Cyclotides: disulfide-rich peptide toxins in plants

1

Cyclotides: disulfide-rich peptide toxins in plants

Yen-Hua Huang, Qingdan Du and David J. Craik*

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

Australia

*Corresponding Author:

Professor David J. Craik

Institute for Molecular Bioscience,

The University of Queensland,

Brisbane, Qld, 4072, Australia

Tel: 61-7-3346 2019

Fax: 61-7-3346 2101

e-mail: [email protected]

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Abstract 1

Cyclotides are a plant-derived family of peptides that comprise approximately 30 amino acid residues, 2

a cyclic backbone and a cystine knot. Due to their unique structure, cyclotides are exceptionally stable 3

to heat or proteolytic degradation and are tolerant to amino acid substitutions in their backbone loops 4

between conserved cysteine residues. Their toxicity to insect pests and their make-up of natural amino 5

acids has led to their applications in eco-friendly crop protection. Furthermore, their stability and cell 6

penetrating properties make cyclotides ideal scaffolds for bioactive epitope grafting. This article gives 7

a brief overview of cyclotide discovery, characterization, distribution, synthesis and mode of action 8

mechanisms. We focus on their toxicities to insect pests and their medical and agricultural 9

applications. 10

Keywords 11

Cyclotides; Stability; Insecticidal peptide; Cytotoxicity; Cell penetrating peptide; Applications 12

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1 Introduction 13

Cyclotides are disulfide-rich peptides from plants that have been known now for nearly two decades 14

(Craik et al., 1999). They typically comprise 30 amino acids and contain the characteristic features of 15

a head-to-tail cyclized backbone and a knotted arrangement of three disulfide bonds. They occur in a 16

wide range of plant tissues, including leaves, flowers, stems and roots, and are thought to be present 17

as natural defense molecules, particularly against insects and nematodes. For this reason, they can be 18

regarded as plant toxins, i.e., molecules that are toxic to certain plant pests. However, most interest in 19

cyclotides has so far related to their exceptional stability and potential as a framework in drug design 20

rather than to their natural pesticidal functions. Here we provide an overview on the discovery, 21

structures and potential biotechnological applications of cyclotides. There have been a number of 22

recent reviews on the discovery and distribution (Gruber, 2010; Weidmann & Craik, 2016), 23

biosynthesis (Conlan et al., 2012; Craik et al., 2018; Qu et al., 2017), biological activities (Craik, 2012; 24

Daly et al., 2011; Göransson et al., 2012), mode of action (Henriques & Craik, 2012; Henriques & 25

Craik, 2017), and applications in drug design (Camarero, 2017; Craik & Du, 2017; de Veer et al., 26

2017; Gould & Camarero, 2017; Gould et al., 2011; Poth et al., 2013; Wang & Craik, 2018) of 27

cyclotides as well as a recent analysis of publication trends in the cyclotide field (Kan & Craik, 2018). 28

The main aim of this article is to explore the context of cyclotides as toxins with interesting 29

biotechnological applications. 30

1.1 History of discovery 31

The first cyclotide discovered was kalata B1 which attracted attention due to its use in an indigenous 32

medicine in Africa (Gran, 1970, 1973a). In this usage, leaves from the Rubiaceae family plant 33

Oldenlandia affinis were boiled by Congolese women to make a tea that was ingested during labor, 34

resulting in accelerated child birth. This discovery was made by a Norwegian physician, Lorents Gran, 35

who observed the accelerated labor while serving on a Red Cross relief mission in the Congo in the 36

1960s. After taking some of the plant material back to Norway he discovered that the uterotonic 37

ingredient was a peptide, which he and colleagues partially characterized and found to comprise 38

approximately 30 amino acids (Gran, 1973a). At that stage, neither the disulfide knotted arrangement 39

nor the head-to-tail cyclized backbone was apparent, but Gran noted that the peptide was very stable 40

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and was one of a number of peptides of similar molecular weight derived from an extract of the plant 41

leaves (Gran, 1973b). 42

The three-dimensional structure of kalata B1 was not elucidated until the mid-1990s, when mass 43

spectrometry and NMR studies were used to delineate the cyclic backbone and the knotted 44

arrangement of disulfide bonds, respectively (Saether et al., 1995). Around the same time, several 45

independent groups reported peptides of similar size with pharmaceutically relevant activities, which 46

had been discovered in the course of plant natural products screening programs. A report from a group 47

at Merck Research Laboratories described cyclopsychotride A, a macrocyclic peptide from a South 48

American plant from the Rubiaceae family, which had neurotensin antagonistic properties (Witherup 49

et al., 1994). In another report, a group at the National Cancer Institute, USA, noted the anti-HIV 50

activities of a series of macrocyclic peptides, which they named circulins because of their head-to-tail 51

cyclic backbone (Gustafson et al., 1994). The circulins were derived from the bark of a Tanzanian 52

tree, also from the Rubiaceae family. Earlier, the discovery of another macrocyclic peptide of similar 53

size from a violet plant (Violaceae family) was reported by an Austrian group, found while looking 54

for saponins with hemolytic activity (Schöpke et al., 1993). 55

Following these initial reports, two groups set up systematic discovery programs to see whether 56

similar peptides occur in related plants. Our group at The University of Queensland (Australia) found 57

a number of examples in plants of Rubiaceae and Violaceae families (Craik et al., 1999), while a 58

group in Sweden focused on plants from the Violaceae family (Claeson et al., 1998; Göransson et al., 59

1999). These combined discoveries in the late 1990s led to the conclusion that there were indeed 60

many other members of this peptide family and the name “cyclotide” was coined to refer to plant-61

derived head-to-tail cyclic peptides that contain a cystine knot motif (see Figure 1) (Craik et al., 1999). 62

1.2 Sequences and nomenclature of cyclotides 63

Currently more than 400 sequences of cyclotides have been reported and are documented in a database 64

called CyBase (www.cybase.org.au), which also contains data on other families of naturally occurring 65

cyclic peptides (Wang et al., 2008b). Figure 1 shows some example sequences from the three currently 66

defined subfamilies of cyclotides and a representative structure of prototypic examples from each 67

subfamily. 68

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Figure 1: Selected sequences and three-dimensional structures from the three subfamilies of 70

cyclotides. A. An alignment of selected cyclotides sequences for which a solution structure has been 71

published. The sequences are aligned starting from the presumed N-terminal cleavage point in the 72

linear precursors. Cyclotides are classified into three subfamilies and listed chronologically according 73

to the publication date of their NMR derived structures. All cyclotides have six loops separated by six 74

conserved cysteines (numbered with Roman numerals I to VI) and are head-to-tail cyclized. The 75

cysteines are highlighted in yellow and the disulfide connectivities (I to IV, II to V and III to VI) are 76

shown at the top of the table with a black line. B. The solution structures (from left to right) of kalata 77

B1 (Rosengren et al., 2003), kalata B8 (Daly et al., 2006) and MCoTI-II (Felizmenio-Quimio et al., 78

2001) from the Möbius, bracelet and trypsin inhibitor subfamilies, respectively. The disulfide bonds 79

are indicated by yellow ball-and-stick structures, the beta sheets by blue arrows and the 310-helix turn 80

by red ribbon. 81

Initially, cyclotides were classified into two major subfamilies: the bracelet and Möbius 82

subfamilies, based on the absence or presence, respectively, of a cis-proline in loop 5 of the circular 83

backbone (the loops being defined as the backbone segments between successive Cys residues, as 84

shown in Figure 1). The origin of this nomenclature is quite straightforward: when all of the backbone 85

peptide linkages of a cyclic peptide exist in the usual trans arrangement, the backbone can be thought 86

of as bracelet-like; however, a three-dimensional structure containing a cis-proline results in a 180° 87

conceptual twist in the circular backbone, which can thus be topologically regarded as a Möbius strip 88

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(Jennings et al., 2005). A third, smaller, subfamily of cyclotides was introduced upon discovery of 89

MCoTI-I and MCoTI-II from the tropical vine Momordica cochinchinensis (Hernandez et al., 2000). 90

Although these two trypsin inhibitor peptides are dissimilar from other cyclotides in sequence, they 91

share the common structural elements of three interlocked disulfide bridges and a cyclic backbone, 92

leading to their classification as cyclotides (Felizmenio-Quimio et al., 2001). They are alternatively 93

referred to as cyclic knottins (Chiche et al., 2004). 94

1.3 Cyclic cystine knot scaffold and structures of cyclotides 95

In addition to the structural elucidation of the prototypic cyclotide, kalata B1, numerous other 96

members of the family have been structurally characterized. Figure 1A shows a sequence alignment 97

of the 18 structurally characterized cyclotides. To date, NMR structures have been determined for: 98

eight cyclotides from the Möbius subfamily, including kalata B1 (Saether et al., 1995), kalata B2 99

(Jennings et al., 2005), cycloviolacin O14 (Ireland et al., 2006a), kalata B7 (Shenkarev et al., 2008), 100

varv F (Wang et al., 2009b), vhl-2 (Daly et al., 2010), Cter M (Poth et al., 2011a), and kalata B12 101

(Wang et al., 2011); ten cyclotides from the bracelet subfamily, including circulin A (Daly et al., 102

1999a), cycloviolacin O1 (Rosengren et al., 2003), palicourein (Barry et al., 2004), vhr1 (Trabi & 103

Craik, 2004), circulin B (Koltay et al., 2005), tricyclon A (Mulvenna et al., 2005), vhl-1 (Chen et al., 104

2005), kalata B8 (Daly et al., 2006), cycloviolacin O2 (Wang et al., 2009a), and kalata B5 (Plan et al., 105

2010); and three cyclotides from the trypsin inhibitor subfamily MCoTI-II (Felizmenio-Quimio et al., 106

2001), MCoTI-I (Kwon et al., 2018) and MCoTI-V (Mylne et al., 2012). 107

As can be seen in the sequence alignment in Figure 1A, loops 1 and 4 are highly conserved in 108

terms of both their number of residues and amino acid composition (Daly et al., 2009). The 109

conservation of these two loops is probably due to the fact that they form the central part of the 110

embedded cystine knot in cyclotides. Additionally, a highly conserved glutamic acid present in loop 111

1 has been shown to take part in a hydrogen-bond network that is important in stabilizing the structure 112

(Rosengren et al., 2003). In fact, this glutamic acid is almost completely conserved in both Möbius 113

and bracelet subfamilies, with the exception of kalata B12 (Plan et al., 2007), where the glutamic acid 114

is substituted for an aspartic acid in the corresponding position. Loop 1 in the trypsin inhibitor 115

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subfamily lacks the Glu residue and contains a larger number of residues than the corresponding loop 116

in the Mobius and bracelet cyclotides. 117

Despite the sequence diversity in the other four loops (i.e. loops 2, 3, 5, and 6), cyclotides from 118

the three subfamilies all share the same structural features of the cyclic backbone and a cystine knot 119

core formed from three disulfide bonds (Figure 1B). The cystine knot motif is surrounded by a small 120

β-hairpin which, in most cyclotides, is combined with a third β-strand to form a distorted triple-121

stranded β-sheet. This so-called cyclic cystine knot (CCK) is a special case of a common motif known 122

as the inhibitor cystine knot (ICK) scaffold (Craik et al., 2001), which is found in a wide range of 123

proteins from plants and animals. Overall, the combination of a head-to-tail cyclic backbone and a 124

cystine knot makes cyclotides extremely resistant to proteolytic breakdown, to high temperatures and 125

to chemical denaturants such as urea or guanidine (Colgrave & Craik, 2004). This stability might 126

explain why they are excellent insecticidal agents in plants, as this stability is presumably important 127

for protein-based natural products that accumulate in leaves without breakdown under harsh 128

environmental conditions. Stability is also the reason why cyclotides have attracted attention from a 129

drug design perspective. 130

1.4 Distribution of cyclotides in the plant kingdom, in individual plants, and within 131

individual tissues 132

So far cyclotides have been discovered in a range of species from five major plant families, i.e. the 133

