Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB2 receptor

We have investigated the binding site of the subtype specific antagonist SR 144528, (N-[(1S)-endo-1,3,3-trimethyl bicycle [2.2.1]heptan-2-yl]-5-(4-chl oro-3-methylphenyl)-1-(4-methoxybenzyl)-pyrazole-3-carboxamide) on the huma n cannabinoid CB2 receptor based on functional studies with mutated recepto rs. Two serine residues in the fourth transmembrane region, Ser(161) and Se r(165), were singly mutated to the cognate cannabinoid CB1 receptor residue , alanine, and each gave receptors with wild-type properties for the cannab inoid agonists CP 55,940 (IR,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phen yl]-4-(3-hydroxypropyl)cycloshexan-1-ol) and WIN 55212-2 (R)-(+)[2,3-dihydr o-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxacin-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 CB2 receptor through a combination of hydrogen bonds and aromatic and hydrophobic interactions. In addition, the replacement by serine of a nearby cannabinoid CB2 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 act ivity of WIN 55212-2, a result compatible with a recently-proposed binding site model for WIN 55212-2. (C) 2000 Elsevier Science B.V. All rights reser ved.