Cyclotides: Overview and Biotechnological Applications

Abstract

Cyclotides are globular microproteins with a unique head-to-tail cyclized backbone, stabilized by three disulfide bonds forming a cystine knot. This unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to chemical, thermal, and biological degradation compared to other peptides of similar size. In addition, cyclotides have been shown to be highly tolerant to sequence variability, aside from the conserved residues forming the cystine knot. Cyclotides can also cross cellular membranes and are able to modulate intracellular protein-protein interactions, both in vitro and in vivo. All of these features make cyclotides highly promising as leads or frameworks for the design of peptide-based diagnostic and therapeutic tools. This article provides an overview on cyclotides and their applications as molecular imaging agents and peptide-based therapeutics.

Keywords: cyclic peptides; cyclotides; cystine knots; drug design; imaging agents.

Figure 1

Primary and tertiary structures of cyclotides belonging to the Möbius (kalata B1, pdb: 1NB1), bracelet (cycloviolacin O1, pdb: 1NBJ) and trypsin inhibitor (MCoTI-II, pdb: 1IB9) subfamilies. The sequence of kalata B8, a hybrid cyclotide isolated from the plant O. affinis is also shown. Conserved Cys and Asp/Asn (required for cyclization) residues are marked in yellow and light blue, respectively. Disulfide connectivities and backbone-cyclization are shown in red and a dark blue line, respectively. Molecular graphics were created using PyMol.

Figure 2

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] Cyclotides from the Fabaceae family of plants isolated recently 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] 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] 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] Cyclotides structures were generated using PyMol and the pdb codes: 2LAM (Cter M), 1NB1 (kalata B1), 1NBJ (cycloviolacin O1) and 1IB9 (MCoTI-II). The structure of cyclotide Phyb A was obtained by homology modeling using the structure of cycloviolacin O1 (pdb: 1NBJ) as template.

Figure 3

General structural features of the cyclic cystine knot (CCK) topology found in all cyclotides. A. Detailed three-dimensional structure of the cyclic cystine knot (CCK) and the connecting loops found in cyclotides. The six Cys residues labeled I through VI whereas loops connecting the different Cys residues are designated as loop 1 through 6, in numerical order from the N- to the C-terminus. B. Möbius cyclotides contain a cis-Pro residue in loop 5 that induces a local 180° backbone twist, whereas bracelet cyclotides do not.

Figure 4

Scheme showing the major steps thought to occur during the biosynthesis of cyclotides. The case for the processing of kalata B1 is shown. It has been proposed that the cyclization step is mediated by an asparaginyl endopeptidase (AEP), a common Cys protease found in plants. The cyclization takes place at the same time as the cleavage of the C-terminal pro-peptide from the cyclotide precursor protein through a transpeptidation reaction. The transpeptidation reaction involves an acyl-transfer step from the acyl-AEP intermediate to the N-terminal residue of the cyclotide domain.^[40] The kalata B1 protein precursor contains an ER signal peptide, an N-terminal pro-region, the N-terminal repeat (NTR), the mature cyclotide domain and a C-terminal flanking region (tail, also known as CTR).

Figure 5

Chemical synthesis of cyclotides by means of an intramolecular native chemical ligation (NCL). This approach requires the chemical synthesis of a linear precursor bearing an N-terminal Cys residue and an α-thioester moiety at the C-terminus. The linear precursor can be cyclized first under reductive conditions and then folded using a redox buffer containing reduced and oxidized glutathione (GSH).^[11] Alternatively, the cyclization and folding can be efficiently accomplished in a ‘single pot’ reaction when the cyclization is carried out in the presence of reduced GSH as the thiol cofactor.^[11]

Figure 6

Heterologous expression of cyclotides by protein trans-splicing (PTS).^–58] In this approach the linear cyclotide precursor is fused in-frame at the C- and N-termini directly to the I_N and I_C polypeptides of the Npu DnaE split intein. None of the additional native C- or N-extein residues were added in this construct. In this work, the native Cys residue located at the beginning of loop 6 of MCoTI-I was used to facilitate backbone cyclization. The N- and C-termini of the linear cyclotide precursor are linked together through a native peptide bond through a transpeptidation reaction mediated by the self-processing domains of the split intein. This approach has been successfully used for the production of bioactive cyclotides in eukaryotic and prokaryotic expression systems.^[–58]

Figure 7

Structure and in vivo activity of the first cyclotide designed to antagonize an intracellular protein-protein interaction in vivo.^[16] A. Solution structure of an engineered cyclotide MCo-PMI (magenta) and its intracellular molecular target, the p53 binding domain of oncogene Hdm2 (blue). The cyclotide binds with low nM affinity to both the p53-binding domains of Hdm2 and HdmX. The overexpression of these two proteins Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. B. Cyclotide MCo-PMI activates the p53 tumor suppressor pathway and blocks tumor growth in a human colorectal carcinoma xenograft mouse model. HCT116 p53^+/+ xenografts mice were treated with vehicle (5% dextrose in water), nutlin 3 (10 mg/kg) or cyclotide (40 mg/kg, 7.6 mmol/kg) by intravenous injection daily for up to 38 days. Tumor volume was monitored by caliper measurement. C. Tumors samples were also subjected to SDS-PAGE and analyzed by western blotting for p53, Hdm2 and p21, indicating activation of p53 on tumor tissue.