Rubiaceae, Violaceae, Cucurbitaceae, Solanaceae, and Fabaceae families (Koehbach et al., 2013a). 134

These represent many economically important plant families and hence cyclotides are potentially of 135

broad interest in plant science. Their distribution within these plant families is highly variable; so far 136

every Violaceous plant examined has been found to contain cyclotides, suggesting that cyclotides are 137

ubiquitous in this family (Burman et al., 2010a). Violaceae comprises approximately 930 species that 138

are distributed both in temperate and tropical zones around the world and include common ornamental 139

plants such as pansies. By contrast, cyclotides occur in less than 5% of the Rubiaceae (Gruber et al., 140

2008) and in only a few members of each of the other plant families, i.e. in two species of the 141

Solanaceae (Poth et al., 2012; Zenoni et al., 2011) and the Cucurbitaceae family (Du et al., 2019; 142

Hernandez et al., 2000), and only one from the Fabaceae (Poth et al., 2011b) so far. Cyclotide-143

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containing plant families, the corresponding orders and the reported number of cyclotides from each 144

plant family are summarized in Figure 2 (Craik, 2013). 145

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Figure 2: Distribution of cyclotides within angiosperms (flowering plants). Cyclotide-bearing 147

plant families and the corresponding orders are highlighted, with the number of cyclotides reported 148

in the literature listed next to the plant images. All cyclotides reported to date were found in core 149

eudicot plants, and only acyclic variants were discovered in a monocot plant from the Poaceae family 150

(labeled with one asterisk) (Nguyen et al., 2013). Small circular peptides distinct from cyclotides in 151

sequence were derived from the Asteraceae family (labeled with two asterisks). The phylogenetic 152

information of angiosperms was obtained from the Angiosperm Phylogeny Website 153

(http://www.mobot.org/MOBOT/research/APweb/). Figure adapted from a previously published article 154

(Craik, 2013). 155

To date, cyclotides have only been discovered in core eudicot plants. It is expected that as more 156

investigations take place, the number of plant families that are reported to contain cyclotides will 157

increase, as will the number of cyclotide-bearing species. However, at this stage it is clear that many 158

plants do not contain cyclotides, but those that do, contain them in large amounts. There remain many 159

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interesting questions as to why some plants have evolved the ability to produce cyclotides but most 160

have apparently not. 161

In the cyclotide-bearing plants that have been examined so far, cyclotides occur in many, 162

indeed probably all, tissues. For example, they occur in leaves, petioles, stems, pedicels, flowers, and 163

roots (Trabi & Craik, 2004). A single plant may contain dozens to hundreds of cyclotides. For example, 164

Viola hederacea has been reported to contain at least 66 cyclotides (Trabi & Craik, 2004) and O. 165

affinis has so far been found to contain 22 cyclotides (Plan et al., 2007). It is expected the number of 166

individual cyclotides known to occur in plants will increase dramatically as more transcriptome and/or 167

genomic studies are published (Koehbach et al., 2013a). Until recently, most discoveries have been 168

made at the peptide rather than nucleic acid level, but this is likely to change in future. Despite this 169

shift to nucleic acids based discovery, new MS-based sequencing approaches for cyclotides show 170

promise for ongoing discoveries at the peptide-level (Parsley et al., 2018). 171

Within a single plant, the distribution and type of cyclotides varies from tissue to tissue. For 172

example, one study showed that the cyclotides present in roots in V. hederacea were typically more 173

hydrophobic than those found in the leaves and flowers (Trabi & Craik, 2004). In only a few cases is 174

the same cyclotide found in multiple plants. For example, varv A is found in four different plants, 175

including O. affinis, Viola odorata, Viola tricolor, and Viola arvensis (Gruber et al., 2008). Similarly, 176

within an individual plant, some cyclotides occur in several tissues and others are specific to a given 177

tissue. For example, vhr1 occurs only in roots in V. hederacea but kalata B1 occurs in leaves, stems 178

and roots in O. affinis (Trabi & Craik, 2004). 179

Imaging studies suggest that there is a non-uniform distribution of cyclotides within a given 180

tissue type. For example, matrix-assisted laser desorption/ionization-mass spectrometric imaging 181

(MALDI-MSI) has been applied to assess the spatial distribution and relative abundances of 182

cyclotides within the leaves of Petunia x hybrida. The study revealed four distinct masses on a P. 183

hybrida leaf, one of them corresponding to Phyb A, which was found in higher abundance within the 184

mid-vein tissue compared with the laminar and peripheral leaf tissues (Poth et al., 2012). Thus, that 185

report demonstrated that cyclotides associate with the vascular section of leaf tissues, suggesting that 186

these peptides may play a role in plant defense through modulating insect herbivory. 187

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2 Biological activities of cyclotides 188

In addition to the reported uterotonic activity of kalata B1 (Gran, 1973a, 1973b), a wide range of other 189

biological activities, including hemolytic activity (Daly et al., 1999a; Ireland et al., 2006a; Schöpke 190

et al., 1993; Tam et al., 1999), neurotensin antagonistic (Witherup et al., 1994), anti-HIV (Bokesch et 191

al., 2001; Chen et al., 2005; Daly et al., 2006; Daly et al., 2004; Gustafson et al., 1994; Gustafson et 192

al., 2000; Hallock et al., 2000; Ireland et al., 2008; Wang et al., 2008a), anti-microbial (Fensterseifer 193

et al., 2015; Gran et al., 2008; Pranting et al., 2010; Tam et al., 1999), protease inhibitory (Hernandez 194

et al., 2000; Quimbar et al., 2013), insecticidal (Jennings et al., 2001), antitumor (Herrmann et al., 195

2008; Lindholm et al., 2002; Svangård et al., 2004; Tang et al., 2010), antifouling (Göransson et al., 196

2004), nematocidal (Colgrave et al., 2008a), molluscicidal (Plan et al., 2008), cell-penetrating 197

(Contreras et al., 2011; Greenwood et al., 2007), immunosuppressive (Gründemann et al., 2012; 198

Gründemann et al., 2013), and prolyl oligopeptidase inhibitory activities (Hellinger et al., 2015) has 199

been reported for cyclotides, as summarized in Table 1. 200

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Table 1. Summary of host defense-related and/or biological activities first reported for cyclotides 201

Activity Exemplary cyclotides First report

Uterotonic kalata B1, B2 and B7 (Gran, 1973a, 1973b)

Hemolytic violapeptide I, circulin A

and B, cyclopsychotride

A, kalata B1, varv A

(Schöpke et al., 1993)

Anti-HIV circulin A and B,

palicourin, kalata B1,

vhl-1

(Gustafson et al., 1994)

Neurotensin antagonist cyclopsychotride A (Witherup et al., 1994)

Anti-microbial circulin A and B,

cyclopsychotride A,

kalata B1, kalata B7,

cycloviolacin O2

(Tam et al., 1999)

Trypsin inhibitor MCoTI-I and MCoTI-II (Hernandez et al., 2000)

Insecticidal kalata B1 and B2 (Jennings et al., 2001)

Antitumor cycloviolacin O2, varv A

and F

(Lindholm et al., 2002)

Antifouling cycloviolacin O2 (Göransson et al., 2004)

Cell internalization MCoTI-II, kalata B1 and

MCoTI-I

(Greenwood et al., 2007)

Nematocidal kalata B1 and B2,

cycloviolacin O2

(Colgrave et al., 2008a)

Molluscicidal cycloviolacin O1, kalata

B1, B2 and B5

(Plan et al., 2008)

Immunosuppressive kalata B1 (Gründemann et al., 2012)

Prolyl oligopeptidase

inhibition

kalata B1, psysol 2 (Hellinger et al., 2015)

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2.1 Toxic activities 202

One of the first activities reported for cyclotides was the ability to cause lysis of human erythrocytes. 203

This hemolytic activity was initially observed in violapeptide I, the first cyclotide discovered from 204

the violet family (Schöpke et al., 1993). Additional macrocyclic peptides were discovered during the 205

course of screening other plants for a range of other biological activities, including anti-HIV and anti-206

neurotensin activities. In one of the first such studies, Gustafson et al. reported the antiviral activities 207

of circulins A and B, bracelet cyclotides isolated from Chassalia parvifolia (Rubiaceae), which 208

demonstrated antiviral cytoprotective effects on various human immunodeficiency virus (HIV) strains 209

at concentrations ranging from 40 to 260 nM (Gustafson et al., 1994). Similarly, screens for 210

neurotensin antagonistic activity led to the discovery of cyclopsychotride A from Psychotria longipes 211

(Rubiaceae) (Witherup et al., 1994), which was later also reported to be active against Gram-positive 212

and Gram-negative bacteria (Tam et al., 1999). 213

The antimicrobial activities of cyclotides were first described by Tam et al. (Tam et al., 1999), 214

whereby synthetically derived kalata B1 was reported to be active against Staphylococcus aureus in 215

the absence of salt in the test solution. The inhibitory activity was abolished under physiological 216

conditions, i.e. in the presence of 100 mM NaCl, and the peptide was inactive against E. coli. By 217

contrast, a later study of native kalata B1 by Gran et al. (Gran et al., 2008) suggested that it had 218

antibiotic effects on E. coli under low or high-salt conditions. In addition to these apparently 219

conflicting reports for in vitro studies, a recent publication reported that cyclotides kalata B2 and 220

cycloviolacin O2 display in vitro antibacterial activities and in vivo efficacy against S. aureus in a 221

subcutaneous wound infection animal model (Fensterseifer et al., 2015). The reported salt dependence 222

for the antimicrobial activity (Tam et al., 1999) suggests that the initial interaction between cyclotides 223

and the microbial surface may be electrostatic, similar to that described for defensins (Oren & Shai, 224

1998). The similarity in size of cyclotides to disulfide-rich defensins is consistent with a functional 225

role of cyclotides in host-defense. 226

There is ongoing interest in the literature in the antimicrobial potential of cyclotides. A recent 227

report on the antibacterial activity of peptide-containing extracts from the flowers of the elderberry 228

tree (Sambucus nigra) noted some partial sequences consistent with known cyclotides but the active 229

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components have not yet been definitely confirmed as cyclotides (Álvarez et al., 2018a). That study 230

and another from the same group suggested potential applications of cyclotides as antimicrobial in 231

aquaculture applications (Álvarez et al., 2018b). Another report demonstrated the use of cyclotides as 232

surface coatings to reduce biofilm formation (Cao et al., 2018). Underpinning such applications, there 233

has also been interest in using computational approaches to predict the preferential cyclotide scaffolds 234

for antimicrobial applications (Balaraman & Ramalingam, 2018) and the potential for the 235

development of drug resistance (Malik et al., 2017; Noonan et al., 2017). 236

Cytotoxic activity with the potential for antitumor applications is another noteworthy property 237

reported in early cyclotide studies. In one study, cycloviolacin O2, isolated from V. odorata, and varv 238

A and varv F, isolated from V. arvensis, displayed cytotoxic activities against ten human tumor cell 239

lines, including immortalized myeloma, T-cell leukemia, small cell lung cancer, lymphoma, and renal 240

adenocarcinoma cell lines, as well as primary ovarian carcinoma cells from cancer patients (Lindholm 241

et al., 2002). The cytotoxicity of varv A and cycloviolacin O2 on healthy human lymphocytes was 242

also evaluated in the same study. Compared to the normal lymphocytes, both peptides displayed ~9-243

fold higher selectivity towards the leukemia cells (Lindholm et al., 2002). Other cyclotides isolated 244

from the Violaceae family were extensively tested against a variety of human cancer cells of varying 245

origin and were reported to have potent activities in vitro (Herrmann et al., 2008; Pinto et al., 2018; 246

