Cyclotides are plant-derived cyclic peptides that have a head-to-tail cyclic backbone and three conserved disulphide bonds that form a cyclic cystine knot motif. They occur in plants from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families, typically with 10-100 cyclotides in a given plant species, in a wide range of tissues, including flowers, leaves, stems, and roots. Some cyclotides are expressed in large amounts (up to 1g kg(-1) wet plant weight) and their natural function appears to be to protect plants from pests or pathogens. This article provides a brief overview of their discovery, distribution in plants, and applications. In particular, their exceptional stability has led to their use as peptide-based scaffolds in drug design applications. They also have potential as natural 'ecofriendly' insecticides, and as protein engineering frameworks.

Ultrastable cyclic peptide frameworks offer great potential for drug design due to their improved bioavailability compared to their linear analogues. Using the sunflower trypsin inhibitor-1 (SFTI-1) peptide scaffold in combination with systematic N-methylation of the grafted pharmacophore led to the identification of novel subtype selective melanocortin receptor (MCR) agonists. Multiple bicyclic peptides were synthesized and tested toward their activity at MC1R and MC3–5R. Double N-methylated compound 18 showed a pKi of 8.73 ± 0.08 (Ki = 1.92 ± 0.34 nM) and a pEC50 of 9.13 ± 0.04 (EC50 = 0.75 ± 0.08 nM) at the human MC1R and was over 100 times more selective for MC1R. Nuclear magnetic resonance structural analysis of 18 emphasized the role of peptide bond N-methylation in shaping the conformation of the grafted pharmacophore. More broadly, this study highlights the potential of cyclic peptide scaffolds for epitope grafting in combination with N-methylation to introduce receptor subtype selectivity in the context of peptide-based drug discovery.

Among the various molecules that plants produce for defense against pests and pathogens, cyclotides stand out as exceptionally stable and structurally unique. These ribosomally synthesized peptides are around 30 amino acids in size, and are stabilized by a head-to-tail cyclic peptide backbone and three disulfide bonds that form a cystine knot. They occur in certain plants of the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, and Solanaceae families, with an individual plant producing up to hundreds of different cyclotides. Aside from being exploitable as crop protection agents based on their natural pesticidal activities, cyclotides are amenable to repurposing by chemists for use as drug leads or as tools in chemical biology. Their macrocyclic peptide backbone and knotted arrangement of three disulfide bonds engenders cyclotides with resistance to proteolytic degradation, high temperatures, and chemical chaotropes. Furthermore, their small size makes them accessible to synthesis using solid-phase peptide chemistry and so non-natural cyclotides can be designed and synthesized for a variety of applications. Our focus here is on cyclotides as tools in chemical biology, and there are four main areas of application that have appeared in the literature so far: (i) cyclotides as probes of membrane binding; (ii) cyclotides as probes of biosynthetic pathways for peptide cyclization; (iii) cyclotides as probes of protease specificity and function; and (iv) cyclotides as probes of receptor binding and specificity, with the potential for them to be developed as drug leads. The main methods used in these studies include solid-phase peptide chemistry for synthesis and NMR spectroscopy for structural characterization, as well as a wide range of biochemical and biophysical techniques for probing intermolecular interactions. In addition, cyclotides have been examined in diverse biological assays, ranging from enzyme inhibition to cell penetration, intracellular targeting and cytotoxicity. The main finding to have emerged from studies over the past decade is that cyclotides are exceptionally stable under a variety of conditions (in assay buffers, biological fluids, membranes, and recombinant expression systems). Furthermore, they are structurally very well-defined and amenable to sequence substitutions that can introduce new desired biological activities, generally without compromising their exceptional stability. Both features contribute to their use as peptide-based frameworks in drug design. Finally, they occupy a size niche between traditional small-molecule drugs (<500 Da in molecular weight) and protein-based biologics (>5000 Da) and thus can probe receptors, membranes, and protein-protein interactions in different ways to what is possible with either small molecules or biologics. Overall, cyclotides are an exciting class of peptides that have great potential as ultrastable chemical biology probes in a variety of applications. They have the advantage of specificity (typical of proteins) combined with the synthetic accessibil...

No abstract