Cyclotides As Drug Design Scaffolds David J Craik* and Junqiao Du

Cyclotides as drug design scaffolds

David J Craik* and Junqiao Du

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072,

Australia

To whom correspondence should be addressed

Email: [email protected]

Highlights

 Cyclotides are ultra-stable and tolerant to sequence substitutions in all loops

 Linear peptide sequences grafted in to a cyclotide scaffold are stabilized

 Sequences grafted in to a cyclotide scaffold generally maintain biological activity

 Cyclotides can penetrate cells to deliver bioactive sequences to intracellular targets

 To date 26 studies of grafted cyclotides have been reported

 Applications include cancer, pain, cardiovascular disease, obesity and others

Abstract

Cyclotides are ultra-stable peptides derived from plants. They are around 30 amino acids in size and are characterized by their head-to-tail cyclic backbone and cystine knot. Their exceptional stability and tolerance to sequence substitutions has led to their use as frameworks in drug design. This article describes recent developments in this field, particularly developments over the last two years relating to the grafting of bioactive peptide sequences into the cyclic cystine knot framework of cyclotides to stabilize the sequences. Grafted cyclotides have now been developed that interact with protein or enzyme targets, both extracellular and intracellular, as well as with cell surface receptors and membranes.

Introduction

Cyclotides [1,2] are disulfide-rich peptides from plants that have a head-to-tail cyclic backbone and cystine knot arrangement of three conserved disulfide bonds, which are linked CysI-CysIV, CysII-CysV and CysIII-CysVI. The combination of the cystine knot motif and cyclic backbone is referred to as a cyclic cystine knot (CCK) and is responsible for the exceptional stability of cyclotides. Their natural function is thought to be plant defence molecules, as they are expressed in a wide range of plant tissues and have activities against insects [3], nematodes [4], and molluscs [5], driven principally by their ability to form pores in membranes [6]. However, the exceptional stability of cyclotides has generated additional interest in their use in protein engineering and drug design applications.

Figure 1 shows the sequence and structure of the prototypical cyclotide kalata B1, originally discovered based on its presence in a plant extract used as a uterotonic medicine in the Congo region of Africa in the 1960s. In that application women boiled the above-ground parts of Oldenlandia affinis plants to make a tea which was ingested during labour to accelerate childbirth [7]. This application implies two things: (i) that the peptide survives boiling; and (ii) it survives the digestive tract and is absorbed sufficiently to exert its uterotonic effect. The structure of kalata B1 was first determined in 1995 [8], revealing the CCK motif, which helped to explain the exceptional stability of kalata B1 and its associated favourable biopharmaceutical properties. A wide range of other cyclotides have been discovered since in plants from the Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae and Fabaceae plant families [2] and cyclotide sequences are documented and regularly updated in the publicly accessible database CyBase [9]. It is conservatively estimated that the cyclotide family might comprise more than 50,000 members [10].

Fig 1 here

Several other properties, aside from their exceptional stability, have driven pharmaceutical interest in cyclotides. These include their tolerance to substitutions in their backbone loops, their potential for oral bioactivity [11], and most recently, the finding that some cyclotides are able to penetrate cells and 3

thereby potentially interact with intracellular pharmaceutical targets [12,13]. There are currently 380 published papers primarily focused on cyclotides and much of this literature has been extensively reviewed over the last decade. Thus, here we do not attempt to discuss their discovery, natural functions, structural characterisation, or biosynthesis, which have been extensively reviewed elsewhere.

(Table 1 provides a list of selected key reviews [14-24] from the last three years that cover these and other background topics). Rather, our focus here is solely on the use of cyclotides as frameworks in drug design [25-40]. These drug design applications have been underpinned by a number of fundamental studies on the synthesis and structural characterisation of cyclotides over the last decade, as schematically illustrated in Figure 1. Furthermore, studies on the cell penetration properties of cyclotides over the last few years have contributed to the possibility of using engineered cyclotides against intracellular pharmaceutical targets.

