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 (CBR) selective inverse agonists ()- and ()-, which were derived from privileged agonist HU-308 by introduction of a phenyl group at the -dimethylheptyl side chain. Epimer ()- exhibits high affinity for CBR with = 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 CBR independent of linker and fluorophore substitution. Ligands ()-, ()-, and their derivatives act as inverse agonists in CBR-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 phosphorylation and Ca-release assays. Confocal fluorescence imaging experiments with ()- (Alexa488) and ()- (Alexa647) probes employing BV-2 microglial cells visualized CBR expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp258 and thereby stabilize CBR in its inactive state.