What are cyclotides?
Cyclotides or macrocycles are small, disulfide-rich mini proteins or peptides found in plants. Their unique structure results in their high stability. Most proteins unfold when heated or exposed to stomach acid, however cyclotides are highly stabile. According to Deegala et al. (2025) more than 760 cyclotides have been identified in five major families, making them the largest known group of cyclic peptides.
In nature, plant cyclotides act like pesticides. When an insect or a caterpillar eats the plant, the cyclotides bind to the membranes in the insect's gut and create holes, eventually causing the cells to burst. Beyond plant defense, the indestructible nature of cyclotides makes them stable scaffolds for drug design. Three solved 3D structures of cyclotides are shown in Figure 1.
Figure 1: Solved Structures of Cyclotides
| Cycloviolacin O1 1NBJ | Kalata B1 1NB1 | MCoTI-II |
| 
| 
| 
|
| PDB ID 1NBJ A Chain A, cycloviolacin O1 | PDB ID 1NB1 A Chain A, kalata B1 | PDB ID 4GUX D Chain D, Trypsin inhibitor 2 |
(Rosengren et al. 2003)
The high stability of a cyclotide is a result of the Cyclic Cystine Knot (CCK) motif defined by two main features:
(1) Head-to-Tail Cyclization in which the protein backbone is a continuous, unbroken circle with no free "ends" for digestive enzymes to recognize, and
(2) the Cystine Knot with six cysteine residues forming three disulfide bonds in which two bonds form a ring, and the third passes through the middle, effectively "locking" the structure in place. Because of this, cyclotides can withstand boiling water and highly acidic environments without losing their shape or function.
Table 1: Potential use of cyclotides
| Use | Notes |
| Drug Delivery & Development | A cyclotide scaffold allows "grafting" of medicinal sequences into the scaffold for the design of drugs that can be taken orally without being destroyed by the stomach. Their rigid, circular structure allows them to act as scaffolds for grafting, making them ideal for developing stable, orally active drugs that target intracellular proteins. |
| Bioimaging | Design of molecular imaging agents for ultrasound, positron emission tomography (PET), or single-photon emission computed tomography (SPECT) imaging as well as others, as suggested by Gould & Camarero. |
| Anti-viral & Therapeutic, Anti-HIV/Cancer | Some cyclotides inhibit HIV entry or target cancer cell membranes. Potential treatments for HIV (CXCR4 antagonism), dengue virus (protease inhibition), and multiple sclerosis (immunomodulation). |
| Agriculture | Cyclotides allow the design and production of potentially eco-friendly, peptide-based sprays to protect crops from pests. Used as potent insecticides and anthelmintics (worm control) due to their natural role in protecting plants against pests. |
| Oncology | Investigated for anti-tumor and anti-metastatic properties, including inhibiting tumor angiogenesis and acting as CTLA-4 antagonists. |
| Other Uses | Evaluated for use in ultrasound and PET imaging of tumors, and for treating obesity, diabetes, and inflammation. |
In many cysteine rich peptides, the disulfide bonds form a cystine knot in which an embedded ring is formed by the first two disulfide bonds and their connecting backbone segments is penetrated by the third (i.e. III-VI) disulfide bond. This type of cystine knot motif is found in a wide variety of proteins, ranging from growth factors to toxins, occurring in a wide range of organisms. In the case of the Kalata B1 cyclic peptide, because of the resistance of the oxidized molecule to enzymatic cleavage it was not possible to determine its disulfide connectivity chemically, but NMR data suggested a I-IV, II-V, III-VI connectivity.
How are cyclotides synthesized?
The synthesis of cyclotides can be challenging because of their unique circular backbone and the Cystine Knot motif. These structural features makes them very stable against heat and degradation by enzymes, hence, one cannot just synthesize them like a standard linear protein.
A successfully synthesis generally follows a three-stage process:
[1] Solid-Phase Peptide Synthesis (SPPS) of the selected linear peptide,
[2] Head-to-Tail Cyclization, and
[3] Oxidative Folding.
Because of their high stability, NMR analysis is needed to assign the cysteine bonds.