The development of chemical methods for the synthesis of cyclotides has been centrally important to their applications as drug design scaffolds. Specifically, solid phase peptide synthesis and native chemical ligation [41-46] is routinely used to assemble the cyclic backbone of cyclotides, which are well suited to this approach due to their multiple Cys residues. Recombinant methods have also been utilised to make cyclotides [47], as have chemoenzymatic cyclisation approaches [48,49]. These technologies typically are used to make engineered or grafted cyclotides in which one of the native loop sequences is replaced by a loop having a desired bioactivity. Structural studies have been important in the design and validation stages of these applications. Such structural studies of cyclotides have mainly involved NMR methods and there are currently 49 entries for cyclotides or related molecules in the PDB, 42 of which were done with NMR and seven with X-ray crystallography.

Table 1 here

Over the last decade there have been 52 papers in the literature focusing primarily on drug design applications of cyclotides, including 25 ‘grafting’ papers [11-13,50-71] where the aim is to introduce a 4

desired activity into a cyclotide framework, 11 papers concerning the synthesis/optimisation of the

CCK framework [72-82] and 16 reviews on drug design applications of cyclotides [25-40]. This information is summarised in Figure 1, which provides a schematic illustration of how inputs from fundamental studies of cyclotide synthesis, structure and cell penetration, combined with inputs from the literature on the sequences of bioactive peptides have underpinned applications used to design grafted cyclotides to hit specific targets.

Figure 2 summarises the grafting concept and highlights that a variety of target receptor types and cellular locations of targets have been explored, ranging from extracellular enzymes to cell surface receptors, membranes, and even intracellular targets, including kinases [13] or protein:protein interactions [12]. In all of these applications just two subfamilies of cyclotide frameworks have been used. Cyclotides are actually divided into three subfamilies, the Möbius, bracelet, and trypsin inhibitor subfamilies. Möbius and bracelet cyclotides are most similar to each other, differing mainly by the presence or absence of a cis-Pro peptide bond in loop 5 of the backbone sequence whereas the trypsin inhibitor cyclotides have quite different sequences. Because bracelet cyclotides are more difficult to correctly fold, almost all grafting studies have focused on Möbius or trypsin inhibitor scaffolds. The fact that bracelet cyclotides cannot practically be used as grafting frameworks is not a major limitation, given the availability of the two other complementary scaffolds, but the development of efficient folding pathways for bracelet cyclotides in future would be valuable to evaluate their potential applications. Recent studies have attempted to unravel the reasons for the difficult folding of bracelet cyclotides by examining chimeric bracelet/Möbius cyclotides [44-46] and a range of other mutated cyclotides.

Fig 2 here

Table 2 provides an analysis of the grafted cyclotides that have been reported in the literature to date, showing the epitope sizes and grafting locations (i.e. loops) used for the two utilised CCK framework subfamilies (Möbius and trypsin inhibitor). The former subfamily framework tends to be used for 5

extracellular targets and for cases when membrane binding might be advantageous, and the latter for intracellular targets or those involving enzyme inhibition. The table emphasises the vast range of applications of grafted cyclotides, from cancer to metabolic disease, pain and multiple sclerosis. It also emphasises the versatility of the CCK framework and its tolerance to loop substitutions of vastly varying content and size. For example, each and every one of the six backbone loops of the CCK framework has been used to incorporate bioactive epitopes, and the sizes of inserted epitopes have ranged from 1-21 amino acids (Table 2). We note that the grafts involving just single residue substitutions can equivalently be regarded as point mutations, and that such mutations have, in some cases, been used to optimize properties of the framework rather than introducing a target bioactivity per se. For example, single Lys substitutions in kalata B1 were used to increase membrane targeting for enhancing the nematocidal activities of cyclotides [83]. Nevertheless, in Table 2 we classify selected examples of these single-residue substitutions as examples of ‘grafting’ to highlight the generality and limits of the grafting approach.

Table 2 here

It is clear from Table 2 that most grafting studies have focused on single loop grafts, with typical epitope sizes of 6-8 amino acids. Loop 6 has been the most commonly grafted loop and loop 6 grafted cyclotides typically fold well for either Mobius or trypsin inhibitor frameworks. Loop 1 has so far only been used as a grafting site for trypsin inhibitor cyclotides, mainly owing to the fact that this is the reactive site loop of MCoTI peptides and thus naturally accommodates protease inhibitory activity, which can be tweaked for quite a wide range of different proteases. For example, loop 1 grafts have been used to make inhibitors of matriptase, foot-and-mouth-disease virus 3C protease, β-tryptase,

Bcr_Abl kinase and FXIIa (Table 2). Other trends apparent from Table 2 are that, so far, loops 2 and 3 have been relatively underrepresented in grafting studies, and that it is possible to include unnatural chirality in grafts. For example, the development of agonists of the MCR4 receptor as potential treatments for obesity involved insertion of the epitope GHfRWG into loop 6 of kalata B1 (‘f’ denotes

D-Phe). It is also worth noting that this example illustrates that agonists, as well as antagonists of receptors, can be developed using cyclotide grafting technology.

