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 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 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 ERK_1/2 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 Trp258^6.48 and thereby stabilize CB₂R in its inactive state.

中文翻译：

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翻转 GPCR 开关：基于结构的选择性大麻素受体 2 反向激动剂的开发

我们报告了合理设计具有定制功能响应的 G 蛋白偶联受体 (GPCR) 配体的蓝图。本研究公开了基于结构的 2 型大麻素受体 (CB ₂ R) 选择性反向激动剂 ( S )- 1和 ( R )- 1，它们是通过在优先激动剂 HU-308 上引入苯基而衍生的。宝石-二甲基庚基侧链。Epimer ( R )- 1对 CB ₂ R 表现出高亲和力，K _d = 39.1 nM，可作为合成多种探针的平台。值得注意的是，这些荧光探针首次保留了其反向激动剂功能、高亲和力以及对 CB ₂ R 的选择性，而不受接头和荧光团取代的影响。配体 ( S )- 1、 ( R )- 1及其衍生物在 CB ₂ R 介导的 cAMP 以及 G 蛋白募集测定中充当反向激动剂，并且不会触发 β-抑制蛋白 - 受体结合。_{此外，在活细胞ERK 1/2}磷酸化和Ca ²⁺释放测定中未检测到受体激活。使用 ( R ) -7 (Alexa488) 和 ( R ) -9 (Alexa647) 探针使用 BV-2 小胶质细胞进行共焦荧光成像实验，观察到内源水平表达的 CB ₂ R。最后，分子动力学模拟证实了初始对接数据，其中反向激动剂限制拨动开关Trp258 ^6.48的运动，从而将CB ₂ R 稳定在其非活性状态。