., Colgrave, M. L., Philip, R., Kerenga, B., Daly, N. L., Anderson, M. A., and Craik, D. J. (2011) Discovery of cyclotides in the fabaceae plant family provides new insights into the cyclization, evolution, and distribution of circular proteins. ACS chemical biology 6, 345–355. (26) Felizmenio-Quimio, M. E., Daly, N. L., and Craik, D. J. (2001) Circular proteins in plants: solution structure of a novel macrocyclic trypsin inhibitor from Momordica cochinchinensis. J. Biol. Chem. 276, 22875–22882. (27) Craik, D. J., Daly, N. L., Mulvenna, J., Plan, M. R., and Trabi, M. (2004) Discovery, structure and biological activities of the cyclotides. Curr. Protein Pept. Sci. 5, 297–315. (28) Mahatmanto, T., Mylne, J. S., Poth, A. G., Swedberg, J. E., Kaas, Q., Schaefer, H., and Craik, D. J. (2015) The evolution of momordica cyclic peptides. Mol. Biol. Evol. 32, 392–405. (29) Heitz, A., Hernandez, J. F., Gagnon, J., Hong, T. T., Pham, T. T., Nguyen, T. M., LeNguyen, D., and Chiche, L. (2001) Solution structure of the squash trypsin inhibitor MCoTI-II. A new family for cyclic knottins. Biochemistry 40, 7973–7983. (30) Craik, D. J., Cemazar, M., Wang, C. K. L., and Daly, N. L. (2006) The cyclotide family of circular miniproteins: nature's combinatorial peptide template. Biopolymers 84, 250–266. (31) Wang, C. K. L., Kaas, Q., Chiche, L., and Craik, D. J. (2008) CyBase: a database of cyclic protein sequences and structures, with applications in protein discovery and engineering. Nucleic Acids Res. 36, D206–10. (32) Chan, L. Y., Gunasekera, S., Henriques, S. T., Worth, N. F., Le, S.-J., Clark, R. J.,