Discovery and Studies of Cyclotide
| During a Red Cross Relief Mission in Democratic Republic of Congo in the 1960s, Dr. Lorents Gran, observed that women in that region used ‘kalata-kalata’, a medicinal tea made from the leaves of the plant Oldenlandia affinis to induce labour and facilitate smooth and painless deliveries. The purified active ingredient of O. affinis was identified as a peptide, named ‘kalata B1.’ Saether et al. (1995) determined the structure of Kalata B1 as a cyclic cystine knot motif peptide now known as a cyclotide. A few years later, Daly et al. (1999) reported the chemical synthesis of the cyclic peptide kalata B1. As reported by Craik et al. (1999), these cyclic peptides were first discovered in plants from the Rubiaceae (coffee) and Violaceae (violet) families but are also found in a range of other plants from the Cucurbitaceae (cucurbit) and Fabaceae (legume) families and appear to be widely distributed within the plant kingdom. | 
Oldenlandia affinis subsp. fugax. (WIKI commons). |
Craik et al. determined the three-dimensional structure of one of these peptides, cycloviolacin O1, using (1)H NMR spectroscopy. The structure consists of a distorted triple-stranded beta-sheet and a cystine-knot arrangement of the disulfide bonds similar to the structure of kalata B1 and circulin A. Gran et al. (2000) reported that peptides found in medicinal plants from the northern Congo/Brazzaville and south-western Central African Republic, included Kalata-peptide B1, have antimicrobial activity and that they may allow the design of new peptide antibiotics.
Rosengren et al. (2003) analyzed the topological of this unique protein family observing rings with a circular backbone, twists in a cis-peptide bond in the Möbius cyclotides, and knots or knotted arrangements of the disulfide bonds. In mathematics, a Möbius strip, Möbius band, or Möbius loop is a surface formed by attaching the ends of a strip of paper together with a half-twist. Trabi et al. (2004) studied expression patterns cyclic peptides in various Viola species (Violaceae) and found that all tissue types examined contained complex mixtures of cyclotides. There were at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Trabi et a. proposed that cyclotides constitute a new family of plant defense peptides. A study done by Plan et al. (2007) expanded the number of known cyclotides in the Oldenlandia affinis (Rubiaceae) plant to 17 and characterized nine new sequences (kalata B9–B17). In addition, the research group identified five derivatives that contained oxidation products of the conserved tryptophan and showed that the formation of these derivatives is catalyzed by exposure to sunlight. In addition, two “linear” cyclotide analogues were described as well.
Daly et al. (2009) reviewed the discovery of cyclotides, describing their unique structural features and range of bioactivities, and also discussed their applications in drug design. Poth et al. (2011) studied seed extracts of the butterfly pea (C. ternatea) and discovered and characterized 12 novel cyclotides in the legume, pea, or bean family (Fabaceae), the third-largest family of flowering plants. Craik (2012) provided an overview of pesticidal and toxic activities of cyclotides as well as discussing a possible common mechanism of action involving the disruption of biological membranes in pest species, and describing methods useful for the production of cyclotides for novel pesticidal agents.
Daly et al. (2013) reported that the trypsin inhibitor peptide MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity. MCoTI-I and II isolated from the seeds of Momordica cochinchinensis are members of the squash trypsin inhibitor family. Because of its proteolytic stability, MCoTI-II is considered as a valuable template for the design of novel drug leads. The study revealed that the cyclization and active site loops of MCoTI-II are flexible in solutions, but when bound to trypsin, the active site loop converges to a single well-defined conformation. As a result, the cyclization loop, not present in acyclic homologues, facilitates a potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin.
Nawae et al. (2014) performed molecular dynamics simulation to reveal that Trp19 in loop 5 of both monomeric and tetrameric Kalata B1 is a key residue for initial anchoring in the membrane binding process to also facilitate the formation of kB1 tetramers. Knowing the role of each amino acid in the bioactivity of the cyclotide should enable a better design of therapeutic cyclotides with less toxicity.
Craik & Du (2017) describes recent developments relating to the grafting of bioactive peptide sequences into the cyclic cystine knot framework of cyclotides to stabilize the peptides. These newly grafted cyclotides interact with protein or enzyme targets, both extracellular and intracellular, as well as with cell surface receptors and membranes. Gould & Camarero (2017) provides an overview on cyclotides and their biotechnological applications as molecular imaging agents and peptide-based therapeutics. Nworu et al. (2017) studied the antiplasmodial as well as anti-inflammatory activities of whole extracts and cyclotide-rich fractions of Oldenlandia affinis. The dichloromethane-methanol extract studied showed a significant (p<0.05) reduction in mean parasitaemia in both the suppressive and curative models of Plasmodium berghei infection in mice, explaining the traditional use of the herb, Oldenlandia affinis for the treatment of malaria fever.