Although most grafting studies have involved single loop substitutions, the simultaneous grafting of two or more of the six CCK loops is possible, and has been used in cases where the target epitope is particularly large, or in cases where dual targeting or dual functionality is desired [68]. An example of the former is the grafting of sequences from myelin oligodendrocyte glycoprotein (MOG) into kalata

B1 for the development of drug leads for multiple sclerosis [61]. In that case, a 22-amino acid epitope spanned both loops 5 and 6 of kalata B1, with a Cys residue incorporated into the grafted epitope in a position that allowed the native disulfide bond to be retained in the CCK scaffold. An example of dual targeting was recently reported for an anti-obesity cyclotide, with the epitopes YwKV (‘w’ denotes D-

Trp) and YHLNQPF grafted into loops 5 and 6, respectively, of MCoTI-II [68]. In our opinion, such dual targeting will be an area of increasing application in future, and represents a particular advantage of the cyclotide framework over some other peptide-based modalities.

More complex grafting approaches involving modification of the cystine knot core or cyclic backbone are also possible in combination with grafting. For example, one study to develop a NS2B-NS3 Dengue protease inhibitor involved the use of a two-disulfide kalata B1 variant in which the CysII-CysV disulfide bond was removed via Cys residue substitutions and grafts were effectively inserted into loops 2 and 5, along with single point mutations in other loops. The authors of that study reported the presence of multiple isomers of the resultant two-disulfide product, and the exact structure of the final product was unclear, but at least the principles of simultaneously using several different engineering approaches is illustrated by this study [54]. Another example involved the use of a hybrid cystine knot peptide that combined two different inhibitory sequences and was cyclised via hydrazone formation.

This engineered cyclotide had high inhibitory activity against trypsin (Ki = 0.1 nM) and tryptase (Ki = 1 nM) [55]. 7

The size of cyclotides (around 30 amino acids, with a MW of 3000 Da) fills a gap between traditional small molecule drugs (MW <500 Da) and larger biologics (MW > 5000 Da) such as antibodies [84] and to some extent this gives them some advantages over both; being bigger than small molecules they are more suited for blocking protein: protein interactions, and being smaller than antibodies they have better cell penetrating properties. Thus, cyclotides potentially can access targets that are inaccessible to either small molecules or antibodies. Of particular current interest is the use of cyclotides as carriers to deliver bioactive cargoes inside cells. The first published example exemplifying this principle involved the use of a grafted trypsin inhibitor cyclotide (MCoTI-I) to modulate the p53 HDM2 intracellular protein-protein interaction to reduce tumour growth in a mouse xenograft model [12]. Another recent example was the use of grafted MCoTI-II to targeting the intracellular SET protein implicated in leukaemia [13].

Finally, since the natural function of cyclotides is as host defence agents, with a common mechanism of action involving membrane binding and disruption, there is an opportunity to use controlled membrane targeting of cyclotides for peptide epitope delivery. Recent studies have shown that the membrane binding of kalata B1 is mediated by interactions with phosphatidylethanolamine [15] and thus lipid selectivity might in principle be used to selectively target cyclotides to cells with particular lipid compositions on their surface. Supporting the proposal of selectivity in membrane targeting, another study showed that cyclotides from different subfamilies (Möbius and bracelet) differed in their orientation when binding to membranes [85]. It has also been reported that cycloviolacin O2 (a bracelet cyclotide) has stronger cytotoxicity against various cancer cell lines than kalata B1 or kalata B2

(Möbius subfamily members) and was more potent towards anionic DOPC/DOPA liposomes than zwitterionic DOPC, suggesting a degree of cell specificity related to the number of positive charged residues in the cyclotide framework [86].