Svangård et al., 2004; Tang et al., 2010), suggesting that these plant-derived peptides might be 247

promising cytotoxic compounds with potential in cancer treatment. Since cycloviolacin O2 showed 248

promising results in cytotoxicity testing against lymphoma, leukemia, small cell lung cancer and 249

colon carcinoma cell lines in vitro at concentrations in the low micromolar range, a follow-up study 250

on the antitumor effects of the peptide was done in vivo using mouse xenograft models with hollow 251

fibers containing various human cancer cell lines implanted subcutaneously (Burman et al., 2010b). 252

The maximum tolerated dose was determined for single-dose injection (1.5 mg/kg) and repeated-dose 253

injection (0.5 mg/kg) prior to a hollow fiber study and xenograft study, respectively. Animals 254

implanted with a hollow fiber encapsulated with tumor cells received a single dose of cycloviolacin 255

O2 at 1 mg/kg one day after the implantation. Xenografted animals were treated with cycloviolacin 256

O2 by intravenous injection at 0.5 mg/kg daily up to 14 days. With no significant antitumor affects 257

14

observed, repeated dosing of cycloviolacin O2 at 1 mg/kg was reported to give a local-inflammatory 258

reaction at the injection site fter 2-3 doses and acute toxicity was observed after administration of the 259

cyclotide at 2 mg/kg. This negative result might be due to a low distribution of peptide at the site of 260

the implants or to intrinsically weak in vivo antitumor activity of the peptide. A recent study on the 261

selectivity of cyclotides against cells further demonstrated that cycloviolacin O2, kalata B1 and kalata 262

B2 are toxic towards both non-transformed (skin and PBMC) and transformed (cervical cancer, 263

melanoma and leukemia) cells and they exert their effects through targeting cell membranes 264

containing phosphatidylethanolamine (PE) phospholipids, causing subsequent membrane disruption 265

(Henriques et al., 2014). The finding of cyclotides interacting preferentially with PE-phospholipids is 266

in agreement with an earlier report by Burman et al (Burman et al., 2011). 267

Cyclotides possess potent intrinsic insecticidal activities, and thus have potential applications 268

in agriculture, as first reported by Jennings et al (Jennings et al., 2001). In that study, purified kalata 269

B1 was incorporated into an artificial diet and fed to larvae of Helicoverpa punctigera, a serious pest 270

of cotton crops worldwide. Half of the kalata B1 treated larvae died and none of the survivors 271

developed past the first instar stage of larval development. A later study demonstrated that cyclotides 272

damage the gut epithelium of H. armigera by inducing disruption of the microvilli, causing blebbing 273

of epithelial cells, swelling of the columnar cells and ultimately rupture of the cells (Barbeta et al., 274

2008). 275

Since cyclotides possess insecticidal activity, it has been of interest to examine their activity 276

against other agricultural pests to understand their potentially broader role as natural pesticides. The 277

insecticidal activities of kalata B1 and a suite of alanine mutants against adult Drosophila 278

melanogaster were assessed and the disruption of the development of first and second instar larvae 279

through to adult D. melanogaster was demonstrated (Simonsen et al., 2008). In another series of 280

studies conducted by Colgrave et al., a range of cyclotides was found to inhibit the larval development 281

of two economically important sheep gastrointestinal nematodes, Haemonchus contortus and 282

Trichostrongylus colubriformis (Colgrave et al., 2008b; Wang et al., 2008a), as well as canine and 283

human hookworms (Colgrave et al., 2009). Kalata B1 (IC50 = 2.2 µM) showed equipotent activity to 284

the commercially used anthelmintic drugs, levamisole (IC50 = 8.9 µM) and naphthalophos (IC50 = 7.5 285

15

µM) against the larval life stage, confirming a potential role for cyclotides as natural anthelmintic 286

control agents. In a later study, kalata B1, B6 and cycloviolacin O14 were shown to have a pronounced 287

effect on the viability of larval and adult life stages of the dog hookworm Ancylostoma caninum and 288

inhibited larval development of the human hookworm Necator americanus (Colgrave et al., 2009). 289

Subsequent investigations of other natural cyclotides extracted from V. odorata were reported 290

and identified examples with up to 18-fold greater potency than kalata B1 in larval development 291

assays against H. contortus, with the most potent cyclotide being cycloviolacin O2 (Colgrave et al., 292

2008b). Modification of cycloviolacin O2 by acetylation of the two lysine residues to mask their 293

charge resulted in a marked decrease in anthelmintic activity for this Viola-derived peptide to a level 294

comparable to kalata B1 (Colgrave et al., 2008b). A correlation was also observed between the 295

number of charged residues present in cyclotide sequences and their anthelmintic activity, suggesting 296

that the net charge of a cyclotide is probably an important determinant of anthelmintic activity. 297

Most recently, the antifungal activities of extracts from Viola odorata were reported (Slazak et al., 298

2018) and suggest a role for cyclotides from this plant against microbial pests. Specifically, 299

cycloviolacin O2, O3, O13, and O19 (cyO2, O3, O13 and O19) isolated from Viola odorata were 300

evaluated for activity against five model plant fungal pathogens, namely Fusarium oxysporum, 301

Fusarium graminearum, Fusarium culmorum, Mycosphaerella fragariae, and Botrytis cinerea, and 302

two Viola-derived pathogens, namely Colletotrichum utrechtense and Alternaria alternate. All tested 303

cyclotides displayed antifungal activity. CyO3 exhibited the most potent activity with minimal 304

inhibitory concentrations (MICs) ranging from 0.8 to 12.5 μM; while cyO13 exhibited the lowest 305

activities with MICs ranging from 3 to 25 μM. All cyclotides displayed low micromolar activity 306

against A. alternate, a fungal pathogen also isolated from Viola odorata. Figure 3 schematically 307

illustrates the range of host defense-related activities of cyclotides. 308

309

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Figure 3: Examples of pesticidal and toxic activities of native cyclotides. Cyclotides have been 310

reported to possess potent activities against various pests, including the nematode H. contortus 311

(nematocidal)(Colgrave et al., 2008a), budworm H. punctigera (insecticidal) (Jennings et al., 2001), 312

rice pest Pomacea canaliculata (molluscicidal) (Plan et al., 2008), and fouling barnacles Balanus 313

improvisus (antifouling) (Göransson et al., 2004). The reported toxic effects of cyclotides against the 314

human immunodeficiency virus (anti-HIV) (Gustafson et al., 1994) and a range of cancer cell lines 315

(anti-tumor) (Lindholm et al., 2002) highlight the potential therapeutic and agrochemical applications 316

of these peptides. Therapeutic activities that are not linked with the toxic effects of cyclotides, e.g. 317

immunosuppressive activity (Gründemann et al., 2012), are not included in this figure. 318

2.2 Pharmaceutical activities 319

In addition to their toxic or host-defense activities, a range of naturally occurring cyclotides possess 320

activities of pharmacological and pharmaceutical relevance beyond the uterotonic, anti-HIV and 321

neurotensin antagonist activities noted already. In a recent study that nicely links the original 322

17

indigenous medical applications of the cyclotide-bearing plant O. affinis with the latest 323

pharmacological studies, the oxytocic activity of kalata B7, a Möbius cyclotide closely related to 324

kalata B1 was reported and the possible molecular target underlying the mechanism of the uterotonic 325

activity was revealed. Pharmacological studies showed that kalata B7 is a partial agonist of both the 326

oxytocin receptor and vasopressin V1A receptor and a structural analysis suggested that loop 3 of 327

kalata B7, which displays moderate sequence homology to human oxytocin, could be responsible for 328

the observed uterostimulant effects on uterine smooth muscle cells (Koehbach et al., 2013b). 329

The discovery of the anti-proliferative activity of cyclotides on primary activated human 330

lymphocytes suggested the potential use of these peptides as immunosuppressant drugs. The 331

inhibitory effects of kalata B1 on the proliferation of human peripheral blood mononuclear cells 332

(PBMC) at non-cytotoxic concentrations was first reported by Gründemann et al. (Gründemann et al., 333

2012). This research led to further investigations of the immunosuppressive properties of cyclotides, 334

whereby an analog of kalata B1 [T20K] was shown to attenuate the interleukin-2 (IL-2) secretion and 335

the expression of IL-2 surface receptor on activated T-lymphocytes (Gründemann et al., 2013). 336

Further mechanistic studies, described in the same report, using several kalata B1 analogs with single 337

point mutations determined the cyclotide motif accountable for the anti-proliferative activity. The 338

recent progression of [T20K]kalata B1 to Phase I clinical trials for multiple sclerosis further illustrates 339

the potential applications of cyclotides in immunopharmacology and immunosuppression 340

(Gründemann et al., 2019). 341

2.3 Cell penetrating properties 342

Delivery of peptide-based drugs to intracellular targets is one of the holy grails of drug development. 343

Cyclotides triggered interest as potential frameworks for intracellular drug delivery after Greenwood 344

et al. reported the internalization of the cyclotide MCoTI-II into mammalian cells by endocytosis 345

(Greenwood et al., 2007). In that study, cells treated with biotinylated cyclotides were fixed and 346

stained with avidin-FITC before the internalization was evaluated using confocal fluorescence 347

microscopy. Although internalization studies in such systems need careful analysis to avoid possible 348

artifacts, the study provided the first indication of the cell penetrating ability of a cyclotide. In a later 349

study, the cellular uptake of fluorescently labeled MCoTI-II and kalata B1 were explored using live-350

18

cell confocal imaging techniques and their affinity for phospholipids was examined on model 351

membrane systems by surface plasmon resonance or on PIP strips™ membranes (Cascales et al., 352

2011). It was confirmed that MCoTI-II and kalata B1 are both able to penetrate cells but that they 353

probably cross cell membranes through different pathways. That study categorized these two peptides 354

and a smaller cyclic sunflower peptide, SFTI-1, as a new class of cell-penetrating peptides, referred 355

to as cyclic cell-penetrating peptides (Cascales et al., 2011). A contemporaneous study, using real 356

time confocal microscopy, showed that MCoTI-I, a cyclotide with ~95% sequence similarity to 357

MCoTI-II, internalized into HeLa cells predominantly through a temperature-dependent active 358

endocytic pathway (Contreras et al., 2011). Overall, these independent reports confirm that cyclotides 359

have the potential to be used as stable scaffolds for delivering therapeutically significant peptide 360

epitopes into cells and this topic is likely to be an active area of future cyclotide research. 361

3 Synthesis, structure-activity relationships and mode of action of cyclotides 362

Research on cyclotides has led to a number of impacts in the field of biological chemistry, including 363

the development of approaches to the chemical and biological synthesis of cyclic peptides, which has 364

opened up technologies for deriving structure-activity studies of cyclotides and for their applications 365

as drug design frameworks. 366

3.1 Chemical and biological syntheses of cyclotides 367

Approaches for the chemical synthesis of cyclotides were first described in the late 1990’s (Daly et 368

al., 1999b; Tam & Lu, 1998). In Tam and Lu’s report (Tam & Lu, 1998), the backbone cyclization of 369

circulin B and cyclopsychotride A was achieved by adapting native chemical ligation (Dawson et al., 370

1994), where an N-terminal cysteine and a C-terminal thioester of peptide precursors synthesized 371

using Boc-chemistry solid phase peptide synthesis (SPPS) reacted to form a thioester that 372

subsequently underwent a spontaneous acyl transfer reaction to produce a native amide bond between 373

the two termini. In that study orthogonal protection of pairs of Cys residues was used to direct the 374

oxidation in a stepwise manner to form the desired disulfide connectivities. Daly et al. (Daly et al., 375

