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 (CB2R) 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 CB2R with Kd = 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 CB2R independent of linker and fluorophore substitution. Ligands (S)-1, (R)-1, and their derivatives act as inverse agonists in CB2R-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 ERK1/2 phosphorylation and Ca2+-release assays. Confocal fluorescence imaging experiments with (R)-7 (Alexa488) and (R)-9 (Alexa647) probes employing BV-2 microglial cells visualized CB2R expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp2586.48 and thereby stabilize CB2R in its inactive state.