Conclusions

Cyclotides are now a widely-studied family of plant proteins and are gaining acceptance as potential drug design scaffolds. No cyclotides have yet reached human clinical trials but, in our opinion, there is a good possibility that this may occur in the not too distant future. The biggest challenge in the field at the moment is increasing the oral bioavailability of peptide-based leads. Although there are several reported cases where cyclotides have oral activity, there is limited literature on the actual oral bioavailability and so more quantitative studies reporting such biopharmaceutical parameters are expected over coming years. This information will make a valuable addition to the wealth of information currently available on the stability and pharmaceutical ‘graftability’ of cyclotides.

Acknowledgements

Work on cyclotides in our laboratory is supported by the Australian Research Council (DP150100443) and the NHMRC, Australia (APP1084604). DJC is an ARC Australian Laureate Fellow

(FL150100146).

Figure captions

Figure 1. Structure of the prototypic cyclotide kalata B1 and schematic overview of drug design applications. A. Peptide sequence in one-letter amino acid code showing the disulfide connectivities.

B. Three dimensional structure (PDBID: 1NB1) showing the cystine knot arrangement of the three conserved disulfide bonds and the labelling of backbone loops between successive Cys residues.

Cyclotides range in size form 28-37 amino acids, with the variation reflecting different loop lengths. It is estimated that the cyclotide family will comprise approximately 50,000 members [10]. C. Schematic illustration of how recent fundamental studies on the synthesis, structures, and (for intracellular targets) cell penetration, have underpinned applications in drug design. The numbers of papers referred to in each box include papers published from 2006 onwards on the indicated topic.

Figure 2. Schematic illustration of the grafting process to highlight the diverse range of targets accessible to cyclotides. These targets range from: (A) cell surface receptors [51], to (B) cell membranes [15], to (C) extracellular targets (e.g. FMDV 3C protease [52]), to (D) intracellular targets

[12,13]. Two types of cyclotide scaffolds are shown: kalata B1 as a representative of the Mӧbius subfamily, and MCoTI-II as a representative of the trypsin inhibitor subfamily. The grafting process involves insertion of a bioactive epitope into one of these stable CCK frameworks. In the examples shown, loop 6 has been used to incorporate a bioactive helix.

Table 1. Selected recent (post 2014) reviews on cyclotides.

Topic Title Ref

General Review Chemistry and biology of cyclotides: Circular plant peptides [18]

outside the box

Historical accounts Joseph Rudinger memorial lecture: Discovery and applications [16]

of cyclotides

Discovery Discovery, structure, function, and applications of cyclotides: [24]

Circular proteins from plants

Sequencing Primary Structural Analysis of Cyclotides [19]

Structure Structural Studies of Cyclotides [20]

Bioactivity Circling the enemy: Cyclic proteins in plant defence [14]

Synthesis Chemical and Biological Production of Cyclotides [22]

Biosynthesis Cyclotide biosynthesis [17]

Membrane binding Importance of the cell membrane on the mechanism of action [15]

of cyclotides

Membrane binding The increasing role of phosphatidylethanolamine as a lipid [23]

receptor in the action of host defence peptides

Biotechnological Cyclotides in a Biotechnological Context: Opportunities and [21]

applications Challenges

Drug design There have been sixteen reviews on this topic in since 2006, [25-

applications with most focussing in the use of the CCK as a scaffold, 40]

including three [38-40] post 2014 reviews

Table 2. Summary of published grafting studies using CCK framework as drug design scaffold

Biological Activity Loop Grafted sequence Potency Application Ref /Dose Proof-of-concept studya 5 DK - Confirm plasticity of CCK scaffold [50] KNK VEGF-A antagonist 2 RRKRRR Anti-angiogenesis with the potential [51] 3 RRKRRR to reduce blood vessel growth in 3 NTRRKRRRG 12b tumours. 5 RRKRRR 5 RRKRRRV 6 RRKRRR Dengue NS2B-NS3 2&5 SEESRRG&RRSRc 1.39±0.35d Dengue Fever [54] protease inhibitor 3.03±0.75d

