Cyclotides: macrocyclic peptides with applications in drug design and agriculture

Abstract

Cyclotides are disulfide-rich peptides from plants that are exceptionally stable as a result of their unique cyclic cystine knot structural motif. Their natural role is thought to be as plant defence agents, most notably against insect pests, but they also have potential applications in drug design and agriculture. This article identifies gaps in current knowledge on cyclotides and suggests future directions for research into this fascinating family of ultra-stable mini-proteins.

Fig. 1

Schematic overview of the prototypic cyclotide kalata B1 and two precursor proteins, Oak1 and Oak4. (Oak refers to O ldenlandia a ffinis kalata). a Sequence of kalata B1 showing disulfide connectivities, loops between conserved Cys residues and the ligation point (arrow) involved in peptide cyclisation. b A ribbon model of kalata B1 highlighting the CCK motif of three disulfide bonds and cyclic backbone. c A surface-rendered model of kalata B1 showing the hydrophobic patch (green) which associates with membranes. d Precursor proteins for kalata B1 (Oak1) and kalata B2 (Oak4). Oak4 is shown to highlight the fact that some cyclotide genes encode multiple copies of cyclotides (as is the case for kalata B2) [16]. The cyclotide C-terminal residue (Asn or Asp), thought to be targeted by AEP, is marked with an arrow

Fig. 2

Overview of potential pharmaceutical and agricultural applications of cyclotides. Oldenlandia affinis (centre) represents an example of a plant that naturally produces cyclotide genes (green arrow) and corresponding cyclotide peptides (red arrow). The upper (red) panel of the diagram represents pharmaceutical applications that involve grafting biologically active peptide epitopes (e.g., the indicated helical peptide) into the CCK framework of natural cyclotides. These modified cyclotides can be synthesised using solid phase peptide synthesis, chemo-enzymatic synthesis or inteins. Alternatively, pharmaceutically modified cyclotides might, in future, be produced (“pharmed”) in plants or in plant cell culture [71] as shown in the middle of the diagram, by transformation with genes encoding modified cyclotides. The lower (green) panel of the diagram represents agricultural applications, whereby cyclotide gene sequences can be expressed in crop plants (e.g., corn as indicated) to confer resistance to pests. The dashed arrows are shown to highlight the linkage between pharmaceutical and agricultural applications schematically. For example, lessons learned from synthetic chemical grafting studies to produce pharmaceutically active cyclotides can be combined with lessons learned from the recombinant expression of insecticidal cyclotide genes in plants, to undertake molecular pharming studies, i.e., the production of pharmaceutically active cyclotides in plants