1999b) used a similar approach for the synthesis of kalata B1 but without orthogonal protection of 376

Cys residues and showed it was possible to readily isolate the correctly folded product from the 377

19

mixture of disulfide isomers. Overall, the native chemical ligation-based synthesis method using Boc-378

chemistry has been applied to the cyclization of a wide range of macrocyclic peptides and has proven 379

to be valuable in the routine production of cyclotides (Clark & Craik, 2010). Various Fmoc-based 380

SPPS methods to generate the thioester precursors of cyclotides for head-to-tail cyclization via native 381

chemical ligation (Gunasekera et al., 2013; Taichi et al., 2013), bacterial expression of recombinant 382

linear precursors of cyclotide followed by in vitro cyclization (Cowper et al., 2013), and a more direct 383

strategy for backbone cyclization using Fmoc-based SPPS (Cheneval et al., 2014) have also been 384

reported in recent years. 385

Several enzyme-mediated approaches for cyclization of cyclotides have been explored, 386

including intein-mediated biosynthetic methods (Camarero et al., 2007; Jagadish et al., 2015; Kimura 387

et al., 2006) and sortase A-catalyzed backbone cyclization (Jia et al., 2014). The intein-mediated 388

backbone cyclization of kalata B1 was achieved by recombinantly expressing linear cyclotide 389

precursors fused to a Met and an engineered intein unit at their N and C termini, respectively. The 390

fusion proteins were cleaved by endogenous Met aminopeptidase and underwent intein-mediated 391

protein splicing in E. coli which resulted in linear cyclotide precursors with a C-terminal α-thioester 392

and an N-terminal Cys required for native chemical ligation-based cyclization in vitro (Kimura et al., 393

2006). The intein-mediated in vivo production of cyclotides MCoTI-II and MCoTI-I was reported by 394

the same group, in live bacterial cells (Camarero et al., 2007) and yeast cells (Jagadish et al., 2015), 395

respectively. These reports demonstrated the applicability of recombinant expression of natively 396

folded cyclotides in microorganisms and the possibility of producing large combinatorial cyclotide-397

based libraries for screening. More recently, a chemo-enzymatic approach was developed to 398

synthesize cyclic disulfide-rich peptides, including kalata B1, cyclic α-conotoxin Vc1.1 and SFTI, 399

whereby the chemically synthesized linear peptide precursors containing a sortase A recognition motif 400

at the C-terminus were cyclized by sortase A in vitro without significant perturbation to the overall 401

peptide fold (Jia et al., 2014). 402

In parallel with the development of new methodologies for the efficient synthesis of cyclotides, 403

there is a growing effort towards understanding the biosynthetic mechanism of these macrocyclic 404

peptides in plants. Since an Asn is highly conserved at the C-terminus of the cyclotide domain, 405

20

asparaginyl endopeptidase (AEP), a cysteine protease with specificity for Asn, has been implicated 406

in the cyclization of cyclotides in vivo (Saska et al., 2007). In this key study, kalata B1 was expressed 407

transiently in Nicotiana benthamiana transformed with the precursor of kalata B1, and a reduction in 408

the yield of backbone-cyclized kalata B1 was observed with AEP-gene silencing constructs, providing 409

an initial correlation between AEP activity and cyclization yield of cyclotides in plants (Saska et al., 410

2007). A recent publication reported the discovery of the AEP homolog butelase 1 in the cyclotide- 411

propeptides with a His-Val sequence at the C terminus in vitro (Nguyen et al., 2014). Butelase 1 has 412

been shown to cyclize peptides of varying lengths (from 14 to 34 residues) and sequences, including 413

kalata B1, SFTI, cyclic conotoxin MrlA, and antimicrobial peptide histatin-3 at a low enzyme-to-414

peptide ratio (1:400) within 48 min. This finding suggests that butelase 1 could be developed as an 415

alternative approach to complement the current chemical and biological methodologies in producing 416

macrocyclic peptides. 417

Another impact deriving from cyclotide research has been the development of methodologies 418

for crystalizing disulfide-rich peptides. Of the many structures of cyclotides published, until recently, 419

only one involved X-ray crystallography because cyclotides along with other disulfide-rich peptides 420

are notoriously difficult to crystallize. However, Wang et al. (Wang et al., 2014) demonstrated that 421

the use of racemic crystallography dramatically improved crystallization rates and determined crystal 422

structures for a series of cyclic disulfide-rich peptides, ranging from SFTI-1 (14 amino acids with one 423

disulfide bond) to a cyclized conotoxin (22 amino acids with two disulfide bonds) to kalata B1 (29 424

amino acids with three disulfide bonds). Although this technology was demonstrated for cyclic 425

molecules, it should be equally applicable to the crystallization of a wide range of acyclic disulfide-426

rich peptides. 427

3.2 Structure-activity relationships 428

The ability to chemically synthesize cyclotides has facilitated a wide range of mutagenesis studies 429

and structure-activity relationship studies that reveal the importance of the circular backbone for 430

maintenance of cyclotide structural integrity. In one early study by Daly et al. (Daly & Craik, 2000), 431

six acyclic permutants of kalata B1, with the backbone opened in each of the six inter-cysteine loops, 432

were synthesized and their overall folds were compared with that of the cyclic native peptide. A native 433

21

fold could not be achieved in acyclic mutants having a break of the backbone in either loops 1 or 4, 434

which are the loops forming the embedded ring in the cystine knot. This result suggests that the cystine 435

knot is essential in stabilizing the intermediates formed during the oxidative folding of cyclotides. 436

The overall folds of the four other acyclic analogs of kalata B1, with a break in one of loops 2, 3, 5, 437

or 6, were found to be very similar to that of their parent peptide, showing that a cyclic backbone is 438

not essential for a native-like fold. Although these four acyclic analogs of kalata B1 retained a native-439

like conformation, their lack of hemolytic activity suggests that the circular backbone is functionally 440

important (Daly & Craik, 2000). Furthermore, the three-dimensional solution structures of a synthetic 441

acyclic permutant of kalata B1 with most of loop 6 removed and a naturally occurring linear cyclotide, 442

violacin A (with a discontinuous loop 6), showed that a backbone discontinuity renders structures 443

more flexible than in their cyclic counterparts (Barry et al., 2003; Daly & Craik, 2000; Ireland et al., 444

2006b). These combined findings indicate that the circular backbone is crucial to both the structure 445

rigidity and activity of cyclotides (Barry et al., 2003; Daly & Craik, 2000). 446

The ability to chemically synthesize cyclotides has also facilitated a wide range of 447

mutagenesis studies that have helped to delineate their mode of action, as described in the following 448

section. 449

3.3 The mode of action of cyclotides 450

The mode of action of cyclotides may vary depending on the particular biological activity but in 451

general is strongly dependent on their unique structural features. The cystine knot structural motif 452

effectively results in the exclusion of non-Cys side-chains from the core region of cyclotides, 453

promoting the surface exposure of hydrophobic residues, some of which are clustered together to form 454

a hydrophobic patch. Several characteristic biophysical properties derive from this surface-exposed 455

hydrophobic patch, including late elution on RP-HPLC and weak self-association. These properties 456

have potential implications for the mode of action of cyclotides as cytotoxic agents since they provide 457

clues as to how these molecules might interact with and form pores in membranes. In this regard, the 458

oligomerization and self-association properties of cyclotides have been investigated using analytical 459

ultracentrifugation techniques. For instance, kalata B2 was observed to self-associate into tetramers 460

and octamers (Nourse et al., 2004), but not dimers. In one model of the geometry of the tetramer 461

22

proposed in that study, the oppositely charged residues Arg-24 and Asp-25 in kalata B2 create an 462

exposed bipolar patch at one end of the molecule, which was postulated to facilitate intermolecular 463

ionic self-interaction and potentially play a role in the formation of membrane-spanning pores. A later 464

experimental NMR study suggested an alternative model for the self-association in solution based on 465

interaction between the hydrophobic patches of kalata B2 (Rosengren et al., 2013). The significance 466

of the solution-state oligomers to the function of cyclotides remains unknown, and similarly it is not 467

known if cyclotides form aggregates in their membrane bound states, but a wide range of studies do 468

suggest that membrane interactions are intimately associated with cyclotide functions. 469

Synthetic and mutagenesis-based studies have contributed significantly to defining the 470

membrane-interacting hypothesis for the mode of action of kalata B1. For instance, a comparison of 471

enantiomer forms of a peptide ligand provides a definitive means to indicate whether a chiral protein 472

receptor is involved in its biological function or whether it acts via a (largely achiral) membrane 473

disruption mechanism. Colgrave et al. (Colgrave et al., 2008a) showed that the nematocidal activity 474

of the mirror-image stereoisomer of kalata B1 was similar to the wild-type peptide, suggesting that 475

the mechanism of action is probably via membrane interaction rather than by a chiral (i.e. protein-476

based) receptor interaction. The self-association behaviour of cyclotides and the comparable 477

bioactivity of the all D-enantiomer of kalata B1 to the native L-form are key pieces of evidence which 478

suggest that the mechanism of action may be via membrane interaction. The membrane-based 479

mechanism of action of cyclotides was supported by an early surface plasmon resonance study which 480

demonstrated that kalata B1 and B6 bind selectively to phosphatidylethanolamine (PE)-containing 481

model membranes (Kamimori et al., 2005). Further support for the membrane-binding properties of 482

native cyclotides derived from the observation that cycloviolacin O2 induced leakage of both calcein-483

loaded HeLa cells and a lipid model in the form of palmitoyloleoylphosphatidylcholine (POPC) 484

liposomes (Svangård et al., 2007). NMR studies by Shenkarev et al. showed that the binding of kalata 485

B1 and kalata B7 to dodecylphosphocholine (DPC) micelles is modulated by both electrostatic and 486

hydrophobic interactions (Shenkarev et al., 2008; Shenkarev et al., 2006). Varv F was shown to bind 487

to DPC micelles and its overall conformation remained unchanged upon binding (Wang et al., 2009b). 488

23

The nematocidal activity of a suite of alanine mutants of kalata B1 was examined and the 489

residues critical for activity against helminths correlated with those significant for insecticidal activity 490

against D. melanogaster (Simonsen et al., 2008). Residues critical for the biological activities of 491

kalata B1 were clustered on one side of the molecule, named ‘the bioactive patch’. Since membrane 492

interaction involving oligomerization was speculated to be responsible for the insecticidal activities 493

of kalata B1, the whole suite of alanine mutants of kalata B1 was screened against a range of model 494

membranes encapsulated with self-quenching dye for their lytic activities (Huang et al., 2009). The 495

leakage study confirmed that the bioactive patch of kalata B1 plays a critical role in its lytic, as well 496

as its insecticidal and hemolytic activities. In addition, kalata B1 was observed to have a preference 497

for phospholipids containing PE headgroups compared to model membranes containing only 498

zwitterionic or anionic phospholipids (Huang et al., 2009). Results from patch-clamp experiments 499

suggested that kalata B1 induced leakage via pore formation on reconstituted asolectin (soybean 500

lecithin), when compared with a membrane-inactive mutant of kalata B1 (V25A) (Huang et al., 2009). 501

A later study of lysine mutants of kalata B1 revealed that a single lysine substitution on a face opposite 502

to the bioactive patch improved its nematocidal activity (Huang et al., 2010). Furthermore, Colgrave 503

et al. observed increasing uptake of the radiolabel [3H]inulin of ligated adult nematodes upon kalata 504

B1 treatment, providing evidence to support the conclusion that the anthelmintic effect of the 505

cyclotide was due to increased permeability of the external membrane of the nematodes (Colgrave et 506

al., 2010). Together, these various mutagenesis studies and electrophysiological recordings provide 507

mechanistic insights into how kalata B1 exerts its effects on different organisms. 508

