Human Cannabinoid CB2 Research Articles

Exploration of the pharmacophore of 3-alkyl-5-arylimidazolidinediones as new CB(1) cannabinoid receptor ligands and potential antagonists: synthesis, lipophilicity, affinity, and molecular modeling.

A set of 29 3-alkyl 5-arylimidazolidinediones (hydantoins) with affinity for the human cannabinoid CB(1) receptor was studied for their lipophilicity and conformational properties in order to delineate a pharmacophore. These molecules constitute a new template for cannabinoid receptor recognition, since (a) their structure differs from that of classical cannabinoid ligands and (b) antagonism is the mechanism of action of at least three compounds (20, 21, and 23). Indeed, in the [(35)S]-GTP gamma S binding assay using rat cerebellum homogenates, they behave as antagonists without any inverse agonism component. Using a set of selected compounds, experimental lipophilicity was measured by RP-HPLC and calculated by a fragmental method (CLOGP) and a conformation-dependent method (CLIP based on the molecular lipophilicity potential). These approaches revealed two models which differentiate the binding mode of nonpolar and polar hydantoins and which could explain, at least for compounds 20, 21, and 23, the mechanism of action of this new family of cannabinoid ligands.

Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB 2 receptor

We have investigated the binding site of the subtype specific antagonist SR 144528, ( N-[(1 S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)-pyrazole-3-carboxamide) on the human cannabinoid CB 2 receptor based on functional studies with mutated receptors. Two serine residues in the fourth transmembrane region, Ser 161 and Ser 165, were singly mutated to the cognate cannabinoid CB 1 receptor residue, alanine, and each gave receptors with wild-type properties for the cannabinoid agonists CP 55,940 (1 R,3 R,4 R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol) and WIN 55212-2 ( R)-(+)[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3- de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone, which SR 144528 completely failed to antagonise. Molecular modelling studies show that SR 144528 interacts with residues in transmembrane domains 3, 4, and 5 of the cannabinoid CB 2 receptor through a combination of hydrogen bonds and aromatic and hydrophobic interactions. In addition, the replacement by serine of a nearby cannabinoid CB 2 receptor-specific residue, Cys 175 resulted in wild-type receptor properties with CP 55,940, loss of SR 144528 binding and eight-fold reduced binding and activity of WIN 55212-2, a result compatible with a recently-proposed binding site model for WIN 55212-2.

Agonist selective regulation of G proteins by cannabinoid CB(1) and CB(2) receptors.

We have examined the ligand regulation and G protein selectivity of the human cannabinoid CB(1) and CB(2) receptors by an in situ reconstitution technique directly measuring G protein activation. Membranes from Spodoptera frugiperda cells expressing CB(1) and CB(2) receptors were chaotrope extracted to denature endogenous GTP-binding proteins. The ability of the receptors to catalyze the GDP-GTP exchange of each G protein was then examined with purified bovine brain G(i) and G(o). Activation of CB(1) receptors produced a high-affinity saturable interaction for both G(i) and G(o). Agonist stimulation of CB(2) receptors also resulted in a high-affinity saturable interaction with G(i). In contrast, CB(2) receptors did not interact efficiently with G(o). G protein activation was then examined with a diverse group of ligands. For the interaction of CB(2) receptors with G(i), HU210 was the only compound tested that demonstrated maximal activation. In contrast, WIN55,212 (64%), anandamide (42%), and Delta(9)-tetrahydrocannabinol (Delta(9)-THC) (44%) all initiated submaximal levels of G protein activation. For CB(1) receptor-catalyzed activation of G(i), HU210, WIN55,212, and anandamide all elicited maximal activation, whereas Delta(9)-THC (56 +/- 6%) caused only partial G(i) activation. In contrast, only HU210 effected maximal CB(1) stimulation of G(o), with anandamide, WIN55, 212, and Delta(9)-THC all stimulating between 60 and 75% compared with HU210. These data demonstrate that different agonists induce different conformations of the CB(1) receptor, which in turn can distinguish between different G proteins. Our data thus demonstrate agonist-selective G protein signaling by the CB(1) receptor and suggest that therapeutic agents may be designed to regulate individual G protein-signaling pathways selectively.

Dual intracellular signaling pathways mediated by the human cannabinoid CB 1 receptor

It has long been established that the cannabinoid CB 1 receptor transduces signals through a pertussis toxin-sensitive G i/G o inhibitory pathway. Although there have been reports that the cannabinoid CB 1 receptor can also mediate an increase in cyclic AMP levels, in most cases the presence of an adenylyl cyclase costimulant or the use of very high amounts of agonist was necessary. Here, we present evidence for dual coupling of the cannabinoid CB 1 receptor to the classical pathway and to a pertussis toxin-insensitive adenylyl cyclase stimulatory pathway initiated with low quantities of agonist in the absence of any costimulant. Treatment of Chinese hamster ovary (CHO) cells expressing the cannabinoid CB 1 receptor with the cannabinoid CP 55,940, {(−)- cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans-4-(3-hydroxypropyl) cyclohexan-1-ol} resulted in cyclic AMP accumulation in a dose–response manner, an accumulation blocked by the cannabinoid CB 1 receptor-specific antagonist SR 141716A, { N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide hydrochloride}. In CHO cells coexpressing the cannabinoid CB 1 receptor and a cyclic AMP response element (CRE)-luciferase reporter gene system, CP 55,940 induced luciferase expression by a pathway blocked by the protein kinase A inhibitor N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89). Under the same conditions the peripheral cannabinoid CB 2 receptor proved to be incapable of inducing cAMP accumulation or luciferase activity. This incapacity allowed us to study the luciferase activation mediated by CB 1/CB 2 chimeric constructs, from which we determined that the first and second internal loop regions of the cannabinoid CB 1 receptor were involved in transducing the pathway leading to luciferase gene expression.

AM630 is an inverse agonist at the human cannabinoid CB 1 receptor

The present investigation examines WIN 55,212-2 and AM630 at the cloned human cannabinoid CB 1 receptor stably expressed in Chinese hamster ovary (CHO) cells. The effect of various concentrations of WIN 55,212-2 and AM630 on basal [ 35S]GTPγS binding to cell membranes was determined. WIN 55,212-2 (100 μM) stimulated basal [ 35S]GTPγS binding 77.9% with an EC 50 value of 0.36 μM. Conversely, AM630 (100 μM) inhibited basal [ 35S]GTPγS binding by 20.9% with an EC 50 value of 0.90 μM. These results show that WIN 55,212-2 is an agonist and AM630 is an inverse agonist in this system.

SR141716A is an inverse agonist at the human cannabinoid CB 1 receptor

The effects of R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3- de]-1,4-benzoxazin-yl]-(1-napthalenyl)methanone mesylate (WIN 55,212-2) and N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A) on guanosine-5′- O-(3-[ 35 S ]thio)triphosphate ([ 35 S ]GTP γS) binding to membranes isolated from human cannabinoid CB 1 receptor-transfected Chinese hamster ovary (CHO) cells were examined. WIN 55,212-2 stimulated [ 35 S ]GTP γS binding 76.3% above basal levels whereas SR141716A produced a 22.3% decrease in basal [ 35 S ]GTP γS binding. These findings demonstrate that WIN 55,212-2 is an agonist and SR141716A is an inverse agonist in this system.