Agonist ligand-CB1 receptor binding motifs: An affinity label and site-directed mutagenesis study

Date of Completion

January 2001


Health Sciences, Pharmacology




For centuries, man has exploited the Cannabis sativa plant for its therapeutic utility, primarily for analgesia and an associated euphoria. Four decades ago the main psychoactive component, Δ 9-tetrahydrocannabinol, of cannabis was isolated. Since then the field of cannabinoid chemistry and pharmacology has grown tremendously. Many of the observed effects of cannabinoids can now be attributed to specific interactions with a series of receptor proteins. A G-protein coupled receptor, the CB1 cannabinoid receptor, has been cloned and is responsible for many of the centrally-mediated effects of these ligands. With the discovery and characterization of CB1, came the identification of several endogenous lipid-derived cannabinoid ligands behaving as neurotransmitters. Additionally, other classes of ligands, structurally distinct from the endogenous ligands and the plant-derived classical cannabinoids have been shown to specifically interact with CB1. ^ In an effort to design therapeutically useful cannabinoids it is of importance to determine elements of ligand recognition at CB1. To facilitate this, a series of classical cannabinoid covalent probes were designed and synthesized. Covalent probes are ligands possessing pharmacophores rendering them specific towards a particular target designed to bind irreversibly to that target. Irreversible binding of the covalent probe permits analysis of a ligand-receptor interaction without ligand dissociation. Investigation of the binding properties of covalent probes at CB1 has potential to identify binding motifs of the various cannabinoids as well as implicate particular amino acids involved with ligand recognition. ^ Specifically, the studies undertaken in this dissertation sought to characterize classical cannabinoid covalent probes for their ability to bind to CB1, both reversibly and irreversibly. These ligands were recognized with high affinity and were successful at specifically covalently attaching to CB1. A more detailed analysis using these probes has shown that structurally dissimilar cannabinoid agonists [3H]-CP55940 and [3H]-WIN55212-2 bind CB1 in distinct motifs. Lastly, one covalent probe implicated the involvement of a particular amino acid in transmembrane-helix 6 that may be critical in covalent binding. Site-directed mutagenesis was employed to characterize the role of this residue in the binding of covalent and non-covalent ligands. The point mutation revealed an essential role for this residue in covalent binding. ^