Analysis of natural and synthetic cannabinoid receptor ligands via [32P]GTPase assay

Two cannabinoid receptors are known, the cannabinoid receptor 1 (CB₁R) and the cannabinoid receptor 2 (CB₂R). Both regulate important effects, such as appetite and motor coordination (CB₁R), as well as inflammation and host defence (CB₂R). Finding new ligands at CBRs is of high importance treating diseases such as multiple sclerosis, obesity or immune deficiencies. Regarding natural products it was recently shown that alkamides of Echinacea bind to CB₂Rs [1, 2].

We established a highly-sensitive test system using the baculovirus/Sf9 cell system to evaluate the potency of natural and synthetic compounds. To study ligands we used the [³²P] GTPase assay. Coexpressing CBRs with Gα_i2-, Gβ₁γ₂- and RGS4-protein resulted in the highest GTPase activation (CB₁R: 70%, CB₂R: 83% vs. blank value: membrane and solvent without ligand). CP 55,940 (5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol), a synthetic compound derived from the natural ligand Δ⁹-THC, is a strong partial agonist at CBRs (nM range). Anandamide (arachidonoylglycerol), the endogenous agonist, is a full (CB₁R)/partial (CB₂R) agonist and WIN 55212,2 (R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]-pyrrolo-[1,2,3,-de]-1,4-benzoxazin-yl]-(1-naphtalenyl)-methanone-mesylate) is a high potency agonist at CB₂Rs. AM 251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyrazole-3-carboxamide) is a selective CB₁R inverse agonist/antagonist and AM 630 (6-iodo-2-methyl-1-[2–4(morpholinyl)-ethyl]-[1H-indol-3-yl](4-methoxyphenyl)methanone) is a selective CB₂R antagonist.

At present we are studying Δ⁹-THC and alkamides by [³²P] GTPase assay and are characterising the binding properties of all ligands by [³H] CP 55,940 binding.

References: [1] Raduner, S. et al. (2006) J. Biol. Chem. 281: 14192–206. [2] Wölkart, K. et al. (2005) Planta Med. 71: 701–5.