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]
1
1 Abstract
2 Cyclotides are a plant-derived family of peptides that comprise approximately 30 amino acid residues,
3 a cyclic backbone and a cystine knot. Due to their unique structure, cyclotides are exceptionally stable
4 to heat or proteolytic degradation and are tolerant to amino acid substitutions in their backbone loops
5 between conserved cysteine residues. Their toxicity to insect pests and their make-up of natural amino
6 acids has led to their applications in eco-friendly crop protection. Furthermore, their stability and cell
7 penetrating properties make cyclotides ideal scaffolds for bioactive epitope grafting. This article gives
8 a brief overview of cyclotide discovery, characterization, distribution, synthesis and mode of action
9 mechanisms. We focus on their toxicities to insect pests and their medical and agricultural
10 applications.
11 Keywords
12 Cyclotides; Stability; Insecticidal peptide; Cytotoxicity; Cell penetrating peptide; Applications
2
13 1 Introduction
14 Cyclotides are disulfide-rich peptides from plants that have been known now for nearly two decades
15 (Craik et al., 1999). They typically comprise 30 amino acids and contain the characteristic features of
16 a head-to-tail cyclized backbone and a knotted arrangement of three disulfide bonds. They occur in a
17 wide range of plant tissues, including leaves, flowers, stems and roots, and are thought to be present
18 as natural defense molecules, particularly against insects and nematodes. For this reason, they can be
19 regarded as plant toxins, i.e., molecules that are toxic to certain plant pests. However, most interest in
20 cyclotides has so far related to their exceptional stability and potential as a framework in drug design
21 rather than to their natural pesticidal functions. Here we provide an overview on the discovery,
22 structures and potential biotechnological applications of cyclotides. There have been a number of
23 recent reviews on the discovery and distribution (Gruber, 2010; Weidmann & Craik, 2016),
24 biosynthesis (Conlan et al., 2012; Craik et al., 2018; Qu et al., 2017), biological activities (Craik, 2012;
25 Daly et al., 2011; Göransson et al., 2012), mode of action (Henriques & Craik, 2012; Henriques &
26 Craik, 2017), and applications in drug design (Camarero, 2017; Craik & Du, 2017; de Veer et al.,
27 2017; Gould & Camarero, 2017; Gould et al., 2011; Poth et al., 2013; Wang & Craik, 2018) of
28 cyclotides as well as a recent analysis of publication trends in the cyclotide field (Kan & Craik, 2018).
29 The main aim of this article is to explore the context of cyclotides as toxins with interesting
30 biotechnological applications.
31 1.1 History of discovery
32 The first cyclotide discovered was kalata B1 which attracted attention due to its use in an indigenous
33 medicine in Africa (Gran, 1970, 1973a). In this usage, leaves from the Rubiaceae family plant
34 Oldenlandia affinis were boiled by Congolese women to make a tea that was ingested during labor,
35 resulting in accelerated child birth. This discovery was made by a Norwegian physician, Lorents Gran,
36 who observed the accelerated labor while serving on a Red Cross relief mission in the Congo in the
37 1960s. After taking some of the plant material back to Norway he discovered that the uterotonic
38 ingredient was a peptide, which he and colleagues partially characterized and found to comprise
39 approximately 30 amino acids (Gran, 1973a). At that stage, neither the disulfide knotted arrangement
40 nor the head-to-tail cyclized backbone was apparent, but Gran noted that the peptide was very stable 3
41 and was one of a number of peptides of similar molecular weight derived from an extract of the plant
42 leaves (Gran, 1973b).
43 The three-dimensional structure of kalata B1 was not elucidated until the mid-1990s, when mass
44 spectrometry and NMR studies were used to delineate the cyclic backbone and the knotted
45 arrangement of disulfide bonds, respectively (Saether et al., 1995). Around the same time, several
46 independent groups reported peptides of similar size with pharmaceutically relevant activities, which
47 had been discovered in the course of plant natural products screening programs. A report from a group
48 at Merck Research Laboratories described cyclopsychotride A, a macrocyclic peptide from a South
49 American plant from the Rubiaceae family, which had neurotensin antagonistic properties (Witherup
50 et al., 1994). In another report, a group at the National Cancer Institute, USA, noted the anti-HIV
51 activities of a series of macrocyclic peptides, which they named circulins because of their head-to-tail
52 cyclic backbone (Gustafson et al., 1994). The circulins were derived from the bark of a Tanzanian
53 tree, also from the Rubiaceae family. Earlier, the discovery of another macrocyclic peptide of similar
54 size from a violet plant (Violaceae family) was reported by an Austrian group, found while looking
55 for saponins with hemolytic activity (Schöpke et al., 1993).
56 Following these initial reports, two groups set up systematic discovery programs to see whether
57 similar peptides occur in related plants. Our group at The University of Queensland (Australia) found
58 a number of examples in plants of Rubiaceae and Violaceae families (Craik et al., 1999), while a
59 group in Sweden focused on plants from the Violaceae family (Claeson et al., 1998; Göransson et al.,
60 1999). These combined discoveries in the late 1990s led to the conclusion that there were indeed
61 many other members of this peptide family and the name “cyclotide” was coined to refer to plant-
62 derived head-to-tail cyclic peptides that contain a cystine knot motif (see Figure 1) (Craik et al., 1999).
63 1.2 Sequences and nomenclature of cyclotides
64 Currently more than 400 sequences of cyclotides have been reported and are documented in a database
65 called CyBase (www.cybase.org.au), which also contains data on other families of naturally occurring
66 cyclic peptides (Wang et al., 2008b). Figure 1 shows some example sequences from the three currently
67 defined subfamilies of cyclotides and a representative structure of prototypic examples from each
68 subfamily. 4
69
70 Figure 1: Selected sequences and three-dimensional structures from the three subfamilies of 71 cyclotides. A. An alignment of selected cyclotides sequences for which a solution structure has been 72 published. The sequences are aligned starting from the presumed N-terminal cleavage point in the 73 linear precursors. Cyclotides are classified into three subfamilies and listed chronologically according 74 to the publication date of their NMR derived structures. All cyclotides have six loops separated by six 75 conserved cysteines (numbered with Roman numerals I to VI) and are head-to-tail cyclized. The 76 cysteines are highlighted in yellow and the disulfide connectivities (I to IV, II to V and III to VI) are 77 shown at the top of the table with a black line. B. The solution structures (from left to right) of kalata 78 B1 (Rosengren et al., 2003), kalata B8 (Daly et al., 2006) and MCoTI-II (Felizmenio-Quimio et al., 79 2001) from the Möbius, bracelet and trypsin inhibitor subfamilies, respectively. The disulfide bonds 80 are indicated by yellow ball-and-stick structures, the beta sheets by blue arrows and the 310-helix turn 81 by red ribbon. 82 Initially, cyclotides were classified into two major subfamilies: the bracelet and Möbius
83 subfamilies, based on the absence or presence, respectively, of a cis-proline in loop 5 of the circular
84 backbone (the loops being defined as the backbone segments between successive Cys residues, as
85 shown in Figure 1). The origin of this nomenclature is quite straightforward: when all of the backbone
86 peptide linkages of a cyclic peptide exist in the usual trans arrangement, the backbone can be thought
87 of as bracelet-like; however, a three-dimensional structure containing a cis-proline results in a 180°
88 conceptual twist in the circular backbone, which can thus be topologically regarded as a Möbius strip
5
89 (Jennings et al., 2005). A third, smaller, subfamily of cyclotides was introduced upon discovery of
