Two Affinity Sites of the Cannabinoid Subtype 2 Receptor Identified by a Novel Homogeneous Binding Assay

Endocannabinoids act on G protein-coupled receptors that are considered potential targets for a variety of diseases. There are two different cannabinoid receptor types: ligands for cannabinoid type 2 receptors (CB(2)Rs) show more promise than those for cannabinoid type 1 receptors (CB(1)Rs) because they lack psychotropic actions. However, the complex pharmacology of these receptors, coupled with the lipophilic nature of ligands, is delaying the translational success of medications targeting the endocannabinoid system. We here report the discovery and synthesis of a fluorophore-conjugated CB2R-selective compound, CM-157 (3-[[4-[2-tert-butyl-1-(tetrahydropyran-4-ylmethyl) benzimidazol-5- yl] sulfonyl-2-pyridyl] oxy] propan-1-amine), which was useful for pharmacological characterization of CB2R by using a time-resolved fluorescence resonance energy transfer assay. This methodology does not require radiolabeled compounds and may be undertaken in homogeneous conditions and in living cells (i.e., without the need to isolate receptor-containing membranes). The affinity of the labeled compound was similar to that of the unlabeled molecule. Time-resolved fluorescence resonance energy transfer assays disclosed a previously unreported second affinity site and showed conformational changes in CB2R forming receptor heteromers with G protein-coupled receptor GPR55, a receptor for l-alpha-lysophosphatidylinositol. The populations displaying subnanomolar and nanomolar affinities were undisclosed in competitive assays using a well known cannabinoid receptor ligand, AM630 (1-[2-(morpholin-4-yl) ethyl]-2-methyl-3( 4-methoxybenzoyl)-6-iodoindole), and TH-chrysenediol, not previously tested on binding to cannabinoid receptors. Variations in binding parameters upon formation of dimers with GPR55 may reflect decreases in binding sites or alterations of the quaternary structure of the macromolecular G protein-coupled receptor complexes. In summary, the homogeneous binding assay described here may serve to better characterize agonist binding to CB2R and to identify specific properties of CB2R on living cells.