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Campbell, J. H., Craik, D. J., and Daly, N. L. (2011) Engineering pro-angiogenic peptides using stable, disulfide-rich cyclic scaffolds. Blood 118, 6709–6717. (33) Ji, Y., Majumder, S., Millard, M., Borra, R., Bi, T., Elnagar, A. Y., Neamati, N., Shekhtman, A., and Camarero, J. A. (2013) In Vivo Activation of the p53 Tumor Suppressor Pathway by an Engineered Cyclotide. J. Am. Chem. Soc. 135, 11623–11633. (34) Poth, A. G., Chan, L. Y., and Craik, D. J. (2013) Cyclotides as grafting frameworks for protein engineering and drug design applications. Biopolymers 100, 480–491. (35) Cascales, L., Henriques, S. T., Kerr, M. C., Huang, Y.-H., Sweet, M. J., Daly, N. L., and Craik, D. J. (2011) Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides. J. Biol. Chem. 286, 36932–36943. (36) Greenwood, K. P., Daly, N. L., Brown, D. L., Stow, J. L., and Craik, D. J. (2007) The cyclic cystine knot miniprotein MCoTI-II is internalized into cells by macropinocytosis. Int. J. Biochem. Cell Biol. 39, 2252–2264. (37) D'Souza, C., Henriques, S. T., Wang, C. K., Cheneval, O., Chan, L. Y., Bokil, N. J., Sweet, M. J., and Craik, D. J. (2016) Using the MCoTI-II Cyclotide Scaffold To Design a Stable Cyclic Peptide Antagonist of SET, a Protein Overexpressed in Human Cancer. Biochemistry 55, 396– 405. (38) Svangård, E., Göransson, U., Smith, D., Verma, C., Backlund, A., Bohlin, L., and Claeson, P. (2003) Primary and 3-D modelled structures of two cyclotides fromViola odorata. Phytochemistry 64, 135–142. (39) Barry, D. G., Daly, N. L., Clark, R. J., Sando, L., and Craik, D. J. (2003) Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity. Biochemistry 42, 6688–6695. (40) Daly, N. L., Gustafson, K. R., and Craik, D. J. (2004) The role of the cyclic peptide backbone in the anti-HIV activity of the cyclotide kalata B1. FEBS Lett. 574, 69–72. (41) 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., and Craik, D. J. (2009) Combined X-ray and NMR analysis of the stability of the cyclotide cystine knot fold that underpins its insecticidal activity and potential use as a drug scaffold. J. Biol. Chem. 284, 10672–10683. (42) Craik, D. J., and Daly, N. L. (2007) NMR as a tool for elucidating the structures of circular and knotted proteins. Mol Biosyst 3, 257–265. (43) Daly, N. L., Clark, R. J., and Craik, D. J. (2003) Disulfide folding pathways of cystine knot proteins. Tying the knot within the circular backbone of the cyclotides. J. Biol. Chem. 278, 6314–6322. (44) Cemazar, M., Joshi, A., Daly, N. L., Mark, A. E., and Craik, D. J. (2008) The structure of a two-disulfide intermediate assists in elucidating the oxidative folding pathway of a cyclic cystine knot protein. Structure 16, 842–851. (45) Shenkarev, Z. O., Nadezhdin, K. D., Sobol, V. A., Sobol, A. G., Skjeldal, L., and Arseniev, A. S. (2006) Conformation and mode of membrane interaction in cyclotides. Spatial structure of kalata B1 bound to a dodecylphosphocholine micelle. FEBS J. 273, 2658–2672. (46) Mylne, J. S., Wang, C. K., van der Weerden, N. L., and Craik, D. J. (2010) Cyclotides are a component of the innate defense of Oldenlandia affinis. Biopolymers 94, 635–646. (47) Barbeta, B. L., Marshall, A. T., Gillon, A. D., Craik, D. J., and Anderson, M. A. (2008) Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proc. Natl. Acad. Sci. U.S.A. 105, 1221–1225. (48) Jennings, C. V., Rosengren, K. J., Daly, N. L., Plan, M., Stevens, J., Scanlon, M. J., Waine,