90 MCoTI-I and MCoTI-II from the tropical vine Momordica cochinchinensis (Hernandez et al., 2000).
91 Although these two trypsin inhibitor peptides are dissimilar from other cyclotides in sequence, they
92 share the common structural elements of three interlocked disulfide bridges and a cyclic backbone,
93 leading to their classification as cyclotides (Felizmenio-Quimio et al., 2001). They are alternatively
94 referred to as cyclic knottins (Chiche et al., 2004).
95 1.3 Cyclic cystine knot scaffold and structures of cyclotides
96 In addition to the structural elucidation of the prototypic cyclotide, kalata B1, numerous other
97 members of the family have been structurally characterized. Figure 1A shows a sequence alignment
98 of the 18 structurally characterized cyclotides. To date, NMR structures have been determined for:
99 eight cyclotides from the Möbius subfamily, including kalata B1 (Saether et al., 1995), kalata B2
100 (Jennings et al., 2005), cycloviolacin O14 (Ireland et al., 2006a), kalata B7 (Shenkarev et al., 2008),
101 varv F (Wang et al., 2009b), vhl-2 (Daly et al., 2010), Cter M (Poth et al., 2011a), and kalata B12
102 (Wang et al., 2011); ten cyclotides from the bracelet subfamily, including circulin A (Daly et al.,
103 1999a), cycloviolacin O1 (Rosengren et al., 2003), palicourein (Barry et al., 2004), vhr1 (Trabi &
104 Craik, 2004), circulin B (Koltay et al., 2005), tricyclon A (Mulvenna et al., 2005), vhl-1 (Chen et al.,
105 2005), kalata B8 (Daly et al., 2006), cycloviolacin O2 (Wang et al., 2009a), and kalata B5 (Plan et al.,
106 2010); and three cyclotides from the trypsin inhibitor subfamily MCoTI-II (Felizmenio-Quimio et al.,
107 2001), MCoTI-I (Kwon et al., 2018) and MCoTI-V (Mylne et al., 2012).
108 As can be seen in the sequence alignment in Figure 1A, loops 1 and 4 are highly conserved in
109 terms of both their number of residues and amino acid composition (Daly et al., 2009). The
110 conservation of these two loops is probably due to the fact that they form the central part of the
111 embedded cystine knot in cyclotides. Additionally, a highly conserved glutamic acid present in loop
112 1 has been shown to take part in a hydrogen-bond network that is important in stabilizing the structure
113 (Rosengren et al., 2003). In fact, this glutamic acid is almost completely conserved in both Möbius
114 and bracelet subfamilies, with the exception of kalata B12 (Plan et al., 2007), where the glutamic acid
115 is substituted for an aspartic acid in the corresponding position. Loop 1 in the trypsin inhibitor
6
116 subfamily lacks the Glu residue and contains a larger number of residues than the corresponding loop
117 in the Mobius and bracelet cyclotides.
118 Despite the sequence diversity in the other four loops (i.e. loops 2, 3, 5, and 6), cyclotides from
119 the three subfamilies all share the same structural features of the cyclic backbone and a cystine knot
120 core formed from three disulfide bonds (Figure 1B). The cystine knot motif is surrounded by a small
121 β-hairpin which, in most cyclotides, is combined with a third β-strand to form a distorted triple-
122 stranded β-sheet. This so-called cyclic cystine knot (CCK) is a special case of a common motif known
123 as the inhibitor cystine knot (ICK) scaffold (Craik et al., 2001), which is found in a wide range of
124 proteins from plants and animals. Overall, the combination of a head-to-tail cyclic backbone and a
125 cystine knot makes cyclotides extremely resistant to proteolytic breakdown, to high temperatures and
126 to chemical denaturants such as urea or guanidine (Colgrave & Craik, 2004). This stability might
127 explain why they are excellent insecticidal agents in plants, as this stability is presumably important
128 for protein-based natural products that accumulate in leaves without breakdown under harsh
129 environmental conditions. Stability is also the reason why cyclotides have attracted attention from a
130 drug design perspective.
131 1.4 Distribution of cyclotides in the plant kingdom, in individual plants, and within
132 individual tissues
133 So far cyclotides have been discovered in a range of species from five major plant families, i.e. the
134 Rubiaceae, Violaceae, Cucurbitaceae, Solanaceae, and Fabaceae families (Koehbach et al., 2013a).
135 These represent many economically important plant families and hence cyclotides are potentially of
136 broad interest in plant science. Their distribution within these plant families is highly variable; so far
137 every Violaceous plant examined has been found to contain cyclotides, suggesting that cyclotides are
138 ubiquitous in this family (Burman et al., 2010a). Violaceae comprises approximately 930 species that
139 are distributed both in temperate and tropical zones around the world and include common ornamental
140 plants such as pansies. By contrast, cyclotides occur in less than 5% of the Rubiaceae (Gruber et al.,
141 2008) and in only a few members of each of the other plant families, i.e. in two species of the
142 Solanaceae (Poth et al., 2012; Zenoni et al., 2011) and the Cucurbitaceae family (Du et al., 2019;
143 Hernandez et al., 2000), and only one from the Fabaceae (Poth et al., 2011b) so far. Cyclotide- 7
144 containing plant families, the corresponding orders and the reported number of cyclotides from each
145 plant family are summarized in Figure 2 (Craik, 2013).
146
147 Figure 2: Distribution of cyclotides within angiosperms (flowering plants). Cyclotide-bearing 148 plant families and the corresponding orders are highlighted, with the number of cyclotides reported 149 in the literature listed next to the plant images. All cyclotides reported to date were found in core 150 eudicot plants, and only acyclic variants were discovered in a monocot plant from the Poaceae family 151 (labeled with one asterisk) (Nguyen et al., 2013). Small circular peptides distinct from cyclotides in 152 sequence were derived from the Asteraceae family (labeled with two asterisks). The phylogenetic 153 information of angiosperms was obtained from the Angiosperm Phylogeny Website 154 (http://www.mobot.org/MOBOT/research/APweb/). Figure adapted from a previously published article 155 (Craik, 2013). 156 To date, cyclotides have only been discovered in core eudicot plants. It is expected that as more
157 investigations take place, the number of plant families that are reported to contain cyclotides will
158 increase, as will the number of cyclotide-bearing species. However, at this stage it is clear that many
159 plants do not contain cyclotides, but those that do, contain them in large amounts. There remain many
8
160 interesting questions as to why some plants have evolved the ability to produce cyclotides but most
161 have apparently not.
