Cyclotide C, partial

The cholecystokinin-2 receptor (CCK2R) is a G protein-coupled receptor (GPCR) that is expressed in peripheral tissues and the central nervous system and constitutes a promising target for drug development in several diseases, such as gastrointestinal cancer. The search for ligands of this receptor over the past years mainly resulted in the discovery of a set of distinct synthetic small molecule chemicals. Here, we carried out a pharmacological screening of cyclotide-containing plant extracts using HEK293 cells transiently-expressing mouse CCK2R, and inositol phosphate (IP1) production as a readout. Our data demonstrated that cyclotide-enriched plant extracts from Oldenlandia affinis, Viola tricolor and Carapichea ipecacuanha activate the CCK2R as measured by the production of IP1. These findings prompted the isolation of a representative cyclotide, namely caripe 11 from C. ipecacuanha for detailed pharmacological analysis. Caripe 11 is a partial agonist of the CCK2R (Emax = 71%) with a moderate potency of 8.5 µM, in comparison to the endogenous full agonist cholecystokinin-8 (CCK-8; EC50 = 11.5 nM). The partial agonism of caripe 11 is further characterized by an increase on basal activity (at low concentrations) and a dextral-shift of the potency of CCK-8 (at higher concentrations) following its co-incubation with the cyclotide. Therefore, cyclotides such as caripe 11 may be explored in the future for the design and development of cyclotide-based ligands or imaging probes targeting the CCK2R and related peptide GPCRs.

The cyclotide T20K inhibits the proliferation of human immune cells and is currently in clinical trials for multiple sclerosis. Here, we provide novel functional data and mechanistic insights into structure-activity relationships of T20K. Analogs with partial or complete reduction of the cystine knot had loss of function in proliferation experiments. Similarly, an acyclic analog of T20K was inactive in lymphocyte bioassays. The lack of activity of non-native peptide analogs appears to be associated with the ability of cyclotides to interact with and penetrate cell membranes, since cellular uptake studies demonstrated fast fractional transfer only of the native peptide into the cytosol of human immune cells. Therefore, structural differences between cyclic and linear native folded peptides were investigated by NMR to elucidate structure-activity relationships. Acyclic T20K had a less rigid backbone and considerable structural changes in loops 1 and 6 compared to the native cyclic T20K, supporting the idea that the cyclic cystine knot motif is a unique bioactive scaffold. This study provides evidence that this structural motif in cyclotides governs bioactivity, interactions with and transport across biological membranes, and the structural integrity of these peptides. These observations could be useful to understand the structure-activity of other cystine knot proteins due to the structural conservation of the cystine knot motif across evolution and to provide guidance for the design of novel cyclic cysteine-stabilized molecules.

The gas-phase linearization of cyclotides via site-selective ring opening at dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to incorporate dehydroalanine into macrocyclic peptide ions, which is easily accomplished through an ion/ion reaction. Triply protonated cyclotide cations are transformed into radical cations via ion/ion reaction with the sulfate radical anion. Subsequent activation of the cyclotide radical cation generates dehydroalanine at a single cysteine residue, which is easily identified by the odd-electron loss of ·SCH2CONH2. The presence of dehydroalanine in cyclotides provides a site-selective ring-opening pathway that, in turn, generates linear cyclotide analogues in the gas phase. Unlike cyclic variants, product ions derived from the linear peptides provide rich sequence information. The sequencing capability of this strategy is demonstrated with four known cyclotides found in Viola inconspicua, where, in each case, greater than 93% sequence coverage was observed. Furthermore, the utility of this method is highlighted by the partial de novo sequencing of an unknown cyclotide with much greater sequence coverage than that obtained with a conventional Glu-C digestion approach. This method is particularly well-suited for cyclotide species that are not abundant enough to characterize with traditional methods.