Many other bioactivities of cyclotides correlate with lipid-binding properties, as supported by 509

detailed biophysical studies using surface plasmon resonance (Henriques et al., 2012; Henriques et 510

al., 2014; Henriques et al., 2011) and isothermal titration calorimetry (ITC) (Wang et al., 2012) on 511

model membranes. An extensive lipid binding study of kalata B1 and a range of its active and inactive 512

mutants using surface plasmon resonance suggested that kalata B1 preferred more rigid membranes 513

containing PE phospholipids and exerted its anti-HIV activities by disrupting the membrane envelope 514

of viral particles (Henriques et al., 2011). Furthermore, a titration of kalata B1 with PE, monitored 515

using 1H NMR chemical shifts, suggested that it interacted specifically with the PE headgroups 516

24

through residues that formed part of the bioactive patch (Henriques et al., 2011). Therefore, the 517

membrane-targeting properties of cyclotides against PE headgroups was proposed as the initial step 518

of their lytic action, followed by membrane insertion with the hydrophobic patch, which leads to local 519

membrane disturbances and eventually membrane disruption (Henriques et al., 2012). More recently, 520

the PE-targeting ability of kalata B1 was also suggested to be responsible for the observed cell 521

internalization of kalata B1 at concentrations lower than the cytotoxicity threshold (Henriques et al., 522

2015). 523

Figure 4 summarizes our current understanding of the proposed mechanism of cell 524

internalization of kalata B1, which involves the binding of the bioactive patch to PE phospholipids in 525

cell membranes via electrostatic interactions, before the hydrophobic face of the cyclotide is inserted 526

into the core of the bilayer. The accumulation of cyclotide on the lipid bilayer causes local membrane 527

disturbances, which eventually leads to cell penetration. 528

529

Figure 4: A schematic representation of the cell internalization of kalata B1. The initial step of 530

cell internalization of kalata B1 is PE-targeting. The bioactive patch (highlighted in red) of kalata B1 531

binds to the headgroup of PE phospholipids in cell membranes via electrostatic interaction (1), 532

followed by the insertion of the hydrophobic face (highlighted in green) into the core of the bilayer 533

(2). The accumulation of cyclotide molecules on the lipid bilayer leads to local membrane 534

disturbances, which eventually leads to cell penetration (3) through: i) endocytosis or ii) membrane 535

translocation by an energy-independent process. Figure adapted from a previously published article 536

(Henriques et al., 2015). 537

538

25

Overall, membrane binding is fundamental for many reported functions of cyclotides, 539

including hemolytic, insecticidal, nematocidal, and anti-HIV activities, as well as cell internalization. 540

However, considering that some cyclotides have been reported to bind to several members of the G 541

protein-coupled receptor family for their oxytocic properties (Koehbach et al., 2013b), there is a 542

possibility that other cyclotides might also exert other activities via modulating cellular receptors 543

separately from or in addition to binding to membranes. 544

545

4 Applications 546

Cyclotides have a range of potential applications in agriculture and medicine based on their 547

exceptional stability and their tolerance to sequence modifications that allow “designer cyclotides” to 548

be made. In this section, we very briefly outline some of those applications. 549

4.1 Medical applications of cyclotides 550

One approach to medical application of cyclotides is to harness some of the toxic properties of natural 551

cyclotides for therapeutic applications, for example, as cytotoxic (anti-cancer agents) or as 552

nematocidal agents with applications for human parasites, such as hookworms. A second area of 553

medical applications is through “designer” cyclotides made by grafting a bioactive epitope into a 554

cyclotide sequence to introduce a new activity of therapeutic relevance not present in the original 555

cyclotide framework. The aim of all of these studies is to take advantage of the stability of the 556

cyclotide framework to stabilize the peptide epitope. These grafting applications have been widely 557

reviewed elsewhere, so we will not discuss them further here except to refer readers to recent reviews 558

on the topic (Camarero, 2017; Craik & Du, 2017; de Veer et al., 2017; Gould & Camarero, 2017; 559

Gould et al., 2011; Poth et al., 2013; Wang & Craik, 2018). There are more than 25 examples of 560

grafted cyclotides for a range of diseases, including cardiovascular disease, cancer, wound healing, 561

pain, inflammation and multiple sclerosis. So far, none of these examples has reached human clinical 562

trials but all are well exemplified by animal studies or at least in vitro testing. 563

26

4.2 Agricultural applications 564

Stimulated by their natural functions as endogenous pesticidal agents in certain plants, 565

cyclotides have attracted attention for potential applications in the protection of crop plants that 566

naturally do not contain them. Such applications include their incorporation into transgenic plants, a 567

topic that is outside the scope of the current article, as well as applications involving external 568

administration onto growing crops or harvested material. The most advanced application involving 569

external application is exemplified with the recent approval of SeroX, an extract from butterfly pea 570

(Clitoria ternatea), as a treatment for insect pests on cotton and macadamia nut crops in Australia. 571

This plant, from the Fabaceae family, contains more than 47 different cyclotides (Gilding et al., 2016), 572

of which the peptide Cter M, at least, has been shown to have insecticidal properties as an isolated 573

peptide (Poth et al., 2011a). The SeroX product is marketed as a spray for cotton at doses as low as 574

2L/hectare by its developer, Innovate Ag, based in Australia. 575

While we will not cover the alternative mode of delivery of cyclotides via the incorporation 576

of transgenes encoding cyclotides into crop plants here, it is useful to note that there have been a 577

number of recent advances in understanding the roles of asparaginyl endopeptidase enzymes in 578

facilitating cyclotide biosynthesis (Harris et al., 2015; Jackson et al., 2018). Additionally, the enzyme 579

kalatase A, which is responsible for the N-terminal processing of cyclotide precursors was recently 580

reported (Rehm et al., 2019). These studies will no doubt be useful in facilitating the production of 581

transgenic plants with high yields of pesticidal cyclotides, thereby engendering these plants with 582

similar levels of insect protection to natural cyclotide-producer plants. 583

5 Outlook and future studies 584

Overall, cyclotides have attracted a great deal of interest, not only for their natural insecticidal 585

activities and their potential as drug scaffolds but also because of their topologically unique structures. 586

These structures engender cyclotides with exceptional stability and thus, in principle, they have 587

advantages over less stable peptides in that they offer potential for a variety of formulation approaches 588

and are stable to long term storage, an important consideration both for pharmaceutical and 589

agricultural applications. 590

27

The cyclotide field is still relatively young and only a small number of groups are currently 591

studying these fascinating cyclic and knotted peptides. Their natural function as insecticidal or 592

nematocidal agents justifiably allows them to be regarded as toxins. Their mechanism of toxicity 593

appears to be mainly related to membrane-binding. Their membrane binding, however, is far from 594

non-specific, and cyclotides exhibit a remarkable preference for PE lipids compared to other lipid 595

types. It is not yet known whether it is this lipid specificity that controls the specificity of different 596

cyclotides for different organisms, but it seems to be a reasonable hypothesis. Also unknown at the 597

moment is why one plant produces so many cyclotides. Is it a strategy for the plant to try and avoid 598

the development of resistance by pests to the chemical defense? Or is it a strategy for simultaneously 599

targeting a wide variety of different pests? These questions will undoubtedly be answered in due 600

course, assisted by advances in technologies for the synthesis of cyclotides. For example, as we have 601

noted, it is now possible to make variants of cyclotides where individual residues or individual loops 602

can be replaced to explore structure-activity studies. Biological methods of producing cyclotides are 603

also improving and promise to accelerate the development of structure-activity relationships. 604

Amongst toxins cyclotides do not have quite the same caché as the deadly toxins from animal 605

venoms, but we hope that this article has convinced readers that they are toxins with a vast range of 606

potential applications in the pharmaceutical and agricultural fields. 607

608

Acknowledgements 609

Work in our laboratory on cyclotides is funded by grants from the Australian Research Council 610

(DP150100443) and the National Health and Medical Research Council (APP1084965 and 611

APP1060225). DJC is an Australian Research Council Laureate Fellow (FL150100146). 612

28

References 613

Álvarez, C. A., Barriga, A., Albericio, F., Romero, M. S., & Guzmán, F. 2018a. Identification of 614

peptides in flowers of sambucus nigra with antimicrobial activity against aquaculture 615

pathogens. Molecules, 23, e1033 616

Álvarez, C. A., Santana, P., Luna, O., Cárdenas, C., Albericio, F., Romero, M. S., & Guzmán, F. 617

2018b. Chemical synthesis and functional analysis of varvA cyclotide. Molecules, 23, e952 618

Balaraman, S., & Ramalingam, R. 2018. The structural and functional reliability of Circulins of 619

Chassalia parvifolia for peptide therapeutic scaffolding. J. Cell. Biochem., 119, 3999-4008 620

Barbeta, B. L., Marshall, A. T., Gillon, A. D., Craik, D. J., & Anderson, M. A. 2008. Plant cyclotides 621

disrupt epithelial cells in the midgut of lepidopteran larvae. Proc. Natl. Acad. Sci. U. S. A., 622

105, 1221-1225 623

Barry, D. G., Daly, N. L., Bokesch, H. R., Gustafson, K. R., & Craik, D. J. 2004. Solution structure 624

of the cyclotide palicourein: Implications for the development of a pharmaceutical framework. 625

Structure, 12, 85-94 626

Barry, D. G., Daly, N. L., Clark, R. J., Sando, L., & Craik, D. J. 2003. Linearization of a naturally 627

occurring circular protein maintains structure but eliminates hemolytic activity. Biochemistry, 628

42, 6688-6695 629

Bokesch, H. R., Pannell, L. K., Cochran, P. K., Sowder, R. C., 2nd, McKee, T. C., & Boyd, M. R. 630

2001. A novel anti-HIV macrocyclic peptide from Palicourea condensata. J. Nat. Prod., 64, 631

249-250 632

Burman, R., Gruber, C. W., Rizzardi, K., Herrmann, A., Craik, D. J., Gupta, M. P., & Göransson, U. 633

2010a. Cyclotide proteins and precursors from the genus Gloeospermum: Filling a blank spot 634

in the cyclotide map of Violaceae. Phytochemistry, 71, 13-20 635

Burman, R., Stromstedt, A. A., Malmsten, M., & Göransson, U. 2011. Cyclotide-membrane 636

interactions: Defining factors of membrane binding, depletion and disruption. Biochim. 637

Biophys. Acta, 1808, 2665-2673 638

29

Burman, R., Svedlund, E., Felth, J., Hassan, S., Herrmann, A., Clark, R. J., Craik, D. J., Bohlin, L., 639

Claeson, P., Göransson, U., & Gullbo, J. 2010b. Evaluation of toxicity and antitumor activity 640

of cycloviolacin O2 in mice. Biopolymers, 94, 626-634 641

Camarero, J. A. 2017. Cyclotides, a versatile ultrastable micro-protein scaffold for biotechnological 642

applications. Bioorg. Med. Chem. Lett., 27, 5089-5099 643

Camarero, J. A., Kimura, R. H., Woo, Y. H., Shekhtman, A., & Cantor, J. 2007. Biosynthesis of a 644

fully functional cyclotide inside living bacterial cells. Chembiochem, 8, 1363-1366 645

Cao, P., Yang, Y., Uche, F. I., Hart, S. R., Li, W.-W., & Yuan, C. 2018. Coupling plant-derived 646

cyclotides to metal surfaces: An antibacterial and antibiofilm study. Int. J. Mol. Sci., 19, 793 647

Cascales, L., Henriques, S. T., Kerr, M. C., Huang, Y. H., Sweet, M. J., Daly, N. L., & Craik, D. J. 648