162 In the cyclotide-bearing plants that have been examined so far, cyclotides occur in many,
163 indeed probably all, tissues. For example, they occur in leaves, petioles, stems, pedicels, flowers, and
164 roots (Trabi & Craik, 2004). A single plant may contain dozens to hundreds of cyclotides. For example,
165 Viola hederacea has been reported to contain at least 66 cyclotides (Trabi & Craik, 2004) and O.
166 affinis has so far been found to contain 22 cyclotides (Plan et al., 2007). It is expected the number of
167 individual cyclotides known to occur in plants will increase dramatically as more transcriptome and/or
168 genomic studies are published (Koehbach et al., 2013a). Until recently, most discoveries have been
169 made at the peptide rather than nucleic acid level, but this is likely to change in future. Despite this
170 shift to nucleic acids based discovery, new MS-based sequencing approaches for cyclotides show
171 promise for ongoing discoveries at the peptide-level (Parsley et al., 2018).
172 Within a single plant, the distribution and type of cyclotides varies from tissue to tissue. For
173 example, one study showed that the cyclotides present in roots in V. hederacea were typically more
174 hydrophobic than those found in the leaves and flowers (Trabi & Craik, 2004). In only a few cases is
175 the same cyclotide found in multiple plants. For example, varv A is found in four different plants,
176 including O. affinis, Viola odorata, Viola tricolor, and Viola arvensis (Gruber et al., 2008). Similarly,
177 within an individual plant, some cyclotides occur in several tissues and others are specific to a given
178 tissue. For example, vhr1 occurs only in roots in V. hederacea but kalata B1 occurs in leaves, stems
179 and roots in O. affinis (Trabi & Craik, 2004).
180 Imaging studies suggest that there is a non-uniform distribution of cyclotides within a given
181 tissue type. For example, matrix-assisted laser desorption/ionization-mass spectrometric imaging
182 (MALDI-MSI) has been applied to assess the spatial distribution and relative abundances of
183 cyclotides within the leaves of Petunia x hybrida. The study revealed four distinct masses on a P.
184 hybrida leaf, one of them corresponding to Phyb A, which was found in higher abundance within the
185 mid-vein tissue compared with the laminar and peripheral leaf tissues (Poth et al., 2012). Thus, that
186 report demonstrated that cyclotides associate with the vascular section of leaf tissues, suggesting that
187 these peptides may play a role in plant defense through modulating insect herbivory.
9
188 2 Biological activities of cyclotides
189 In addition to the reported uterotonic activity of kalata B1 (Gran, 1973a, 1973b), a wide range of other
190 biological activities, including hemolytic activity (Daly et al., 1999a; Ireland et al., 2006a; Schöpke
191 et al., 1993; Tam et al., 1999), neurotensin antagonistic (Witherup et al., 1994), anti-HIV (Bokesch et
192 al., 2001; Chen et al., 2005; Daly et al., 2006; Daly et al., 2004; Gustafson et al., 1994; Gustafson et
193 al., 2000; Hallock et al., 2000; Ireland et al., 2008; Wang et al., 2008a), anti-microbial (Fensterseifer
194 et al., 2015; Gran et al., 2008; Pranting et al., 2010; Tam et al., 1999), protease inhibitory (Hernandez
195 et al., 2000; Quimbar et al., 2013), insecticidal (Jennings et al., 2001), antitumor (Herrmann et al.,
196 2008; Lindholm et al., 2002; Svangård et al., 2004; Tang et al., 2010), antifouling (Göransson et al.,
197 2004), nematocidal (Colgrave et al., 2008a), molluscicidal (Plan et al., 2008), cell-penetrating
198 (Contreras et al., 2011; Greenwood et al., 2007), immunosuppressive (Gründemann et al., 2012;
199 Gründemann et al., 2013), and prolyl oligopeptidase inhibitory activities (Hellinger et al., 2015) has
200 been reported for cyclotides, as summarized in Table 1.
10
201 Table 1. Summary of host defense-related and/or biological activities first reported for cyclotides
Activity Exemplary cyclotides First report
Uterotonic kalata B1, B2 and B7 (Gran, 1973a, 1973b)
Hemolytic violapeptide I, circulin A (Schöpke et al., 1993)
and B, cyclopsychotride
A, kalata B1, varv A
Anti-HIV circulin A and B, (Gustafson et al., 1994)
palicourin, kalata B1,
vhl-1
Neurotensin antagonist cyclopsychotride A (Witherup et al., 1994)
Anti-microbial circulin A and B, (Tam et al., 1999)
cyclopsychotride A,
kalata B1, kalata B7,
cycloviolacin O2
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 (Lindholm et al., 2002)
and F
Antifouling cycloviolacin O2 (Göransson et al., 2004)
Cell internalization MCoTI-II, kalata B1 and (Greenwood et al., 2007)
MCoTI-I
Nematocidal kalata B1 and B2, (Colgrave et al., 2008a)
cycloviolacin O2
Molluscicidal cycloviolacin O1, kalata (Plan et al., 2008)
B1, B2 and B5
Immunosuppressive kalata B1 (Gründemann et al., 2012)
Prolyl oligopeptidase kalata B1, psysol 2 (Hellinger et al., 2015)
inhibition
11
202 2.1 Toxic activities
203 One of the first activities reported for cyclotides was the ability to cause lysis of human erythrocytes.
204 This hemolytic activity was initially observed in violapeptide I, the first cyclotide discovered from
205 the violet family (Schöpke et al., 1993). Additional macrocyclic peptides were discovered during the
206 course of screening other plants for a range of other biological activities, including anti-HIV and anti-
207 neurotensin activities. In one of the first such studies, Gustafson et al. reported the antiviral activities
208 of circulins A and B, bracelet cyclotides isolated from Chassalia parvifolia (Rubiaceae), which
209 demonstrated antiviral cytoprotective effects on various human immunodeficiency virus (HIV) strains
210 at concentrations ranging from 40 to 260 nM (Gustafson et al., 1994). Similarly, screens for
211 neurotensin antagonistic activity led to the discovery of cyclopsychotride A from Psychotria longipes
212 (Rubiaceae) (Witherup et al., 1994), which was later also reported to be active against Gram-positive
213 and Gram-negative bacteria (Tam et al., 1999).