Melanocortin 4 receptor 6 GHFRWG 580 Obesity [58] (MC4R) agonist 6 GHfRWGe 6 HFRWG e Mӧbius 6 HfRWG subfamily g h Bradykinin B1 receptor 6 KRPPGFSPL 49% , 42% Chronic pain & inflammatory pain [11] antagonist 6 QIPGLGPL 38%g, 28%h Neuropilin-1/-2 antagonist 5&6 KPLR& KAPRMVRi 100i, f Inhibit endothelial cell migration, [59] angiogenesis, lymphangiogenesis Immunomodulation 5 RSPFSRV 28.1±16.6j Multiple sclerosis [61] (Experimental autoimmune 5 6 others encephalomyelitis, EAE, 6 6 others mouse model) 5&6 4 others Experimental autoimmune 4 K 20k Multiple sclerosis, T-cell-mediated [71] encephalomyelitis 2 K disorders

HIV gp120 inhibitor 5&6 Sl&GSFLRFLTKG -1067m Anti-HIV [66] 5&6 Sl&GSFLTGQGSF -959m CXCR4 antagonist, 6 YRXCRGpRRBCYXKn 20f,o, 2f,o Anticancer and anti HIV-1 [57] HIV-1 cell entry blocker 6 7 others Hdm2/HdmX antagonist 6 GASRAPTSFAEYWNLLSA 2.3±0.1q & Inhibit tumour growth via [12] 6 GASKAPTSFAEYWNLLSA 9.7±0.9q modulation of intracellular protein- 6 GASKAPTSAAEYWNLLSA protein interaction 6 GASRAPTSAAEYWNLLSA 6 GASRAPTSFAEYZNLLSAp 2.6 ±0.4q FMDV 3C protease 1 Q 41±25d Foot-and-mouth disease [52] inhibitor 1 AKQ 56±35d 1 F+3 othersr > 100d β-tryptase inhibitor 3,5&6 LAG, GPNGF&AKKVHseZs 0.001d Allergic asthma and inflammation [55] 3,5&6 LAG, GPNGF&SHseZDGs 4d disorders

β-tryptase and HLE 1 V >50d, t Inflammatory disease [53] inhibitor 1 6 others &0.021d, t 6 SDGGl 0.009d, t 6 7 others &0.0025d, t u

subfamily Angiogenic 6 SIKVAV 14.6 Cardiovascular disease and/or wound [56] 6 SVVYGLR 13.9u healing Trypsin inhibitor Trypsin and matriptase 6 R 0.00001d,v& Anticancer [60] inhibitor plus 15 others 0.00029d,v CTLA-4 1,3&6 KYSHVP, K&PR 3.7 w Immunotherapy of cancer and other [65] diseases 1,3&6 9 others Synuclein-induced 6 SLATWAVG Explore the feasibility of phenotypic [64] cytotoxicity screening of bioactive cyclotides BCR-ABL kinase inhibitor 1&6 EAIYAAPK&GEAIYAAPFAR 1.3±0.1b Chronic myeloid leukaemia [63] 1 4 others 6 7 others 1&6 2 others Cell migration inhibitor 6 GViTRIRx Anti-angiogenesis [62]