2011. Identification and characterization of a new family of cell-penetrating peptides: Cyclic 649

cell-penetrating peptides. J. Biol. Chem., 286, 36932-36943 650

Chen, B., Colgrave, M. L., Daly, N. L., Rosengren, K. J., Gustafson, K. R., & Craik, D. J. 2005. 651

Isolation and characterization of novel cyclotides from Viola hederaceae: Solution structure 652

and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide. J. Biol. Chem., 280, 22395-653

22405 654

Cheneval, O., Schroeder, C. I., Durek, T., Walsh, P., Huang, Y. H., Liras, S., Price, D. A., & Craik, 655

D. J. 2014. Fmoc-based synthesis of disulfide-rich cyclic peptides. J. Org. Chem., 79, 5538-656

5544 657

Chiche, L., Heitz, A., Gelly, J. C., Gracy, J., Chau, P. T., Ha, P. T., Hernandez, J. F., & Le-Nguyen, 658

D. 2004. Squash inhibitors: From structural motifs to macrocyclic knottins. Curr. Protein Pept. 659

Sci., 5, 341-349 660

Claeson, P., Göransson, U., Johansson, S., Luijendijk, T., & Bohlin, L. 1998. Fractionation protocol 661

for the isolation of polypeptides from plant biomass. J. Nat. Prod., 61, 77-81 662

Clark, R. J., & Craik, D. J. 2010. Native chemical ligation applied to the synthesis and bioengineering 663

of circular peptides and proteins. Biopolymers, 94, 414-422 664

Colgrave, M. L., & Craik, D. J. 2004. Thermal, chemical, and enzymatic stability of the cyclotide 665

kalata B1: The importance of the cyclic cystine knot. Biochemistry, 43, 5965-5975 666

30

Colgrave, M. L., Huang, Y. H., Craik, D. J., & Kotze, A. C. 2010. Cyclotide interactions with the 667

nematode external surface. Antimicrob. Agents Chemother., 54, 2160-2166 668

Colgrave, M. L., Kotze, A. C., Huang, Y. H., O'Grady, J., Simonsen, S. M., & Craik, D. J. 2008a. 669

Cyclotides: Natural, circular plant peptides that possess significant activity against 670

gastrointestinal nematode parasites of sheep. Biochemistry, 47, 5581-5589 671

Colgrave, M. L., Kotze, A. C., Ireland, D. C., Wang, C. K., & Craik, D. J. 2008b. The anthelmintic 672

activity of the cyclotides: Natural variants with enhanced activity. Chembiochem, 11, 1939-673

1945 674

Colgrave, M. L., Kotze, A. C., Kopp, S., McCarthy, J. S., Coleman, G. T., & Craik, D. J. 2009. 675

Anthelmintic activity of cyclotides: In vitro studies with canine and human hookworms. Acta 676

Trop., 109, 163-166 677

Conlan, B. F., Colgrave, M. L., Gillon, A. D., Guarino, R., Craik, D. J., & Anderson, M. A. 2012. 678

Insights into processing and cyclization events associated with biosynthesis of the cyclic 679

peptide kalata B1. J. Biol. Chem., 287, 28037-28046 680

Contreras, J., Elnagar, A. Y., Hamm-Alvarez, S. F., & Camarero, J. A. 2011. Cellular uptake of 681

cyclotide MCoTI-I follows multiple endocytic pathways. J. Control. Release, 155, 134-143 682

Cowper, B., Craik, D. J., & Macmillan, D. 2013. Making ends meet: Chemically mediated 683

circularization of recombinant proteins. Chembiochem, 14, 809-812 684

Craik, D. J. 2012. Host-defense activities of cyclotides. Toxins (Basel), 4, 139-156 685

Craik, D. J. 2013. Joseph Rudinger Memorial Lecture: Discovery and applications of cyclotides. J. 686

Pept. Sci., 19, 393-407 687

Craik, D. J., Daly, N. L., Bond, T., & Waine, C. 1999. Plant cyclotides: A unique family of cyclic and 688

knotted proteins that defines the cyclic cystine knot structural motif. J. Mol. Biol., 294, 1327-689

1336 690

Craik, D. J., Daly, N. L., & Waine, C. 2001. The cystine knot motif in toxins and implications for 691

drug design. Toxicon, 39, 43-60 692

Craik, D. J., & Du, J. 2017. Cyclotides as drug design scaffolds. Curr. Opin. Chem. Biol., 38, 8-16 693

31

Craik, D. J., Lee, M.-H., Rehm, F. B., Tombling, B., Doffek, B., & Peacock, H. 2018. Ribosomally-694

synthesised cyclic peptides from plants as drug leads and pharmaceutical scaffolds. Bioorg. 695

Med. Chem., 26, 2727-2737 696

Daly, N. L., Chen, B., Nguyencong, P., & Craik, D. J. 2010. Structure and activity of the leaf-specific 697

cyclotide vhl-2. Aust. J. Chem., 63, 771-778 698

Daly, N. L., Clark, R. J., Plan, M. R., & Craik, D. J. 2006. Kalata B8, a novel antiviral circular protein, 699

exhibits conformational flexibility in the cystine knot motif. Biochem. J., 393, 619-626 700

Daly, N. L., & Craik, D. J. 2000. Acyclic permutants of naturally occurring cyclic proteins. 701

Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide 702

kalata B1. J. Biol. Chem., 275, 19068-19075 703

Daly, N. L., Gustafson, K. R., & Craik, D. J. 2004. The role of the cyclic peptide backbone in the 704

anti-HIV activity of the cyclotide kalata B1. FEBS Lett., 574, 69-72 705

Daly, N. L., Koltay, A., Gustafson, K. R., Boyd, M. R., Casas-Finet, J. R., & Craik, D. J. 1999a. 706

Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV 707

activity. J. Mol. Biol., 285, 333-345 708

Daly, N. L., Love, S., Alewood, P. F., & Craik, D. J. 1999b. Chemical synthesis and folding pathways 709

of large cyclic polypeptides: Studies of the cystine knot polypeptide kalata B1. Biochemistry, 710

38, 10606-10614 711

Daly, N. L., Rosengren, K. J., & Craik, D. J. 2009. Discovery, structure and biological activities of 712

cyclotides. Adv. Drug Delivery Rev., 61, 918-930 713

Daly, N. L., Rosengren, K. J., Henriques, S. T., & Craik, D. J. 2011. NMR and protein structure in 714

drug design: Application to cyclotides and conotoxins. Eur. Biophys. J., 40, 359-370 715

Dawson, P. E., Muir, T. W., Clark-Lewis, I., & Kent, S. B. 1994. Synthesis of proteins by native 716

chemical ligation. Science, 266, 776-779 717

de Veer, S. J., Weidmann, J., & Craik, D. J. 2017. Cyclotides as tools in chemical biology. Acc. Chem. 718

Res., 50, 1557-1565 719

Du, J. Q., Chan, L. Y., Poth, A. G., & Craik, D. J. 2019. Discovery and characterization of cyclic and 720

acyclic trypsin inhibitors from Momordica dioica. J. Nat. Prod., 82, 293-300 721

32

Felizmenio-Quimio, M. E., Daly, N. L., & Craik, D. J. 2001. Circular proteins in plants: Solution 722

structure of a novel macrocyclic trypsin inhibitor from Momordica cochinchinensis. J. Biol. 723

Chem., 276, 22875-22882 724

Fensterseifer, I. C. M., Silva, O. N., Malik, U., Ravipati, A. S., Novaes, N. R. F., Miranda, P. R. R., 725

Rodrigues, E. A., Moreno, S. E., Craik, D. J., & Franco, O. L. 2015. Effects of cyclotides 726

against cutaneous infections caused by Staphylococcus aureus. Peptides, 63, 38-42 727

Gilding, E. K., Jackson, M. A., Poth, A. G., Henriques, S. T., Prentis, P. J., Mahatmanto, T., & Craik, 728

D. J. 2016. Gene coevolution and regulation lock cyclic plant defence peptides to their targets. 729

New. Phytol., 210, 717-730 730

Göransson, U., Burman, R., Gunasekera, S., Stromstedt, A. A., & Rosengren, K. J. 2012. Circular 731

proteins from plants and fungi. J. Biol. Chem., 287, 27001-27006 732

Göransson, U., Luijendijk, T., Johansson, S., Bohlin, L., & Claeson, P. 1999. Seven novel 733

macrocyclic polypeptides from Viola arvensis. J. Nat. Prod., 62, 283-286 734

Göransson, U., Sjogren, M., Svangård, E., Claeson, P., & Bohlin, L. 2004. Reversible antifouling 735

effect of the cyclotide cycloviolacin O2 against barnacles. J. Nat. Prod., 67, 1287-1290 736

Gould, A., & Camarero, J. A. 2017. Cyclotides: Overview and biotechnological applications. 737

Chembiochem, 18, 1350-1363 738

Gould, A., Ji, Y., Aboye, T. L., & Camarero, J. A. 2011. Cyclotides, a novel ultrastable polypeptide 739

scaffold for drug discovery. Curr. Pharm. Des., 17, 4294-4307 740

Gran, L. 1970. An oxytocic principle found in Oldenlandia affinis DC. Medd. Nor. Farm. Selsk., 12, 741

173-180 742

Gran, L. 1973a. On the effect of a polypeptide isolated from "Kalata-Kalata" (Oldenlandia affinis DC) 743

on the oestrogen dominated uterus. Acta Pharmacol. Toxicol. (Copenh.), 33, 400-408 744

Gran, L. 1973b. Oxytocic principles of Oldenlandia affinis. Lloydia, 36, 174-178 745

Gran, L., Sletten, K., & Skjeldal, L. 2008. Cyclic peptides from Oldenlandia affinis DC. Molecular 746

and biological properties. Chem. Biodivers., 5, 2014-2022 747

33

Greenwood, K. P., Daly, N. L., Brown, D. L., Stow, J. L., & Craik, D. J. 2007. The cyclic cystine 748

knot miniprotein MCoTI-II is internalized into cells by macropinocytosis. Int. J. Biochem. 749

Cell Biol., 39, 2252-2264 750

Gruber, C. W. 2010. Global cyclotide adventure: A journey dedicated to the discovery of circular 751

peptides from flowering plants. Biopolymers, 94, 565-572 752

Gruber, C. W., Elliott, A. G., Ireland, D. C., Delprete, P. G., Dessein, S., Göransson, U., Trabi, M., 753

Wang, C. K., Kinghorn, A. B., Robbrecht, E., & Craik, D. J. 2008. Distribution and evolution 754

of circular miniproteins in flowering plants. Plant Cell, 20, 2471-2483 755

Gründemann, C., Koehbach, J., Huber, R., & Gruber, C. W. 2012. Do plant cyclotides have potential 756

as immunosuppressant peptides? J. Nat. Prod., 75, 167-174 757

Gründemann, C., Stenberg, K. G., & Gruber, C. W. 2019. T20K: An immunomodulatory cyclotide 758

on its way to the clinic. Int. J. Pept. Res. Ther., 25, 9-13 759

Gründemann, C., Thell, K., Lengen, K., Garcia-Kaufer, M., Huang, Y. H., Huber, R., Craik, D. J., 760

Schabbauer, G., & Gruber, C. W. 2013. Cyclotides suppress human T-lymphocyte 761

proliferation by an interleukin 2-dependent mechanism. PLoS One, 8, e68016 762

Gunasekera, S., Aboye, T. L., Madian, W. A., El-Seedi, H. R., & Göransson, U. 2013. Making ends 763

meet: Microwave-accelerated synthesis of cyclic and disulfide rich proteins via in situ 764

thioesterification and native chemical ligation. Int. J. Pept. Res. Ther., 19, 43-54 765

Gustafson, K. R., Sowder II, R. C., Henderson, L. E., Parsons, I. C., Kashman, Y., Cardellina II, J. 766

