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]
2
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
3
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
4
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
5
69
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
6
(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
7
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
8
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
146
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
9
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
10
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
11
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)
12
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
13
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
16
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