LyP1 marker for tumour 1 GNKRTRG >128b, y Anti-tumour and anticancer [69] lymphatics 2 GNKRTRG >128/n.d. b, y 6 GNKRTRG >128 b, y SET antagonist 6 GASKAPASXLRKLXKRLLRDAz 2.9±0.14b, z Anticancer [13] 6 GASKAPASXLRKLXKRLLz 5.0±0.15b, z Aniothensin (1-7) receptor 6 EIVYRX* Lung cancer or myocardial infarction [67] Anti-angiogenic 5&6 YwKV&YHLNQPF# 1#, f Anticancer [68] 7 others FXIIa and FXa inhibitors 1&6 FR&Q 0.1Δ, 0.46Δ Thrombosis, cardiovascular diseases [70] 1&6 SR&Q 2.16Δ, 96 Δ 1&6 FRW&Q 0.51Δ, >100 Δ 1&6 FRW&KQ 0.49 Δ, >100 Δ a b c Proof-of-concept study to demonstrate that loops are tolerant to modification. IC50, half maximal inhibitory concentration (μM). C9, C21 are substituted by E and R (bold), respectively; There are also mutations E7S, T8E, T20R and N29R in loops1,4 and 6. The d two isomers of grafted cyclotides have Kis of 1.39 ± 0.35 μM (isomer B) and 3.03 ± 0.75 μM (isomer C), respectively. Ki, inhibition e f g constant (μM). Residue f refers to D-phenylalanine. EC50, half maximal effective concentration (nM). Inhibition of writhing after intraperitoneal injection (i.p. 1 mg/kg). h Inhibition of writhing under oral administration (p.o. 10 mg/kg). i There are mutations of j V10M in loop2 and N15S, P16L in loop3, the EC50 was tested on HUVEC cells. Cumulative clinical score using experimental autoimmune encephalomyelitis mouse model was significantly lower than the cumulative score of control group. k Oral administration (mg/kg). l Truncated residue/s. m The binding affinity was determined by measuring the non-covalent interaction energy, which is a combination of electrostatic and VDW interaction energies (kJ/mol). n Single letter codes B, X, and p represent the amino acids 2- o naphthylalanine, citruline, and D-proline, respectively. A potent CXCR4 antagonist with EC50 ≈ 20 nM and an efficient HIV-1 cell- p q entry blocker with EC50 ≈ 2 nM. Z=6-chlorotryptophan. Binding affinity of grafted cyclotides to recombinant Hdm2 and HdmX was measured by fluorescence polarisation anisotropy. Cyclotide MCo-PMI-K37R displayed strong affinity for Hdm2 (KD = 2.3 ± 0.1 nM) r and HdmX (KD = 9.7 ± 0.9 nM); MCo-PMI-K37R-F42A had affinity of Hdm2 (KD = 2.6 ± 0.1 nM). Another three cyclotides have mutations K10R, K10V, K10A. s Hse, homoserine; Z, N-terminal glyoxylyl residue. t MCoTI-II[K10V] displayed inhibitory activity of Ki > 50 μM (tryptase) and Ki ≈ 0.021 μM (HLE); MCoTI-II[SDGG truncated] had inhibitory activity of Ki ≈ 0.009 μM (tryptase), u v and Ki ≈ 0.0026 μM (trypsin). The lowest concentration with significant difference compared to control (μM). MCoTI-II[V3R] w displayed inhibition of Ki ≈ 0.00001 μM (trypsin) and Ki ≈ 0.00029 μM (matriptase). Binding constant was estimated using ELISA(μM). x Letter i=D-Isoleucine. y Cytotoxicity of grafted cyclotide in loop 1and loop 6 against normal and cancer cell lines were same as IC50>128 μM (MDA-MB-435S, MDA-MB-231, MM96L, HFF1), the IC50 of grafted cyclotide in loop 2 was >128 μM in

z MDA-MB-435S and not determined in other three cell lines. Letter X represents amino-isobutyric acid. The IC50 was tested against * # K562 cells. Residue X represents L-2,3-diaminopropionic acid. Residue w represents D-tryptophan, the EC50 was tested on HUVEC Δ cells. Inhibition constants of each grafted cyclotide for FXIIa and FXa are Ki ≈ 0.11 μM and 0.46 μM, 2.16 μM and 96 μM, 0.51 μM and >100 μM, 0.49 μM and >100 μM, respectively.

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41. Dawson PE, Muir TW, Clark-Lewis I, Kent SB: Synthesis of proteins by native

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42. Aboye T, Kuang Y, Neamati N, Camarero JA: Rapid parallel synthesis of bioactive

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43. Akcan M, Craik DJ: Synthesis of cyclic disulfide-rich peptides. In Peptide Synthesis

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44. Gunasekera S, Daly N, Clark R, Craik DJ: Dissecting the oxidative folding of circular

cystine knot miniproteins: Development of hybrid cyclotide scaffolds for protein

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45. Aboye TL, Clark RJ, Burman R, Roig MB, Craik DJ, Goransson U: Interlocking

disulfides in circular proteins: toward efficient oxidative folding of cyclotides.

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46. Cheneval O, Schroeder CI, Durek T, Walsh P, Huang Y-H, Liras S, Price DA, Craik DJ:

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47. Aboye TL, Camarero JA: Biological synthesis of circular polypeptides. J. Biol. Chem.

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48. Nguyen GKT, Wang SJ, Qiu YB, Hemu X, Lian YL, Tam JP: Butelase 1 is an Asx-

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**Paper reporting the isolation of the enzyme butelase-1 with broad utility for

cyclising peptides.