H., McMahon, J. B., Buckheit, J. R. W., Pannell, L. K., & Boyd, M. R. 1994. Circulins A and 767

B: Novel HIV-inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia. J. 768

Am. Chem. Soc., 116, 9337-9338 769

Gustafson, K. R., Walton, L. K., Sowder, R. C., Jr., Johnson, D. G., Pannell, L. K., Cardellina, J. H., 770

Jr., & Boyd, M. R. 2000. New circulin macrocyclic polypeptides from Chassalia parvifolia. J. 771

Nat. Prod., 63, 176-178 772

Hallock, Y. F., Sowder, R. C., 2nd, Pannell, L. K., Hughes, C. B., Johnson, D. G., Gulakowski, R., 773

Cardellina, J. H., 2nd, & Boyd, M. R. 2000. Cycloviolins A-D, anti-HIV macrocyclic peptides 774

from Leonia cymosa. J. Org. Chem., 65, 124-128 775

34

Harris, K. S., Durek, T., Kaas, Q., Poth, A. G., Gilding, E. K., Conlan, B. F., Saska, I., Daly, N. L., 776

van der Weerden, N. L., Craik, D. J., & Anderson, M. A. 2015. Efficient backbone cyclization 777

of linear peptides by a recombinant asparaginyl endopeptidase. Nat. Commun., 6, 10199 778

Hellinger, R., Koehbach, J., Puigpinos, A., Clark, R. J., Tarrago, T., Giralt, E., & Gruber, C. W. 2015. 779

Inhibition of human prolyl oligopeptidase activity by the cyclotide psysol 2 isolated from 780

Psychotria solitudinum. J. Nat. Prod., 78, 1073-1082 781

Henriques, S. T., & Craik, D. J. 2012. Importance of the cell membrane on the mechanism of action 782

of cyclotides. ACS Chem. Biol., 7, 626-636 783

Henriques, S. T., & Craik, D. J. 2017. Cyclotide structure and function: The role of membrane binding 784

and permeation. Biochemistry, 56, 669-682 785

Henriques, S. T., Huang, Y. H., Castanho, M. A., Bagatolli, L. A., Sonza, S., Tachedjian, G., Daly, 786

N. L., & Craik, D. J. 2012. Phosphatidylethanolamine binding is a conserved feature of 787

cyclotide-membrane interactions. J. Biol. Chem., 287, 33629-33643 788

Henriques, S. T., Huang, Y. H., Chaousis, S., Sani, M. A., Poth, A. G., Separovic, F., & Craik, D. J. 789

2015. The Prototypic Cyclotide Kalata B1 Has a Unique Mechanism of Entering Cells. Chem. 790

Biol., 22, 1087-1097 791

Henriques, S. T., Huang, Y. H., Chaousis, S., Wang, C. K., & Craik, D. J. 2014. Anticancer and toxic 792

properties of cyclotides are dependent on phosphatidylethanolamine phospholipid targeting. 793

Chembiochem, 15, 1956-1965 794

Henriques, S. T., Huang, Y. H., Rosengren, K. J., Franquelim, H. G., Carvalho, F. A., Johnson, A., 795

Sonza, S., Tachedjian, G., Castanho, M. A., Daly, N. L., & Craik, D. J. 2011. Decoding the 796

membrane activity of the cyclotide kalata B1: The importance of phosphatidylethanolamine 797

phospholipids and lipid organization on hemolytic and anti-HIV activities. J. Biol. Chem., 286, 798

24231-24241 799

Hernandez, J. F., Gagnon, J., Chiche, L., Nguyen, T. M., Andrieu, J. P., Heitz, A., Trinh Hong, T., 800

Pham, T. T., & Le Nguyen, D. 2000. Squash trypsin inhibitors from Momordica 801

cochinchinensis exhibit an atypical macrocyclic structure. Biochemistry, 39, 5722-5730 802

35

Herrmann, A., Burman, R., Mylne, J. S., Karlsson, G., Gullbo, J., Craik, D. J., Clark, R. J., & 803

Göransson, U. 2008. The alpine violet, Viola biflora, is a rich source of cyclotides with potent 804

cytotoxicity. Phytochemistry, 69, 939-952 805

Huang, Y. H., Colgrave, M. L., Clark, R. J., Kotze, A. C., & Craik, D. J. 2010. Lysine-scanning 806

mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of 807

nematocidal activity. J. Biol. Chem., 285, 10797-10805 808

Huang, Y. H., Colgrave, M. L., Daly, N. L., Keleshian, A., Martinac, B., & Craik, D. J. 2009. The 809

biological activity of the prototypic cyclotide kalata b1 is modulated by the formation of 810

multimeric pores. J. Biol. Chem., 284, 20699-20707 811

Ireland, D. C., Colgrave, M. L., & Craik, D. J. 2006a. A novel suite of cyclotides from Viola odorata: 812

Sequence variation and the implications for structure, function and stability. Biochem. J., 400, 813

1-12 814

Ireland, D. C., Colgrave, M. L., Nguyencong, P., Daly, N. L., & Craik, D. J. 2006b. Discovery and 815

characterization of a linear cyclotide from Viola odorata: Implications for the processing of 816

circular proteins. J. Mol. Biol., 357, 1522-1535 817

Ireland, D. C., Wang, C. K., Wilson, J. A., Gustafson, K. R., & Craik, D. J. 2008. Cyclotides as natural 818

anti-HIV agents. Biopolymers, 90, 51-60 819

Jackson, M. A., Gilding, E. K., Shafee, T., Harris, K. S., Kaas, Q., Poon, S., Yap, K., Jia, H., Guarino, 820

R., Chan, L. Y., Durek, T., Anderson, M. A., & Craik, D. J. 2018. Molecular basis for the 821

production of cyclic peptides by plant asparaginyl endopeptidases. Nat. Commun., 9, 2411 822

Jagadish, K., Gould, A., Borra, R., Majumder, S., Mushtaq, Z., Shekhtman, A., & Camarero, J. A. 823

2015. Recombinant expression and phenotypic screening of a bioactive cyclotide against 824

alpha-Synuclein-Induced cytotoxicity in Baker's Yeast. Angew. Chem. Int. Ed. Engl., 54, 825

8390-8394 826

Jennings, C., West, J., Waine, C., Craik, D., & Anderson, M. 2001. Biosynthesis and insecticidal 827

properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis. Proc. Natl. 828

Acad. Sci. U. S. A., 98, 10614-10619 829

36

Jennings, C. V., Rosengren, K. J., Daly, N. L., Plan, M., Stevens, J., Scanlon, M. J., Waine, C., 830

Norman, D. G., Anderson, M. A., & Craik, D. J. 2005. Isolation, solution structure, and 831

insecticidal activity of kalata B2, a circular protein with a twist: Do Mobius strips exist in 832

nature? Biochemistry, 44, 851-860 833

Jia, X., Kwon, S., Wang, C. I., Huang, Y. H., Chan, L. Y., Tan, C. C., Rosengren, K. J., Mulvenna, J. 834

P., Schroeder, C. I., & Craik, D. J. 2014. Semienzymatic cyclization of disulfide-rich peptides 835

using Sortase A. J. Biol. Chem., 289, 6627-6638 836

Kamimori, H., Hall, K., Craik, D. J., & Aguilar, M. I. 2005. Studies on the membrane interactions of 837

the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon 838

resonance. Anal. Biochem., 337, 149-153 839

Kan, M.-W., & Craik, D. J. (2018). Trends in Cyclotide Research In Cyclic Peptides: From 840

Bioorganic Synthesis to Applications (pp. 302-339, chapter 14): The Royal Society of 841

Chemistry. 842

Kimura, R. H., Tran, A. T., & Camarero, J. A. 2006. Biosynthesis of the cyclotide Kalata B1 by using 843

protein splicing. Angew. Chem. Int. Ed. Engl., 45, 973-976 844

Koehbach, J., Attah, A. F., Berger, A., Hellinger, R., Kutchan, T. M., Carpenter, E. J., Rolf, M., 845

Sonibare, M. A., Moody, J. O., Wong, G. K., Dessein, S., Greger, H., & Gruber, C. W. 2013a. 846

Cyclotide discovery in Gentianales revisited-identification and characterization of cyclic 847

cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants. Biopolymers, 848

100, 438-452 849

Koehbach, J., O'Brien, M., Muttenthaler, M., Miazzo, M., Akcan, M., Elliott, A. G., Daly, N. L., 850

Harvey, P. J., Arrowsmith, S., Gunasekera, S., Smith, T. J., Wray, S., Göransson, U., Dawson, 851

P. E., Craik, D. J., Freissmuth, M., & Gruber, C. W. 2013b. Oxytocic plant cyclotides as 852

templates for peptide G protein-coupled receptor ligand design. Proc. Natl. Acad. Sci. U. S. 853

A., 110, 21183-21188 854

Koltay, A., Daly, N. L., Gustafson, K. R., & Craik, D. J. 2005. Structure of circulin B and implications 855

for antimicrobial activity of the cyclotides. Int. J. Pept. Res. Ther., 11, 99-106 856

37

Kwon, S., Duarte, J. N., Li, Z., Ling, J. J., Cheneval, O., Durek, T., Schroeder, C. I., Craik, D. J., & 857

Ploegh, H. L. 2018. Targeted delivery of cyclotides via conjugation to a nanobody. ACS Chem. 858

Biol., 13, 2973-2980 859

Lindholm, P., Göransson, U., Johansson, S., Claeson, P., Gullbo, J., Larsson, R., Bohlin, L., & 860

Backlund, A. 2002. Cyclotides: A novel type of cytotoxic agents. Mol. Cancer Ther., 1, 365-861

369 862

Malik, S. Z., Linkevicius, M., Göransson, U., & Andersson, D. I. 2017. Resistance to the cyclotide 863

cycloviolacin O2 in Salmonella enterica caused by different mutations that often confer cross-864

resistance or collateral sensitivity to other antimicrobial peptides. Antimicrob. Agents 865

Chemother., 61, e00684-00617 866

Mulvenna, J. P., Sando, L., & Craik, D. J. 2005. Processing of a 22 kDa precursor protein to produce 867

the circular protein tricyclon A. Structure, 13, 691-701 868

Mylne, J. S., Chan, L. Y., Chanson, A. H., Daly, N. L., Schaefer, H., Bailey, T. L., Nguyencong, P., 869

Cascales, L., & Craik, D. J. 2012. Cyclic peptides arising by evolutionary parallelism via 870

asparaginyl-endopeptidase–mediated biosynthesis. Plant Cell, 24, 2765-2778 871

Nguyen, G. K., Lian, Y., Pang, E. W., Nguyen, P. Q., Tran, T. D., & Tam, J. P. 2013. Discovery of 872

linear cyclotides in monocot plant Panicum laxum of Poaceae family provides new insights 873

into evolution and distribution of cyclotides in plants. J. Biol. Chem., 288, 3370-3380 874

Nguyen, G. K., Wang, S., Qiu, Y., Hemu, X., Lian, Y., & Tam, J. P. 2014. Butelase 1 is an Asx-875

specific ligase enabling peptide macrocyclization and synthesis. Nat. Chem. Biol., 10, 732-876

738 877

Noonan, J., Williams, W., & Shan, X. 2017. Investigation of antimicrobial peptide genes associated 878

with fungus and insect resistance in maize. Int. J. Mol. Sci., 18, E1938 879

Nourse, A., Trabi, M., Daly, N. L., & Craik, D. J. 2004. A comparison of the self-association behavior 880

of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation. J. Biol. 881

Chem., 279, 562-570 882

Oren, Z., & Shai, Y. 1998. Mode of action of linear amphipathic alpha-helical antimicrobial peptides. 883