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characterization of cytotoxic cyclotides from Viola philippica. Peptides 32, 1719–1723. (80) Burman, R., Svedlund, E., Felth, J., Hassan, S., Herrmann, A., Clark, R. J., Craik, D. J., Bohlin, L., Claeson, P., Göransson, U., and Gullbo, J. (2010) Evaluation of toxicity and antitumor activity of cycloviolacin O2 in mice. Biopolymers 94, 626–634. (81) Chen, B., Colgrave, M. L., Wang, C., and Craik, D. J. (2006) Cycloviolacin H4, a hydrophobic cyclotide from Viola hederaceae. J. Nat. Prod. 69, 23–28. (82) Henriques, S. T., Huang, Y.-H., Chaousis, S., Wang, C. K., and Craik, D. J. (2014) Anticancer and toxic properties of cyclotides are dependent on phosphatidylethanolamine phospholipid targeting. Chembiochem 15, 1956–1965. (83) Kamimori, H., Hall, K. N., Craik, D. J., and Aguilar, M.-I. (2005) Studies on the membrane interactions of the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon resonance. Anal. Biochem. 337, 149–153. (84) Wang, C. K., Wacklin, H. P., and Craik, D. J. (2012) Cyclotides insert into lipid bilayers to form membrane pores and destabilize the membrane through hydrophobic and phosphoethanolamine-specific interactions. J. Biol. Chem. 287, 43884–43898. (85) Huang, Y.-H., Colgrave, M. L., Clark, R. J., Kotze, A. C., and Craik, D. J. (2010) Lysinescanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of nematocidal activity. J. Biol. Chem. 285, 10797–10805. (86) Wang, C. K. L., Clark, R. J., Harvey, P. J., Rosengren, K. J., Cemazar, M., and Craik, D. J. (2011) 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. Biochemistry 50, 4077– 4086. (87) Huang, Y.-H., Colgrave, M. L., Daly, N. L., Keleshian, A., Martinac, B., and Craik, D. J. (2009) The biological activity of the prototypic cyclotide kalata b1 is modulated by the formation of multimeric pores. J. Biol. Chem. 284, 20699–20707. (88) Sando, L., Henriques, S. T., Foley, F., Simonsen, S. M., Daly, N. L., Hall, K. N., Gustafson, K. R., Aguilar, M.-I., and Craik, D. J. (2011) A Synthetic mirror image of kalata B1 reveals that cyclotide activity is independent of a protein receptor. Chembiochem 12, 2456–2462. (89) Nawae, W., Hannongbua, S., and Ruengjitchatchawalya, M. (2014) Defining the membrane disruption mechanism of kalata B1 via coarse-grained molecular dynamics simulations. Sci Rep 4, 3933. (90) Burman, R., Herrmann, A., Tran, R., Kivelä, J.-E., Lomize, A., Gullbo, J., and Göransson, U. (2011) Cytotoxic potency of small macrocyclic knot proteins: structure-activity and mechanistic studies of native and chemically modified cyclotides. Org. Biomol. Chem. 9, 4306– 4314. (91) Herrmann, A., Svangård, E., Claeson, P., Gullbo, J., Bohlin, L., and Göransson, U. (2006) Key role of glutamic acid for the cytotoxic activity of the cyclotide cycloviolacin O2. Cell. Mol. Life Sci. 63, 235–245. (92) Wang, C. K., Colgrave, M. L., Ireland, D. C., Kaas, Q., and Craik, D. J. (2009) Despite a conserved cystine knot motif, different cyclotides have different membrane binding modes. Biophys. J. 97, 1471–1481. (93) Shenkarev, Z. O., Nadezhdin, K. D., Lyukmanova, E. N., Sobol, V. A., Skjeldal, L., and Arseniev, A. S. (2008) Divalent cation coordination and mode of membrane interaction in cyclotides: NMR spatial structure of ternary complex Kalata B7/Mn2+/DPC micelle. J. Inorg. Biochem. 102, 1246–1256. (94) Strömstedt, A. A., Kristiansen, P. E., Gunasekera, S., Grob, N., Skjeldal, L., and Göransson,


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(111) Huang, Y.-H., Henriques, S. T., Wang, C. K., Thorstholm, L., Daly, N. L., Kaas, Q., and Craik, D. J. (2015) Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold. Sci Rep 5, 12974. (112) Contreras, J., Elnagar, A. Y. O., Hamm-Alvarez, S. F., and Camarero, J. A. (2011) Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways. J Control Release 155, 134– 143. (113) Henriques, S. T., Huang, Y.-H., Chaousis, S., Sani, M.-A., Poth, A. G., Separovic, F., and Craik, D. J. (2015) The Prototypic Cyclotide Kalata B1 Has a Unique Mechanism of Entering Cells. Chemistry & Biology 22, 1087–1097. (114) Daly, N. L., and Craik, D. J. (2000) Acyclic permutants of naturally occurring cyclic proteins. Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1. J. Biol. Chem. 275, 19068–19075. (115) D'Souza, C., Henriques, S. T., Wang, C. K., and Craik, D. J. (2014) Structural parameters modulating the cellular uptake of disulfide-rich cyclic cell-penetrating peptides: MCoTI-II and SFTI-1. Eur J Med Chem 88, 10–18. (116) Huang, Y.-H., Chaousis, S., Cheneval, O., Craik, D. J., and Henriques, S. T. (2015) Optimization of the cyclotide framework to improve cell penetration properties. Front Pharmacol 6, 17.


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TABLES.