214 The antimicrobial activities of cyclotides were first described by Tam et al. (Tam et al., 1999),
215 whereby synthetically derived kalata B1 was reported to be active against Staphylococcus aureus in
216 the absence of salt in the test solution. The inhibitory activity was abolished under physiological
217 conditions, i.e. in the presence of 100 mM NaCl, and the peptide was inactive against E. coli. By
218 contrast, a later study of native kalata B1 by Gran et al. (Gran et al., 2008) suggested that it had
219 antibiotic effects on E. coli under low or high-salt conditions. In addition to these apparently
220 conflicting reports for in vitro studies, a recent publication reported that cyclotides kalata B2 and
221 cycloviolacin O2 display in vitro antibacterial activities and in vivo efficacy against S. aureus in a
222 subcutaneous wound infection animal model (Fensterseifer et al., 2015). The reported salt dependence
223 for the antimicrobial activity (Tam et al., 1999) suggests that the initial interaction between cyclotides
224 and the microbial surface may be electrostatic, similar to that described for defensins (Oren & Shai,
225 1998). The similarity in size of cyclotides to disulfide-rich defensins is consistent with a functional
226 role of cyclotides in host-defense.
227 There is ongoing interest in the literature in the antimicrobial potential of cyclotides. A recent
228 report on the antibacterial activity of peptide-containing extracts from the flowers of the elderberry
229 tree (Sambucus nigra) noted some partial sequences consistent with known cyclotides but the active
12
230 components have not yet been definitely confirmed as cyclotides (Álvarez et al., 2018a). That study
231 and another from the same group suggested potential applications of cyclotides as antimicrobial in
232 aquaculture applications (Álvarez et al., 2018b). Another report demonstrated the use of cyclotides as
233 surface coatings to reduce biofilm formation (Cao et al., 2018). Underpinning such applications, there
234 has also been interest in using computational approaches to predict the preferential cyclotide scaffolds
235 for antimicrobial applications (Balaraman & Ramalingam, 2018) and the potential for the
236 development of drug resistance (Malik et al., 2017; Noonan et al., 2017).
237 Cytotoxic activity with the potential for antitumor applications is another noteworthy property
238 reported in early cyclotide studies. In one study, cycloviolacin O2, isolated from V. odorata, and varv
239 A and varv F, isolated from V. arvensis, displayed cytotoxic activities against ten human tumor cell
240 lines, including immortalized myeloma, T-cell leukemia, small cell lung cancer, lymphoma, and renal
241 adenocarcinoma cell lines, as well as primary ovarian carcinoma cells from cancer patients (Lindholm
242 et al., 2002). The cytotoxicity of varv A and cycloviolacin O2 on healthy human lymphocytes was
243 also evaluated in the same study. Compared to the normal lymphocytes, both peptides displayed ~9-
244 fold higher selectivity towards the leukemia cells (Lindholm et al., 2002). Other cyclotides isolated
245 from the Violaceae family were extensively tested against a variety of human cancer cells of varying
246 origin and were reported to have potent activities in vitro (Herrmann et al., 2008; Pinto et al., 2018;
247 Svangård et al., 2004; Tang et al., 2010), suggesting that these plant-derived peptides might be
248 promising cytotoxic compounds with potential in cancer treatment. Since cycloviolacin O2 showed
249 promising results in cytotoxicity testing against lymphoma, leukemia, small cell lung cancer and
250 colon carcinoma cell lines in vitro at concentrations in the low micromolar range, a follow-up study
251 on the antitumor effects of the peptide was done in vivo using mouse xenograft models with hollow
252 fibers containing various human cancer cell lines implanted subcutaneously (Burman et al., 2010b).
253 The maximum tolerated dose was determined for single-dose injection (1.5 mg/kg) and repeated-dose
254 injection (0.5 mg/kg) prior to a hollow fiber study and xenograft study, respectively. Animals
255 implanted with a hollow fiber encapsulated with tumor cells received a single dose of cycloviolacin
256 O2 at 1 mg/kg one day after the implantation. Xenografted animals were treated with cycloviolacin
257 O2 by intravenous injection at 0.5 mg/kg daily up to 14 days. With no significant antitumor affects
13
258 observed, repeated dosing of cycloviolacin O2 at 1 mg/kg was reported to give a local-inflammatory
259 reaction at the injection site fter 2-3 doses and acute toxicity was observed after administration of the
260 cyclotide at 2 mg/kg. This negative result might be due to a low distribution of peptide at the site of
261 the implants or to intrinsically weak in vivo antitumor activity of the peptide. A recent study on the
262 selectivity of cyclotides against cells further demonstrated that cycloviolacin O2, kalata B1 and kalata
263 B2 are toxic towards both non-transformed (skin and PBMC) and transformed (cervical cancer,
264 melanoma and leukemia) cells and they exert their effects through targeting cell membranes
265 containing phosphatidylethanolamine (PE) phospholipids, causing subsequent membrane disruption
266 (Henriques et al., 2014). The finding of cyclotides interacting preferentially with PE-phospholipids is
267 in agreement with an earlier report by Burman et al (Burman et al., 2011).
268 Cyclotides possess potent intrinsic insecticidal activities, and thus have potential applications
269 in agriculture, as first reported by Jennings et al (Jennings et al., 2001). In that study, purified kalata
270 B1 was incorporated into an artificial diet and fed to larvae of Helicoverpa punctigera, a serious pest
271 of cotton crops worldwide. Half of the kalata B1 treated larvae died and none of the survivors
272 developed past the first instar stage of larval development. A later study demonstrated that cyclotides
273 damage the gut epithelium of H. armigera by inducing disruption of the microvilli, causing blebbing
274 of epithelial cells, swelling of the columnar cells and ultimately rupture of the cells (Barbeta et al.,
275 2008).
276 Since cyclotides possess insecticidal activity, it has been of interest to examine their activity
277 against other agricultural pests to understand their potentially broader role as natural pesticides. The
278 insecticidal activities of kalata B1 and a suite of alanine mutants against adult Drosophila
279 melanogaster were assessed and the disruption of the development of first and second instar larvae
280 through to adult D. melanogaster was demonstrated (Simonsen et al., 2008). In another series of
281 studies conducted by Colgrave et al., a range of cyclotides was found to inhibit the larval development
282 of two economically important sheep gastrointestinal nematodes, Haemonchus contortus and
283 Trichostrongylus colubriformis (Colgrave et al., 2008b; Wang et al., 2008a), as well as canine and
284 human hookworms (Colgrave et al., 2009). Kalata B1 (IC50 = 2.2 µM) showed equipotent activity to
285 the commercially used anthelmintic drugs, levamisole (IC50 = 8.9 µM) and naphthalophos (IC50 = 7.5
14
286 µM) against the larval life stage, confirming a potential role for cyclotides as natural anthelmintic
287 control agents. In a later study, kalata B1, B6 and cycloviolacin O14 were shown to have a pronounced
288 effect on the viability of larval and adult life stages of the dog hookworm Ancylostoma caninum and
289 inhibited larval development of the human hookworm Necator americanus (Colgrave et al., 2009).