Biopolymers, 47, 451-463 884

38

Parsley, N. C., Kirkpatrick, C. L., Crittenden, C. M., Rad, J. G., Hoskin, D. W., Brodbelt, J. S., & 885

Hicks, L. M. 2018. PepSAVI-MS reveals anticancer and antifungal cycloviolacins in Viola 886

odorata. Phytochemistry, 152, 61-70 887

Pinto, M. E. F., Najas, J. Z. G., Magalhães, L. G., Bobey, A. F., Mendonça, J. N., Lopes, N. P., Leme, 888

F. M., Teixeira, S. P., Trovó, M., Andricopulo, A. D., Koehbach, J., Gruber, C. W., Cilli, E. 889

M., & Bolzani, V. S. 2018. Inhibition of breast cancer cell migration by cyclotides isolated 890

from Pombalia Calceolaria. J. Nat. Prod., 81, 1203-1208 891

Plan, M. R., Göransson, U., Clark, R. J., Daly, N. L., Colgrave, M. L., & Craik, D. J. 2007. The 892

cyclotide fingerprint in oldenlandia affinis: Elucidation of chemically modified, linear and 893

novel macrocyclic peptides. Chembiochem, 8, 1001-1011 894

Plan, M. R., Rosengren, K. J., Sando, L., Daly, N. L., & Craik, D. J. 2010. Structural and biochemical 895

characteristics of the cyclotide kalata B5 from Oldenlandia affinis. Biopolymers, 94, 647-658 896

Plan, M. R., Saska, I., Cagauan, A. G., & Craik, D. J. 2008. Backbone cyclised peptides from plants 897

show molluscicidal activity against the rice pest Pomacea canaliculata (golden apple snail). J. 898

Agric. Food Chem., 56, 5237-5241 899

Poth, A. G., Chan, L. Y., & Craik, D. J. 2013. Cyclotides as grafting frameworks for protein 900

engineering and drug design applications. Biopolymers, 100, 480-491 901

Poth, A. G., Colgrave, M. L., Lyons, R. E., Daly, N. L., & Craik, D. J. 2011a. Discovery of an unusual 902

biosynthetic origin for circular proteins in legumes. Proc. Natl. Acad. Sci. U. S. A., 108, 903

10127-10132 904

Poth, A. G., Colgrave, M. L., Philip, R., Kerenga, B., Daly, N. L., Anderson, M. A., & Craik, D. J. 905

2011b. Discovery of cyclotides in the fabaceae plant family provides new insights into the 906

cyclization, evolution, and distribution of circular proteins. ACS Chem. Biol., 6, 345-355 907

Poth, A. G., Mylne, J. S., Grassl, J., Lyons, R. E., Millar, A. H., Colgrave, M. L., & Craik, D. J. 2012. 908

Cyclotides associate with leaf vasculature and are the products of a novel precursor in petunia 909

(Solanaceae). J. Biol. Chem., 287, 27033-27046 910

39

Pranting, M., Loov, C., Burman, R., Göransson, U., & Andersson, D. I. 2010. The cyclotide 911

cycloviolacin O2 from Viola odorata has potent bactericidal activity against Gram-negative 912

bacteria. J. Antimicrob. Chemother., 65, 1964-1971 913

Qu, H., Smithies, B. J., Durek, T., & Craik, D. J. 2017. Synthesis and protein engineering applications 914

of cyclotides. Aust. J. Chem., 70, 152-161 915

Quimbar, P., Malik, U., Sommerhoff, C. P., Kaas, Q., Chan, L. Y., Huang, Y. H., Grundhuber, M., 916

Dunse, K., Craik, D. J., Anderson, M. A., & Daly, N. L. 2013. High-affinity cyclic peptide 917

matriptase inhibitors. J. Biol. Chem., 288, 13885-13896 918

Rehm, F. B. H., Jackson, M. A., De Geyter, E., Yap, K., Gilding, E. K., Durek, T., & Craik, D. J. 919

2019. Papain-like cysteine proteases prepare plant cyclic peptide precursors for cyclization. 920

Proc. Natl. Acad. Sci. U. S. A., 116, 7831-7836 921

Rosengren, K. J., Daly, N. L., Harvey, P. J., & Craik, D. J. 2013. The self-association of the cyclotide 922

kalata B2 in solution is guided by hydrophobic interactions. Biopolymers, 100, 453-460 923

Rosengren, K. J., Daly, N. L., Plan, M. R., Waine, C., & Craik, D. J. 2003. Twists, knots, and rings 924

in proteins. Structural definition of the cyclotide framework. J. Biol. Chem., 278, 8606-8616 925

Saether, O., Craik, D. J., Campbell, I. D., Sletten, K., Juul, J., & Norman, D. G. 1995. Elucidation of 926

the primary and three-dimensional structure of the uterotonic polypeptide kalata B1. 927

Biochemistry, 34, 4147-4158 928

Saska, I., Gillon, A. D., Hatsugai, N., Dietzgen, R. G., Hara-Nishimura, I., Anderson, M. A., & Craik, 929

D. J. 2007. An asparaginyl endopeptidase mediates in vivo protein backbone cyclization. J. 930

Biol. Chem., 282, 29721-29728 931

Schöpke, T., Hasan Agha, M. I., Kraft, R., Otto, A., & Hiller, K. 1993. Hämolytisch aktive 932

komponenten aus Viola tricolor L. und Viola arvensis Murray. Sci. Pharm., 61, 145-153 933

Shenkarev, Z. O., Nadezhdin, K. D., Lyukmanova, E. N., Sobol, V. A., Skjeldal, L., & Arseniev, A. 934

S. 2008. Divalent cation coordination and mode of membrane interaction in cyclotides: NMR 935

spatial structure of ternary complex Kalata B7/Mn2+/DPC micelle. J. Inorg. Biochem., 102, 936

1246-1256 937

40

Shenkarev, Z. O., Nadezhdin, K. D., Sobol, V. A., Sobol, A. G., Skjeldal, L., & Arseniev, A. S. 2006. 938

Conformation and mode of membrane interaction in cyclotides. Spatial structure of kalata B1 939

bound to a dodecylphosphocholine micelle. FEBS J., 273, 2658-2672 940

Simonsen, S. M., Sando, L., Rosengren, K. J., Wang, C. K., Colgrave, M. L., Daly, N. L., & Craik, 941

D. J. 2008. Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of 942

residues essential for bioactivity. J. Biol. Chem., 283, 9805-9813 943

Slazak, B., Kapusta, M., Strömstedt, A. A., Słomka, A., Krychowiak, M., Shariatgorji, M., Andrén, 944

P. E., Bohdanowicz, J., Kuta, E., & Göransson, U. 2018. How does the sweet violet (Viola 945

odorata L.) fight pathogens and dests - cyclotides as a comprehensive plant host defense 946

system. Front. Recent Dev. Plant Sci., 9, 1296 947

Svangård, E., Burman, R., Gunasekera, S., Lovborg, H., Gullbo, J., & Göransson, U. 2007. 948

Mechanism of action of cytotoxic cyclotides: Cycloviolacin O2 disrupts lipid membranes. J. 949

Nat. Prod., 70, 643-647 950

Svangård, E., Göransson, U., Hocaoglu, Z., Gullbo, J., Larsson, R., Claeson, P., & Bohlin, L. 2004. 951

Cytotoxic cyclotides from Viola tricolor. J. Nat. Prod., 67, 144-147 952

Taichi, M., Hemu, X., Qiu, Y., & Tam, J. P. 2013. A thioethylalkylamido (TEA) thioester surrogate 953

in the synthesis of a cyclic peptide via a tandem acyl shift. Org. Lett., 15, 2620-2623 954

Tam, J. P., & Lu, Y. A. 1998. A biomimetic strategy in the synthesis and fragmentation of cyclic 955

protein. Protein Sci., 7, 1583-1592 956

Tam, J. P., Lu, Y. A., Yang, J. L., & Chiu, K. W. 1999. An unusual structural motif of antimicrobial 957

peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc. Natl. Acad. Sci. 958

U. S. A., 96, 8913-8918 959

Tang, J., Wang, C. K., Pan, X., Yan, H., Zeng, G., Xu, W., He, W., Daly, N. L., Craik, D. J., & Tan, 960

N. 2010. Isolation and characterization of cytotoxic cyclotides from Viola tricolor. Peptides, 961

31, 1434-1440 962

Trabi, M., & Craik, D. J. 2004. Tissue-specific expression of head-to-tail cyclized miniproteins in 963

Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1. 964

Plant Cell, 16, 2204-2216 965

41

Wang, C. K., Clark, R. J., Harvey, P. J., Johan Rosengren, K., Cemazar, M., & Craik, D. J. 2011. The 966

role of conserved Glu residue on cyclotide stability and activity: a structural and functional 967

study of kalata B12, a naturally occurring Glu to Asp mutant. Biochemistry, 50, 4077-4086 968

Wang, C. K., Colgrave, M. L., Gustafson, K. R., Ireland, D. C., Göransson, U., & Craik, D. J. 2008a. 969

Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis. J. Nat. Prod., 71, 47-970

52 971

Wang, C. K., Colgrave, M. L., Ireland, D. C., Kaas, Q., & Craik, D. J. 2009a. Despite a conserved 972

cystine knot motif, different cyclotides have different membrane binding modes. Biophys. J., 973

97, 1471-1481 974

Wang, C. K., & Craik, D. J. 2018. Designing macrocyclic disulfide-rich peptides for biotechnological 975

applications. Nat. Chem. Biol., 14, 417-427 976

Wang, C. K., Hu, S. H., Martin, J. L., Sjogren, T., Hajdu, J., Bohlin, L., Claeson, P., Göransson, U., 977

Rosengren, K. J., Tang, J., Tan, N. H., & Craik, D. J. 2009b. Combined X-ray and NMR 978

analysis of the stability of the cyclotide cystine knot fold that underpins its insecticidal activity 979

and potential use as a drug scaffold. J. Biol. Chem., 284, 10672-10683 980

Wang, C. K., Kaas, Q., Chiche, L., & Craik, D. J. 2008b. CyBase: A database of cyclic protein 981

sequences and structures, with applications in protein discovery and engineering. Nucleic 982

Acids Res., 36, D206-210 983

Wang, C. K., King, G. J., Northfield, S. E., Ojeda, P. G., & Craik, D. J. 2014. Racemic and quasi-984

racemic X-ray structures of cyclic disulfide-rich peptide drug scaffolds. Angew. Chem. Int. 985

Ed. Engl., 53, 11236-11241 986

Wang, C. K., Wacklin, H. P., & Craik, D. J. 2012. Cyclotides insert into lipid bilayers to form 987

membrane pores and destabilize the membrane through hydrophobic and 988

phosphoethanolamine-specific interactions. J. Biol. Chem., 287, 43884-43898 989

Weidmann, J., & Craik, D. J. 2016. Discovery, structure, function, and applications of cyclotides: 990

circular proteins from plants. J. Exp. Bot., 67, 4801-4812 991

42

Witherup, K. M., Bogusky, M. J., Anderson, P. S., Ramjit, H., Ransom, R. W., Wood, T., & Sardana, 992

M. 1994. Cyclopsychotride A, a biologically active, 31-residue cyclic peptide isolated from 993

Psychotria longipes. J. Nat. Prod., 57, 1619-1625 994

Zenoni, S., D’Agostino, N., Tornielli, G. B., Quattrocchio, F., Chiusano, M. L., Koes, R., Zethof, J., 995

Guzzo, F., Delledonne, M., Frusciante, L., Gerats, T., & Pezzotti, M. 2011. Revealing 996

impaired pathways in the an11 mutant by high-throughput characterization of Petunia axillaris 997

and Petunia inflata transcriptomes. The Plant Journal, 68, 11-27 998

999