Table 1. Sequences of selected cyclotides belonging to the Möbius, bracelet and trypsin inhibitor subfamilies. loop 6 loop 1 loop 2 loop 3 4 loop 5 Möbius I II III IV V

VI

kalata B1

G-LPV

C

G---ET

C

VG-GT

C

NT---PG

C

T

C

-SW-PV

C

TR--N

kalata B2

G-LPV

C

G---ET

C

FG-GT

C

NT---PG

C

S

C

-TW-PI

C

TR--D

kalata B6

G-LPT

C

G---ET

C

FG-GT

C

NT---PG

C

S

C

SSW-PI

C

TR--N

kalata B7

G-LPV

C

G---ET

C

TL-GT

C

YT---QG

C

T

C

-SW-PI

C

KR--D

varv F Bracelet cycloviolacin O1

G-VPI

C

G---ET

C

TL-GT

C

YT---AG

C

S

C

-SW-PV

C

TR--N

G-IP-

C

A---ES

C

VY-IP

C

TVTALLG

C

S

C

-SN-RV

C

Y---N

cycloviolacin O2

G-IP-

C

G---ES

C

VW-IP

C

ISS-AIG

C

S

C

-KS-KV

C

YR--N

kalata B5

G-TP-

C

G---ES

C

VY-IP

C

ISG-VIG

C

S

C

-TD-KV

C

YL--N

kalata B8

GSVLN

C

G---ET

C

LL-GT

C

YT---TG

C

T

C

NKYRV

C

TK--D

Trypsin Inhibitor MCoTI-I

G--GV

C

PKILQR

C

RRDSD

C

PG----A

C

I

C

RGNGY

C

GSGSD

MCoTI-II

G--GV

C

PKILKK

C

RRDSD

C

PG----A

C

I

C

RGNGY

C

GSGSD

loop 6


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FIGURE LEGENDS Figure 1. Cyclotide structure and diversity. (A) Three-dimensional structure of the prototypic cyclotide kB1 (PDB ID:1nb1). Cyclotides have six cysteine residues (labeled I-VI) and the backbone sequences between adjacent cysteines are designated as loops 1-6. The black arrow indicates the direction of the peptide chain N-C. The first and the last residues, Gly and Asn, are indicated with G and N respectively. (B) Diversity wheel of sequence variations within the Möbius subfamily of cyclotides. The innermost circle in grey shows the most frequently observed residues, with the less commonly observed amino acids appearing at positions further outward. The first residue G and the last residue N are shown in black. Residues variations at each position protrude outwards as axial spikes. Residues that are also very common are shaded in light grey.

Figure 2. Insecticidal properties of cyclotides. (A) Helicoverpa larvae on control diet (right) or on artificial diet containing kB1 (left); bar = 1 cm.47 Scanning electron micrographs of midgut epithelia after ingestion of a control diet (B) or a diet containing kB1 (C), bar = 10 µm.21 Figure adapted from references 47 and 21.

Figure 3. Binding of kB1 to phospholipid bilayers is dependent on the presence of PEphospholipids. (A) The amount of kB1 bound to POPC or POPC/POPE (95:5, 90:10 or 80:20) bilayers as monitored by SPR is plotted as a function of the amount of POPE on the chip surface. At 25ºC all lipid systems are in fluid phase. (B) Leakage of vesicles composed of POPC or


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POPC/POPG (80:20) induced by increasing concentrations of kB1. (C) Binding of kB1 to a nitrocellulose membrane (PIP strip) containing various lipids: lysophosphatidic acid (LPA); lysophosphatidylcholine (LPC); phosphatidylethanolamine (PE); phosphatidic acid (PA); phosphatidylserine (PS); sphingosine 1-phosphate (SphP); phosphatidylinositol (PI); PI-3phosphate (PI3P); PI-4 phosphate (PI4P); PI-5-phosphate (PI5P); PI-3,4-diphosphate (PI3,4P2); PI-4,5-phosphate (PI4,5P). Figure adapted from references 63 and 35.

