Discovery of the cyclotide caripe 11 as a ligand of the cholecystokinin-2 receptor

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 (E_max = 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.