290 Subsequent investigations of other natural cyclotides extracted from V. odorata were reported
291 and identified examples with up to 18-fold greater potency than kalata B1 in larval development
292 assays against H. contortus, with the most potent cyclotide being cycloviolacin O2 (Colgrave et al.,
293 2008b). Modification of cycloviolacin O2 by acetylation of the two lysine residues to mask their
294 charge resulted in a marked decrease in anthelmintic activity for this Viola-derived peptide to a level
295 comparable to kalata B1 (Colgrave et al., 2008b). A correlation was also observed between the
296 number of charged residues present in cyclotide sequences and their anthelmintic activity, suggesting
297 that the net charge of a cyclotide is probably an important determinant of anthelmintic activity.
298 Most recently, the antifungal activities of extracts from Viola odorata were reported (Slazak et al.,
299 2018) and suggest a role for cyclotides from this plant against microbial pests. Specifically,
300 cycloviolacin O2, O3, O13, and O19 (cyO2, O3, O13 and O19) isolated from Viola odorata were
301 evaluated for activity against five model plant fungal pathogens, namely Fusarium oxysporum,
302 Fusarium graminearum, Fusarium culmorum, Mycosphaerella fragariae, and Botrytis cinerea, and
303 two Viola-derived pathogens, namely Colletotrichum utrechtense and Alternaria alternate. All tested
304 cyclotides displayed antifungal activity. CyO3 exhibited the most potent activity with minimal
305 inhibitory concentrations (MICs) ranging from 0.8 to 12.5 μM; while cyO13 exhibited the lowest
306 activities with MICs ranging from 3 to 25 μM. All cyclotides displayed low micromolar activity
307 against A. alternate, a fungal pathogen also isolated from Viola odorata. Figure 3 schematically
308 illustrates the range of host defense-related activities of cyclotides.
309
15
310 Figure 3: Examples of pesticidal and toxic activities of native cyclotides. Cyclotides have been 311 reported to possess potent activities against various pests, including the nematode H. contortus 312 (nematocidal)(Colgrave et al., 2008a), budworm H. punctigera (insecticidal) (Jennings et al., 2001), 313 rice pest Pomacea canaliculata (molluscicidal) (Plan et al., 2008), and fouling barnacles Balanus 314 improvisus (antifouling) (Göransson et al., 2004). The reported toxic effects of cyclotides against the 315 human immunodeficiency virus (anti-HIV) (Gustafson et al., 1994) and a range of cancer cell lines 316 (anti-tumor) (Lindholm et al., 2002) highlight the potential therapeutic and agrochemical applications 317 of these peptides. Therapeutic activities that are not linked with the toxic effects of cyclotides, e.g. 318 immunosuppressive activity (Gründemann et al., 2012), are not included in this figure.
319 2.2 Pharmaceutical activities
320 In addition to their toxic or host-defense activities, a range of naturally occurring cyclotides possess
321 activities of pharmacological and pharmaceutical relevance beyond the uterotonic, anti-HIV and
322 neurotensin antagonist activities noted already. In a recent study that nicely links the original
16
323 indigenous medical applications of the cyclotide-bearing plant O. affinis with the latest
324 pharmacological studies, the oxytocic activity of kalata B7, a Möbius cyclotide closely related to
325 kalata B1 was reported and the possible molecular target underlying the mechanism of the uterotonic
326 activity was revealed. Pharmacological studies showed that kalata B7 is a partial agonist of both the
327 oxytocin receptor and vasopressin V1A receptor and a structural analysis suggested that loop 3 of
328 kalata B7, which displays moderate sequence homology to human oxytocin, could be responsible for
329 the observed uterostimulant effects on uterine smooth muscle cells (Koehbach et al., 2013b).
330 The discovery of the anti-proliferative activity of cyclotides on primary activated human
331 lymphocytes suggested the potential use of these peptides as immunosuppressant drugs. The
332 inhibitory effects of kalata B1 on the proliferation of human peripheral blood mononuclear cells
333 (PBMC) at non-cytotoxic concentrations was first reported by Gründemann et al. (Gründemann et al.,
334 2012). This research led to further investigations of the immunosuppressive properties of cyclotides,
335 whereby an analog of kalata B1 [T20K] was shown to attenuate the interleukin-2 (IL-2) secretion and
336 the expression of IL-2 surface receptor on activated T-lymphocytes (Gründemann et al., 2013).
337 Further mechanistic studies, described in the same report, using several kalata B1 analogs with single
338 point mutations determined the cyclotide motif accountable for the anti-proliferative activity. The
339 recent progression of [T20K]kalata B1 to Phase I clinical trials for multiple sclerosis further illustrates
340 the potential applications of cyclotides in immunopharmacology and immunosuppression
341 (Gründemann et al., 2019).
342 2.3 Cell penetrating properties
343 Delivery of peptide-based drugs to intracellular targets is one of the holy grails of drug development.
344 Cyclotides triggered interest as potential frameworks for intracellular drug delivery after Greenwood
345 et al. reported the internalization of the cyclotide MCoTI-II into mammalian cells by endocytosis
346 (Greenwood et al., 2007). In that study, cells treated with biotinylated cyclotides were fixed and
347 stained with avidin-FITC before the internalization was evaluated using confocal fluorescence
348 microscopy. Although internalization studies in such systems need careful analysis to avoid possible
349 artifacts, the study provided the first indication of the cell penetrating ability of a cyclotide. In a later
350 study, the cellular uptake of fluorescently labeled MCoTI-II and kalata B1 were explored using live- 17
351 cell confocal imaging techniques and their affinity for phospholipids was examined on model
352 membrane systems by surface plasmon resonance or on PIP strips™ membranes (Cascales et al.,
353 2011). It was confirmed that MCoTI-II and kalata B1 are both able to penetrate cells but that they
354 probably cross cell membranes through different pathways. That study categorized these two peptides
355 and a smaller cyclic sunflower peptide, SFTI-1, as a new class of cell-penetrating peptides, referred
356 to as cyclic cell-penetrating peptides (Cascales et al., 2011). A contemporaneous study, using real
357 time confocal microscopy, showed that MCoTI-I, a cyclotide with ~95% sequence similarity to
358 MCoTI-II, internalized into HeLa cells predominantly through a temperature-dependent active
359 endocytic pathway (Contreras et al., 2011). Overall, these independent reports confirm that cyclotides
360 have the potential to be used as stable scaffolds for delivering therapeutically significant peptide
361 epitopes into cells and this topic is likely to be an active area of future cyclotide research.
362 3 Synthesis, structure-activity relationships and mode of action of cyclotides
363 Research on cyclotides has led to a number of impacts in the field of biological chemistry, including
364 the development of approaches to the chemical and biological synthesis of cyclic peptides, which has
365 opened up technologies for deriving structure-activity studies of cyclotides and for their applications
366 as drug design frameworks.