Chemical synthesis strategies
Ahangarzadeh et al. (2019) discussed the use of bicyclic peptides as a platform for the design and development of therapeutic drugs covering types, synthesis and applications of bicyclic peptides. Different organisms produce natural bicyclic peptides including actino-mycin D, moroidin, celogentin C, phalloidin, α-amanitin, and theonellamide F.
The research group reviewed and discussed several chemical strategies allowing the synthesis of bicyclic peptides.
(a) Synthesis through an on-resin intramolecular thioester ligation and an off-resin DMSO-mediated disulfide formation.
(b) Synthesis by ring-closing metathesis.
(c) Synthesis by formation of C–C bond through an intramolecular palladium-catalyzed C–H activation process between tryptophan and iodinated tyrosine or phenylalanine.
(d) Synthesis through ring-closing alkyne metathesis.
(e) Synthesis through the formation of an amide bond between the α- or γ-carboxyl group of glutamate and the N-terminus, then subjected to amide-bond formation conditions to perform the second cyclization.
Alignment of sequences from published cyclotide structures
Natural cyclotides are found in the plants
In C.ternatea, O. affinis, V. odorata, M. cochinchinensis, P. hybrida (Gould & Camarero 2017).
| Plant | Flower | Cyclotide |
| Fabaceae C.ternatea, commonly known as Shankhupushpam (conch-shaped flower), Asian pigeonwings, bluebellvine, blue pea, butterfly pea, cordofan pea, or Darwin pea. 2LAM (Cter M). | 
| Cter M 
|
| Rubiaceae O. affinis, a perennial plant, widespread in tropical Africa, Mpumalanga and KwaZulu-Natal, South Africa, Madagascar and the Comoro Islands. 1NB1 (kalata B1). | 
| Kalata B1 
|
| Violaceae V. odorata, commonly known as wood violet, sweet violet, English violet, common violet, florist's violet, or garden violet. 1NBJ (cycloviolacin O1). | 
| Cycloviolacin O1 
|
| Cucurbitacea M. cochinchinensis (Gac fruit) is a perennial, dioecious climbing vine in the Cucurbitaceae family, native to Southeast Asia. 1IB9 (MCoTI-II). | 
| MCoTI-II 
|
| Soonacea P. hybrida, commonly known as the garden petunia. It is the most common hybrid petunia found in home gardens and nurseries. | 
| Phyb A N.A. |
Genetic origin of cyclotides in plants.
Rubiacea and Violaceae plants have dedicated genes for the production of cyclotides. The genes encode protein precursors containing an ER signal peptide, an N-terminal pro-region, the N-terminal repeat (NTR), the mature cyclotide domain and a C-terminal flanking region (CTR).[40 Craik DJ, Malik U. Curr Opin Chem Biol. 2013;17:546–554. [PubMed] [Google Scholar]]
Cyclotides from the Fabaceae family of plants isolated from C. ternatea, are produced from precursor proteins containing an ER signal peptide immediately followed by the cyclotide domain, which is flanked at the C-terminus by a peptide linker and the albumin a-chain. In this case, the cyclotide domain replaces the albumin-1 b-chain.[7] Poth AG, Colgrave ML, Lyons RE, Daly NL, Craik DJ. Proc Natl Acad Sci USA. 2011;108:1027–1032. [PMC free article] [PubMed] [Google Scholar]]
Cyclotides from the trypsin inhibitor subfamily are produced from TIPTOP proteins, which contain a tandem series of cyclic trypsin inhibitors terminating with an acyclic trypsin inhibitor.[38b Mylne JS, Chan LY, Chanson AH, Daly NL, Schaefer H, Bailey TL, Nguyencong P, Cascales L, Craik DJ. Plant Cell. 2012;24:2765–2778. [PMC free article] [PubMed] [Google Scholar]]
The protein precursors for cyclotides from the Solanaceae family are encoded in genes similar to those found in the Rubiacea and Violaceae plants with dedicated precursor proteins that have an ER signal, a pro-region, the linear peptide precursor, and end with a hydrophobic tail.[45a Poth AG, Mylne JS, Grassl J, Lyons RE, Millar AH, Colgrave ML, Craik DJ. J Biol Chem. 2012;287:27033–27046. [PMC free article] [PubMed] [Google Scholar];]
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