Figure 4. The potency of kB1 correlates with its membrane-binding properties. (A) Surface representation of kB1 shown in two views depicting the hydrophobic face (green), the bioactive face and the amendable face (blue). (B) The affinity of kB1 for lipid membranes correlates with its hemolytic activity, as shown for kB1 analogues with higher hemolytic potency (mutations in the amendable face) and for the non-hemolytic analogues (with single mutations in either the bioactive or hydrophobic faces).63 (C) D-kB1 (PDB ID:2jue) is a mirror image of native L-kB1, as determined by 1H NMR. (D) Hemolytic dose-response obtained with D-kB1 and L-kB1. (E) Sensorgrams obtained with 50 µM D-kB1 or L-kB1 injected for 180s (association phase) over POPC/POPE (80:20) lipid bilayers deposited on a L1 chip. Lower hemolysis induced by D-kB1 correlates with its lower affinity for PE-containing membranes compared to L-kB1.88 Figure adapted from references 63 and. 88

Figure 5. A proposed model for the interaction of kB1 with lipid bilayers. 1) The binding of kB1 with membranes is initiated by targeting PE-headgroups in which an electrostatic interaction between PE and the bioactive face is important.63 2) The hydrophobic face of the peptide inserts


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in the membrane.45 3) Local disturbance in the membrane occurs upon insertion of kB1; membrane disruption occurs with peptide overload. Figure adapted from reference.66

Figure 6. Comparison of membrane-binding properties and potency of native cyclotides. (A) Surface representation of Möbius kB2 and bracelet cyO2 in two views showing the conserved bioactive face centered on the Glu residue and the presence of a nearby hydrophobic face. Although with their hydrophobic faces located different regions, Möbius and bracelet cyclotides have identical lipid selectivity as shown in (B) sensorgrams obtained with 30 µM of kB2 or cyO2 over lipid bilayers with defined lipid compositions. The signal is normalized to peptide-tolipid ratio (mol/mol) to better compare the distinct lipid bilayers and peptides. (C) Permeabilization of POPC/POPE (80:20) or POPC vesicles, (D) binding to POPC/POPE (80:20) bilayers, (E) hemolytic dose response and (F) anti-HIV cytoprotective effect induced by kB1, kB2, tcA or cyO2.

Figure 7. Schematic representation of the principle of grafting in the CCK framework. The bioactive sequence is inserted by replacing the native residues with a sequence with a desired activity. MCoTI-II is here used to illustrate the cyclotide framework with the bioactive sequence grafted in loop 6, but active sequences can be inserted into other CCK scaffolds and in other loops, as summarized in a recent review.34


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Biochemistry

Figure 8. Putative mechanistic proposal for the internalization of cyclotides. (A) A possible internalization mechanism for Möbius and bracelet cyclotides is illustrated with kB1. The surface structure of kB1 is shown in two views with the hydrophobic residues colored in in green, Glu7 in blue and Arg28 in red. The bioactive face is delineated by a red line. 1) Internalization is initiated by targeting of the cell membrane through interaction with PE phospholipids and insertion into the outer leaflet; 2) kB1 enters into cells through direct membrane translocation or via endocytosis;113 3) at high concentration the cell membrane is overloaded with peptide and the lipid membrane is disrupted.63,66,87 (B) Internalization of trypsin inhibitor cyclotides. The surface structure of MCoTI-II is shown with positively-charged residues in blue and negatively-charged residues in red. MCoTI-II is attracted to the cell membrane through electrostatic attractions between the positive charges of the peptide and negatively-charged molecules at the surface of the cell membrane. Internalization of MCoTI-II occurs via endocytic pathways such as macropinocytosis.35


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Biochemistry

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Figures

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Biochemistry

Figure 3

Figure 4


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Biochemistry

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Biochemistry

Figure 7

Figure 8


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Biochemistry

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Cyclotide Structure and Function: The Role of Membrane Binding and

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