Flipping the GPCR Switch: Structure-Based Development of Selective Cannabinoid Receptor 2 Inverse Agonists.

We report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses the structure-based design of cannabinoid receptor type 2 (CB 2 R) selective inverse agonists ( S )- 1 and ( R )- 1 , which were derived from privileged agonist HU-308 by introduction of a phenyl group at the gem -dimethylheptyl side chain. Epimer ( R )- 1 exhibits high affinity for CB 2 R with K d = 39.1 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, for the first time these fluorescent probes retain their inverse agonist functionality, high affinity, and selectivity for CB 2 R independent of linker and fluorophore substitution. Ligands ( S )- 1 , ( R )- 1 , and their derivatives act as inverse agonists in CB 2 R-mediated cAMP as well as G protein recruitment assays and do not trigger β-arrestin-receptor association. Furthermore, no receptor activation was detected in live cell ERK 1/2 phosphorylation and Ca 2+ -release assays. Confocal fluorescence imaging experiments with ( R )- 7 (Alexa488) and ( R )- 9 (Alexa647) probes employing BV-2 microglial cells visualized CB 2 R expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp258 6.48 and thereby stabilize CB 2 R in its inactive state.