49. Harris KS, Durek T, Kaas Q, Poth AG, Gilding EK, Conlan BF, Saska I, Daly NL, van

der Weerden NL, Craik DJ, et al.: Efficient backbone cyclization of linear peptides by

a recombinant asparaginyl endopeptidase. Nat. Commun. 2015, 6.

**Paper reporting the recombinant production of the enzyme OaAEP1b with broad

utility for cyclising peptides.

50. Clark RJ, Daly NL, Craik DJ: Structural plasticity of the cyclic-cystine-knot

framework: implications for biological activity and drug design. Biochem. J. 2006,

394:85-93.

51. Gunasekera S, Foley FM, Clark RJ, Sando L, Fabri LJ, Craik DJ, Daly NL: Engineering

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52. Thongyoo P, Roque-Rosell N, Leatherbarrow RJ, Tate EW: Chemical and biomimetic

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53. Thongyoo P, Bonomelli C, Leatherbarrow RJ, Tate EW: Potent inhibitors of β-tryptase

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54. Gao Y, Cui T, Lam Y: Synthesis and disulfide bond connectivity-activity studies of a

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55. Sommerhoff CP, Avrutina O, Schmoldt HU, Gabrijelcic-Geiger D, Diederichsen U,

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56. Chan LY, Gunasekera S, Henriques ST, Worth NF, Le SJ, Clark RJ, Campbell JH, Craik

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57. Aboye TL, Ha H, Majumder S, Christ F, Debyser Z, Shekhtman A, Neamati N, Camarero

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58. Eliasen R, Daly NL, Wulff BS, Andresen TL, Conde-Frieboes KW, Craik DJ: Design,

synthesis, structural and functional characterization of novel melanocortin agonists

based on the cyclotide kalata B1. J. Biol. Chem. 2012, 287:40493-40501.

59. Getz JA, Cheneval O, Craik DJ, Daugherty PS: Design of a cyclotide antagonist of

neuropilin-1 and -2 that potently inhibits endothelial cell migration. ACS Chem. Biol.

2013, 8:1147-1154.

60. Quimbar P, Malik U, Sommerhoff CP, Kaas Q, Chan LY, Huang Y-H, Grundhuber M,

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61. Wang CK, Gruber CW, Cĕmažar M, Siatskas C, Tagore P, Payne N, Sun G, Wang S,

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62. Chan Lai Y, Craik David J, Daly Norelle L: Cyclic thrombospondin-1 mimetics:

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63. Huang Y-H, Henriques ST, Wang CK, Thorstholm L, Daly NL, Kaas Q, Craik DJ:

Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold.

Sci. Rep. 2015, 5:12974.

64. Jagadish K, Gould A, Borra R, Majumder S, Mushtaq Z, Shekhtman A, Camarero JA:

Recombinant expression and phenotypic screening of a bioactive cyclotide against

α-synuclein-induced cytotoxicity in baker's yeast. Angew. Chem. Int. Ed. 2015,

54:8390-8394.

*The first paper to report the recombinant expression of cyclotides inside live yeast

cells.

65. Maaß F, Wustehube-Lausch J, Dickgiessr S, Valldorf B, Reinwarth M, Schmoldt HU,

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66. Sangphukieo A, Nawae W, Laomettachit T, Supasitthimethee U, Ruengjitchatchawalya

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as HIV gp120 inhibitor. PLoS One 2015, 10.

67. Aboye T, Meeks C, Majumder S, Shekhtman A, Rodgers K, Camarero J: Design of a

MCoTI-based cyclotide with angiotensin (1-7)-like activity. Molecules 2016, 21:152.

68. Chan LY, Craik DJ, Daly NL: Dual-targeting anti-angiogenic cyclic peptides as

potential drug leads for cancer therapy. Sci. Rep. 2016, 6:35347.

**An example of dual grafting of a cyclotide scaffold- in this case by grafting two

anti-angiogenic epitopes targeting cancer.

69. Conibear AC, Chaousis S, Durek T, Rosengren KJ, Craik DJ, Schroeder CI: Approaches

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70. Swedberg JE, Mahatmanto T, Abdul Ghani H, de Veer SJ, Schroeder CI, Harris JM,

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71. Thell K, Hellinger R, Sahin E, Michenthaler P, Gold-Binder M, Haider T, Kuttke M,

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cyclic peptide for the treatment of multiple sclerosis. Proc. Natl. Acad. Sci. U. S. A.