367 3.1 Chemical and biological syntheses of cyclotides
368 Approaches for the chemical synthesis of cyclotides were first described in the late 1990’s (Daly et
369 al., 1999b; Tam & Lu, 1998). In Tam and Lu’s report (Tam & Lu, 1998), the backbone cyclization of
370 circulin B and cyclopsychotride A was achieved by adapting native chemical ligation (Dawson et al.,
371 1994), where an N-terminal cysteine and a C-terminal thioester of peptide precursors synthesized
372 using Boc-chemistry solid phase peptide synthesis (SPPS) reacted to form a thioester that
373 subsequently underwent a spontaneous acyl transfer reaction to produce a native amide bond between
374 the two termini. In that study orthogonal protection of pairs of Cys residues was used to direct the
375 oxidation in a stepwise manner to form the desired disulfide connectivities. Daly et al. (Daly et al.,
376 1999b) used a similar approach for the synthesis of kalata B1 but without orthogonal protection of
377 Cys residues and showed it was possible to readily isolate the correctly folded product from the
18
378 mixture of disulfide isomers. Overall, the native chemical ligation-based synthesis method using Boc-
379 chemistry has been applied to the cyclization of a wide range of macrocyclic peptides and has proven
380 to be valuable in the routine production of cyclotides (Clark & Craik, 2010). Various Fmoc-based
381 SPPS methods to generate the thioester precursors of cyclotides for head-to-tail cyclization via native
382 chemical ligation (Gunasekera et al., 2013; Taichi et al., 2013), bacterial expression of recombinant
383 linear precursors of cyclotide followed by in vitro cyclization (Cowper et al., 2013), and a more direct
384 strategy for backbone cyclization using Fmoc-based SPPS (Cheneval et al., 2014) have also been
385 reported in recent years.
386 Several enzyme-mediated approaches for cyclization of cyclotides have been explored,
387 including intein-mediated biosynthetic methods (Camarero et al., 2007; Jagadish et al., 2015; Kimura
388 et al., 2006) and sortase A-catalyzed backbone cyclization (Jia et al., 2014). The intein-mediated
389 backbone cyclization of kalata B1 was achieved by recombinantly expressing linear cyclotide
390 precursors fused to a Met and an engineered intein unit at their N and C termini, respectively. The
391 fusion proteins were cleaved by endogenous Met aminopeptidase and underwent intein-mediated
392 protein splicing in E. coli which resulted in linear cyclotide precursors with a C-terminal α-thioester
393 and an N-terminal Cys required for native chemical ligation-based cyclization in vitro (Kimura et al.,
394 2006). The intein-mediated in vivo production of cyclotides MCoTI-II and MCoTI-I was reported by
395 the same group, in live bacterial cells (Camarero et al., 2007) and yeast cells (Jagadish et al., 2015),
396 respectively. These reports demonstrated the applicability of recombinant expression of natively
397 folded cyclotides in microorganisms and the possibility of producing large combinatorial cyclotide-
398 based libraries for screening. More recently, a chemo-enzymatic approach was developed to
399 synthesize cyclic disulfide-rich peptides, including kalata B1, cyclic α-conotoxin Vc1.1 and SFTI,
400 whereby the chemically synthesized linear peptide precursors containing a sortase A recognition motif
401 at the C-terminus were cyclized by sortase A in vitro without significant perturbation to the overall
402 peptide fold (Jia et al., 2014).
403 In parallel with the development of new methodologies for the efficient synthesis of cyclotides,
404 there is a growing effort towards understanding the biosynthetic mechanism of these macrocyclic
405 peptides in plants. Since an Asn is highly conserved at the C-terminus of the cyclotide domain,
19
406 asparaginyl endopeptidase (AEP), a cysteine protease with specificity for Asn, has been implicated
407 in the cyclization of cyclotides in vivo (Saska et al., 2007). In this key study, kalata B1 was expressed
408 transiently in Nicotiana benthamiana transformed with the precursor of kalata B1, and a reduction in
409 the yield of backbone-cyclized kalata B1 was observed with AEP-gene silencing constructs, providing
410 an initial correlation between AEP activity and cyclization yield of cyclotides in plants (Saska et al.,
411 2007). A recent publication reported the discovery of the AEP homolog butelase 1 in the cyclotide-
412 propeptides with a His-Val sequence at the C terminus in vitro (Nguyen et al., 2014). Butelase 1 has
413 been shown to cyclize peptides of varying lengths (from 14 to 34 residues) and sequences, including
414 kalata B1, SFTI, cyclic conotoxin MrlA, and antimicrobial peptide histatin-3 at a low enzyme-to-
415 peptide ratio (1:400) within 48 min. This finding suggests that butelase 1 could be developed as an
416 alternative approach to complement the current chemical and biological methodologies in producing
417 macrocyclic peptides.
418 Another impact deriving from cyclotide research has been the development of methodologies
419 for crystalizing disulfide-rich peptides. Of the many structures of cyclotides published, until recently,
420 only one involved X-ray crystallography because cyclotides along with other disulfide-rich peptides
421 are notoriously difficult to crystallize. However, Wang et al. (Wang et al., 2014) demonstrated that
422 the use of racemic crystallography dramatically improved crystallization rates and determined crystal
423 structures for a series of cyclic disulfide-rich peptides, ranging from SFTI-1 (14 amino acids with one
424 disulfide bond) to a cyclized conotoxin (22 amino acids with two disulfide bonds) to kalata B1 (29
425 amino acids with three disulfide bonds). Although this technology was demonstrated for cyclic
426 molecules, it should be equally applicable to the crystallization of a wide range of acyclic disulfide-
427 rich peptides.
428 3.2 Structure-activity relationships
429 The ability to chemically synthesize cyclotides has facilitated a wide range of mutagenesis studies
430 and structure-activity relationship studies that reveal the importance of the circular backbone for
431 maintenance of cyclotide structural integrity. In one early study by Daly et al. (Daly & Craik, 2000),
432 six acyclic permutants of kalata B1, with the backbone opened in each of the six inter-cysteine loops,
433 were synthesized and their overall folds were compared with that of the cyclic native peptide. A native 20
434 fold could not be achieved in acyclic mutants having a break of the backbone in either loops 1 or 4,
435 which are the loops forming the embedded ring in the cystine knot. This result suggests that the cystine
436 knot is essential in stabilizing the intermediates formed during the oxidative folding of cyclotides.
437 The overall folds of the four other acyclic analogs of kalata B1, with a break in one of loops 2, 3, 5,
438 or 6, were found to be very similar to that of their parent peptide, showing that a cyclic backbone is
439 not essential for a native-like fold. Although these four acyclic analogs of kalata B1 retained a native-
440 like conformation, their lack of hemolytic activity suggests that the circular backbone is functionally
441 important (Daly & Craik, 2000). Furthermore, the three-dimensional solution structures of a synthetic
442 acyclic permutant of kalata B1 with most of loop 6 removed and a naturally occurring linear cyclotide,
443 violacin A (with a discontinuous loop 6), showed that a backbone discontinuity renders structures
444 more flexible than in their cyclic counterparts (Barry et al., 2003; Daly & Craik, 2000; Ireland et al.,
445 2006b). These combined findings indicate that the circular backbone is crucial to both the structure
446 rigidity and activity of cyclotides (Barry et al., 2003; Daly & Craik, 2000).
