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

Abstract

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.

Figure 1

Analytical profile of cyclotide-containing plant extracts. Analytical RP-HPLC chromatograms of the extracts of C. ipecacuanha (a) with magnification of the cyclotide eluting region (inset), O. affinis (c), and V. tricolor (e). MALDI-TOF MS of the extracts of C. ipecacuanha (b), O. affinis (d), and V. tricolor (f), including labelling of identified major cyclotides according to their monoisotopic [M + H]⁺ ions (in parentheses) and common name (according to the CyBase).

Figure 2

Cytotoxicity of plant extracts and purified cyclotides. Viability of HEK293 cells was determined using the cell counting kit-8 after 2 h incubation with varying concentrations (as indicated in the graphs) of (a) extracts of C. ipecacuanha (Caripe), (b) O. affinis (Oaff), (c) V. tricolor (Vitri), as well as (d) purified caripe 11. Cell viability was calculated as percentage using an equation described in the Materials and Methods section using absorbance at 450 nm. Data represent technical triplicates (n = 2) and were expressed as mean ± SD.

Figure 3

Pharmacological activity of the plant extracts at the CCK₂R. Agonist efficacy of the plant extracts of C. ipecacuanha (Caripe), O. affinis (Oaff), and V. tricolor (Vitri) were measured by IP1 quantification (fold difference over baseline). Receptor activation was presented as ‘fold difference over baseline’, and data were expressed as mean ± SD (n = 4–5, except Caripe 300 µg/mL is n = 2). Stimulation buffer (not shown) and CCK-8 peptide (1 and 3 nM, n = 3–4) were used as negative and positive controls, respectively.

Figure 4

Analytical profile of native caripe 11 cyclotide isolated from C. ipecacuanha. The quality control using RP-HPLC (a) and MALDI TOF MS (b) with yielded 99.8% purity, whereas labeled m/z refers to monoisotopic [M + H]⁺ ion.

Figure 5

Partial agonist activity of caripe 11 at the CCK₂R. (a) Concentration–response curves of the effect of CCK-8 and caripe 11 upon activation of the receptor via measurement of the intracellular formation of IP1. Potency and efficacy values are listed in Table 2. (b) Concentration–response curves of CCK-8 alone and in combination with caripe 11 (10 µM). Stimulation buffer and CCK-8 peptide were used as negative and positive controls with 0 and 100% efficacy, respectively. Each experiment was carried out in technical triplicate and three (n = 3) biological repeats. All data are shown as mean ± SD.