2016, 113:3960-3965.

*A paper reporting oral activity for a cyclotide active in an animal model of

multiple sclerosis.

72. Kimura RH, Tran A-T, Camarero JA: Biosynthesis of the cyclotide kalata B1 by using

protein splicing. Angew. Chem. Int. Ed. 2006, 118:987-990.

73. Werle M, Schmitz T, Haung H-L, Wentzel A, Kolmar H, Bernkop-Schnurch A: The

potential of cystine-knot microproteins as novel pharmacophoric scaffolds in organ

peptide drug delivery. J. Drug Target. 2006, 14:137-146.

74. Aboye TL, Clark RJ, Craik DJ, Göransson U: Ultra stable peptide scaffolds for protein

engineering - synthesis and folding of the circular cystine knotted cyclotide

cycloviolacin O2. ChemBioChem 2008, 9:103-113.

75. Avrutina O, Schmoldt HU, Gabrijelcic-Geiger D, Wentzel A, Frauendorf H, Sommerhoff

CP, Diederichsen U, Kolmar H: Head-to-tail cyclized cystine-knot peptides by a

combined recombinant and chemical route of synthesis. ChemBioChem 2008, 9:33-

37.

76. Austin J, Wang W, Puttamadappa S, Shekhtman A, Camarero JA: Biosynthesis and

biological screening of a genetically encoded library based on the cyclotide MCoTI-

I. ChemBioChem 2009, 10:2663-2670.

77. Getz JA, Rice JJ, Daugherty PS: Protease-resistant peptide ligands from a knottin

scaffold library. ACS Chem. Biol. 2011, 6:837-844.

78. Glotzbach B, Reinwarth M, Weber N, Fabritz S, Tomaszowski M, Fittler H, Christmann

A, Avrutina O, Kolmar H: Combinatorial optimization of cystine-knot peptides

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79. Jagadish K, Borra R, Lacey V, Majumder S, Shekhtman A, Wang L, Camarero JA:

Expression of fluorescent cyclotides using protein trans-splicing for easy monitoring

of cyclotide–protein interactions. Angew. Chem. Int. Ed. 2013, 52:3126-3131.

*This paper showed that cyclotides containing the non-natural amino acid AziF can

be labeled to monitor cyclotide-protein interactions.

80. Koehbach J, O’Brien M, Muttenthaler M, Miazzo M, Akcan M, Elliott AG, Daly NL,

Harvey PJ, Arrowsmith S, Gunasekera S, et al.: Oxytocic plant cyclotides as templates

for peptide G protein-coupled receptor ligand design. Proc. Natl. Acad. Sci. U. S. A.

2013, 110:21183-21188.

81. Gray K, Elghadban S, Thongyoo P, Owen KA, Szabo R, Bugge TH, Tate EW,

Leatherbarrow RJ, Ellis V: Potent and specific inhibition of the biological activity of

the type-II transmembrane serine protease matriptase by the cyclic microprotein

MCoTI-II. Thromb. Haemost. 2014, 112:402-411.

82. Senthilkumar B, Kumar P, Rajasekaran R: In-silico template selection of in-vitro

evolved kalata B1 of Oldenlandia affinis for scaffolding peptide-based drug design.

J. Cell. Biochem. 2016, 117:66-73.

83. Huang YH, Colgrave ML, Clark RJ, Kotze AC, Craik DJ: Lysine-scanning mutagenesis

reveals a previously unidentified amendable face of the cyclotide kalata B1 for the

optimisation of nematocidal activity. J. Biol. Chem. 2010, 285:10797-10805.

84. Craik DJ, Fairlie DP, Liras S, Price D: The future of peptide-based drugs. Chem. Biol.

Drug Des. 2013, 81:136-147.

*A recent review on the potential of peptides, including cyclotides, in drug

development.

85. Wang CK, Colgrave ML, Ireland DC, Kaas Q, Craik DJ: Despite a conserved cystine

knot motif, different cyclotides have different membrane binding modes. Biophys. J.

2009, 97:1471-1481.

86. Burman R, Stromstedt AA, Malmsten M, Göransson U: Cyclotide-membrane

interactions: Defining factors of membrane binding, depletion and disruption.

Biochim. Biophys. Acta - Biomembr. 2011, 1808:2665-2673.