447 The ability to chemically synthesize cyclotides has also facilitated a wide range of
448 mutagenesis studies that have helped to delineate their mode of action, as described in the following
449 section.
450 3.3 The mode of action of cyclotides
451 The mode of action of cyclotides may vary depending on the particular biological activity but in
452 general is strongly dependent on their unique structural features. The cystine knot structural motif
453 effectively results in the exclusion of non-Cys side-chains from the core region of cyclotides,
454 promoting the surface exposure of hydrophobic residues, some of which are clustered together to form
455 a hydrophobic patch. Several characteristic biophysical properties derive from this surface-exposed
456 hydrophobic patch, including late elution on RP-HPLC and weak self-association. These properties
457 have potential implications for the mode of action of cyclotides as cytotoxic agents since they provide
458 clues as to how these molecules might interact with and form pores in membranes. In this regard, the
459 oligomerization and self-association properties of cyclotides have been investigated using analytical
460 ultracentrifugation techniques. For instance, kalata B2 was observed to self-associate into tetramers
461 and octamers (Nourse et al., 2004), but not dimers. In one model of the geometry of the tetramer 21
462 proposed in that study, the oppositely charged residues Arg-24 and Asp-25 in kalata B2 create an
463 exposed bipolar patch at one end of the molecule, which was postulated to facilitate intermolecular
464 ionic self-interaction and potentially play a role in the formation of membrane-spanning pores. A later
465 experimental NMR study suggested an alternative model for the self-association in solution based on
466 interaction between the hydrophobic patches of kalata B2 (Rosengren et al., 2013). The significance
467 of the solution-state oligomers to the function of cyclotides remains unknown, and similarly it is not
468 known if cyclotides form aggregates in their membrane bound states, but a wide range of studies do
469 suggest that membrane interactions are intimately associated with cyclotide functions.
470 Synthetic and mutagenesis-based studies have contributed significantly to defining the
471 membrane-interacting hypothesis for the mode of action of kalata B1. For instance, a comparison of
472 enantiomer forms of a peptide ligand provides a definitive means to indicate whether a chiral protein
473 receptor is involved in its biological function or whether it acts via a (largely achiral) membrane
474 disruption mechanism. Colgrave et al. (Colgrave et al., 2008a) showed that the nematocidal activity
475 of the mirror-image stereoisomer of kalata B1 was similar to the wild-type peptide, suggesting that
476 the mechanism of action is probably via membrane interaction rather than by a chiral (i.e. protein-
477 based) receptor interaction. The self-association behaviour of cyclotides and the comparable
478 bioactivity of the all D-enantiomer of kalata B1 to the native L-form are key pieces of evidence which
479 suggest that the mechanism of action may be via membrane interaction. The membrane-based
480 mechanism of action of cyclotides was supported by an early surface plasmon resonance study which
481 demonstrated that kalata B1 and B6 bind selectively to phosphatidylethanolamine (PE)-containing
482 model membranes (Kamimori et al., 2005). Further support for the membrane-binding properties of
483 native cyclotides derived from the observation that cycloviolacin O2 induced leakage of both calcein-
484 loaded HeLa cells and a lipid model in the form of palmitoyloleoylphosphatidylcholine (POPC)
485 liposomes (Svangård et al., 2007). NMR studies by Shenkarev et al. showed that the binding of kalata
486 B1 and kalata B7 to dodecylphosphocholine (DPC) micelles is modulated by both electrostatic and
487 hydrophobic interactions (Shenkarev et al., 2008; Shenkarev et al., 2006). Varv F was shown to bind
488 to DPC micelles and its overall conformation remained unchanged upon binding (Wang et al., 2009b).
22
489 The nematocidal activity of a suite of alanine mutants of kalata B1 was examined and the
490 residues critical for activity against helminths correlated with those significant for insecticidal activity
491 against D. melanogaster (Simonsen et al., 2008). Residues critical for the biological activities of
492 kalata B1 were clustered on one side of the molecule, named ‘the bioactive patch’. Since membrane
493 interaction involving oligomerization was speculated to be responsible for the insecticidal activities
494 of kalata B1, the whole suite of alanine mutants of kalata B1 was screened against a range of model
495 membranes encapsulated with self-quenching dye for their lytic activities (Huang et al., 2009). The
496 leakage study confirmed that the bioactive patch of kalata B1 plays a critical role in its lytic, as well
497 as its insecticidal and hemolytic activities. In addition, kalata B1 was observed to have a preference
498 for phospholipids containing PE headgroups compared to model membranes containing only
499 zwitterionic or anionic phospholipids (Huang et al., 2009). Results from patch-clamp experiments
500 suggested that kalata B1 induced leakage via pore formation on reconstituted asolectin (soybean
501 lecithin), when compared with a membrane-inactive mutant of kalata B1 (V25A) (Huang et al., 2009).
502 A later study of lysine mutants of kalata B1 revealed that a single lysine substitution on a face opposite
503 to the bioactive patch improved its nematocidal activity (Huang et al., 2010). Furthermore, Colgrave
504 et al. observed increasing uptake of the radiolabel [3H]inulin of ligated adult nematodes upon kalata
505 B1 treatment, providing evidence to support the conclusion that the anthelmintic effect of the
506 cyclotide was due to increased permeability of the external membrane of the nematodes (Colgrave et
507 al., 2010). Together, these various mutagenesis studies and electrophysiological recordings provide
508 mechanistic insights into how kalata B1 exerts its effects on different organisms.
509 Many other bioactivities of cyclotides correlate with lipid-binding properties, as supported by
510 detailed biophysical studies using surface plasmon resonance (Henriques et al., 2012; Henriques et
511 al., 2014; Henriques et al., 2011) and isothermal titration calorimetry (ITC) (Wang et al., 2012) on
512 model membranes. An extensive lipid binding study of kalata B1 and a range of its active and inactive
513 mutants using surface plasmon resonance suggested that kalata B1 preferred more rigid membranes
514 containing PE phospholipids and exerted its anti-HIV activities by disrupting the membrane envelope
515 of viral particles (Henriques et al., 2011). Furthermore, a titration of kalata B1 with PE, monitored
516 using 1H NMR chemical shifts, suggested that it interacted specifically with the PE headgroups
23
517 through residues that formed part of the bioactive patch (Henriques et al., 2011). Therefore, the
518 membrane-targeting properties of cyclotides against PE headgroups was proposed as the initial step
519 of their lytic action, followed by membrane insertion with the hydrophobic patch, which leads to local
520 membrane disturbances and eventually membrane disruption (Henriques et al., 2012). More recently,
521 the PE-targeting ability of kalata B1 was also suggested to be responsible for the observed cell
522 internalization of kalata B1 at concentrations lower than the cytotoxicity threshold (Henriques et al.,
523 2015).
