Ligand-assisted protein structure characterization as a method for exploring the binding and functional motifs of ligands on the CB2 cannabinoid receptor 2

This dissertation describes an investigation of the G-protein coupled human cannabinoid receptor 2 (GPCR hCB2) binding sites and mechanism of activation utilizing Ligand-Assisted Protein Structure (LAPS), an experimental method developed within the Center for Drug Discovery. Coupled primarily to G i/o proteins, CB2 mediates numerous physiological responses and are important pharmaceutical targets. The aim is to study the covalent interactions between covalent probes specifically designed to activate/deactivate the receptor and hCB2. We utilize these probes to determine binding motifs at the receptor active site(s) and investigate functional consequences of covalent binding. We found that a distinct binding motif of individual covalent probes can lead to distinct, functional pathways (agonist or antagonist) for CB2.;The isothiocyanato (--NCS) is an electrophilic covalent group that specifically targets cysteine residues. AM841 is a CB2 megagonist that has an NCS group at the terminal carbon (C7') of the C3-akyl tail end. AM4073 is an analog of AM841 that has the NCS group at the C11 headgroup position. AM4099 has two NCS groups at the C7' and the C11 positions respectively. The two new analogs were identified to irreversibly bind to C2.59(89), and function as agonists. The helix-2 cysteine is discovered to be a unique binding site for cannabinergic ligand recognition.;A C3'-functionalized adamantyl series of compounds contain an adamantyl group at the 3'-carbon of the phenolic ring and covalent groups associated to the adamantyl unit. AM994 carrying a methylene-NCS chain was identified to react irreversibly with C7.38(284) and C7.42(288) on helix 7 of hCB2 and induce an inverse agonist state on CB2.;The azido (--N3) is a photoactivatable group capable of covalent bond formation at the receptor site upon 245-nm ultra-violet (UV) irradiation. Two aliphatic azides, AM993 and AM8135 were tested through LAPS but contrary to our expectations, the two compounds do not form covalent attachment with any cysteine in the transmembrane region on the intact hCB2. We suggest future experiments to answer this question.;An array of classical cannabinergic ligands incorporating heteroaroyl groups at the C3 position were employed as probes to the cannabinoid receptors. Compound 41, a classical cannabinergic carrying a benzothiophenone moiety, was determined to have the best affinity and selectivity to mCB2.