524 Figure 4 summarizes our current understanding of the proposed mechanism of cell
525 internalization of kalata B1, which involves the binding of the bioactive patch to PE phospholipids in
526 cell membranes via electrostatic interactions, before the hydrophobic face of the cyclotide is inserted
527 into the core of the bilayer. The accumulation of cyclotide on the lipid bilayer causes local membrane
528 disturbances, which eventually leads to cell penetration.
529
530 Figure 4: A schematic representation of the cell internalization of kalata B1. The initial step of 531 cell internalization of kalata B1 is PE-targeting. The bioactive patch (highlighted in red) of kalata B1 532 binds to the headgroup of PE phospholipids in cell membranes via electrostatic interaction (1), 533 followed by the insertion of the hydrophobic face (highlighted in green) into the core of the bilayer 534 (2). The accumulation of cyclotide molecules on the lipid bilayer leads to local membrane 535 disturbances, which eventually leads to cell penetration (3) through: i) endocytosis or ii) membrane 536 translocation by an energy-independent process. Figure adapted from a previously published article 537 (Henriques et al., 2015). 538 24
539 Overall, membrane binding is fundamental for many reported functions of cyclotides,
540 including hemolytic, insecticidal, nematocidal, and anti-HIV activities, as well as cell internalization.
541 However, considering that some cyclotides have been reported to bind to several members of the G
542 protein-coupled receptor family for their oxytocic properties (Koehbach et al., 2013b), there is a
543 possibility that other cyclotides might also exert other activities via modulating cellular receptors
544 separately from or in addition to binding to membranes.
545
546 4 Applications
547 Cyclotides have a range of potential applications in agriculture and medicine based on their
548 exceptional stability and their tolerance to sequence modifications that allow “designer cyclotides” to
549 be made. In this section, we very briefly outline some of those applications.
550 4.1 Medical applications of cyclotides
551 One approach to medical application of cyclotides is to harness some of the toxic properties of natural
552 cyclotides for therapeutic applications, for example, as cytotoxic (anti-cancer agents) or as
553 nematocidal agents with applications for human parasites, such as hookworms. A second area of
554 medical applications is through “designer” cyclotides made by grafting a bioactive epitope into a
555 cyclotide sequence to introduce a new activity of therapeutic relevance not present in the original
556 cyclotide framework. The aim of all of these studies is to take advantage of the stability of the
557 cyclotide framework to stabilize the peptide epitope. These grafting applications have been widely
558 reviewed elsewhere, so we will not discuss them further here except to refer readers to recent reviews
559 on the topic (Camarero, 2017; Craik & Du, 2017; de Veer et al., 2017; Gould & Camarero, 2017;
560 Gould et al., 2011; Poth et al., 2013; Wang & Craik, 2018). There are more than 25 examples of
561 grafted cyclotides for a range of diseases, including cardiovascular disease, cancer, wound healing,
562 pain, inflammation and multiple sclerosis. So far, none of these examples has reached human clinical
563 trials but all are well exemplified by animal studies or at least in vitro testing.
25
564 4.2 Agricultural applications
565 Stimulated by their natural functions as endogenous pesticidal agents in certain plants,
566 cyclotides have attracted attention for potential applications in the protection of crop plants that
567 naturally do not contain them. Such applications include their incorporation into transgenic plants, a
568 topic that is outside the scope of the current article, as well as applications involving external
569 administration onto growing crops or harvested material. The most advanced application involving
570 external application is exemplified with the recent approval of SeroX, an extract from butterfly pea
571 (Clitoria ternatea), as a treatment for insect pests on cotton and macadamia nut crops in Australia.
572 This plant, from the Fabaceae family, contains more than 47 different cyclotides (Gilding et al., 2016),
573 of which the peptide Cter M, at least, has been shown to have insecticidal properties as an isolated
574 peptide (Poth et al., 2011a). The SeroX product is marketed as a spray for cotton at doses as low as
575 2L/hectare by its developer, Innovate Ag, based in Australia.
576 While we will not cover the alternative mode of delivery of cyclotides via the incorporation
577 of transgenes encoding cyclotides into crop plants here, it is useful to note that there have been a
578 number of recent advances in understanding the roles of asparaginyl endopeptidase enzymes in
579 facilitating cyclotide biosynthesis (Harris et al., 2015; Jackson et al., 2018). Additionally, the enzyme
580 kalatase A, which is responsible for the N-terminal processing of cyclotide precursors was recently
581 reported (Rehm et al., 2019). These studies will no doubt be useful in facilitating the production of
582 transgenic plants with high yields of pesticidal cyclotides, thereby engendering these plants with
583 similar levels of insect protection to natural cyclotide-producer plants.
584 5 Outlook and future studies
585 Overall, cyclotides have attracted a great deal of interest, not only for their natural insecticidal
586 activities and their potential as drug scaffolds but also because of their topologically unique structures.
587 These structures engender cyclotides with exceptional stability and thus, in principle, they have
588 advantages over less stable peptides in that they offer potential for a variety of formulation approaches
589 and are stable to long term storage, an important consideration both for pharmaceutical and
590 agricultural applications.
26
591 The cyclotide field is still relatively young and only a small number of groups are currently
592 studying these fascinating cyclic and knotted peptides. Their natural function as insecticidal or
593 nematocidal agents justifiably allows them to be regarded as toxins. Their mechanism of toxicity
594 appears to be mainly related to membrane-binding. Their membrane binding, however, is far from
595 non-specific, and cyclotides exhibit a remarkable preference for PE lipids compared to other lipid
596 types. It is not yet known whether it is this lipid specificity that controls the specificity of different
597 cyclotides for different organisms, but it seems to be a reasonable hypothesis. Also unknown at the
598 moment is why one plant produces so many cyclotides. Is it a strategy for the plant to try and avoid
599 the development of resistance by pests to the chemical defense? Or is it a strategy for simultaneously
600 targeting a wide variety of different pests? These questions will undoubtedly be answered in due
601 course, assisted by advances in technologies for the synthesis of cyclotides. For example, as we have
602 noted, it is now possible to make variants of cyclotides where individual residues or individual loops
603 can be replaced to explore structure-activity studies. Biological methods of producing cyclotides are
604 also improving and promise to accelerate the development of structure-activity relationships.
605 Amongst toxins cyclotides do not have quite the same caché as the deadly toxins from animal
606 venoms, but we hope that this article has convinced readers that they are toxins with a vast range of
607 potential applications in the pharmaceutical and agricultural fields.
608
609 Acknowledgements
610 Work in our laboratory on cyclotides is funded by grants from the Australian Research Council
611 (DP150100443) and the National Health and Medical Research Council (APP1084965 and
612 APP1060225). DJC is an Australian Research Council Laureate Fellow (FL150100146).
27
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