Cannabinoid CB2 receptor (CB2R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB2R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB2R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB2R activation by selective agonists and highlights the role of lipophilicity in CB2R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.

SUL-compounds are protectants from cold-induced ischemia and mitochondrial dysfunction. We discovered that adding SUL-121 to renal grafts during warm machine reperfusion elicits a rapid improvement in perfusion parameters. Therefore, we investigate the molecular mechanisms of action in porcine intrarenal arteries (PIRA). Porcine kidneys were stored on ice overnight and perfusion parameters were recorded during treatment with SUL-compounds. Agonist-induced vasoconstriction was measured in isolated PIRA after pre-incubation with SUL-compounds. Receptor binding and calcium transients were assessed in α1-adrenoceptor (α1-AR) transgenic CHO cells. Molecular docking simulation was performed using Schrödinger software. Renal pressure during warm reperfusion was reduced by SUL-121 (−11.9 ± 2.50 mmHg) and its (R)-enantiomer SUL-150 (−13.2 ± 2.77 mmHg), but not by the (S)-enantiomer SUL-151 (−1.33 ± 1.26 mmHg). Additionally, SUL-150 improved renal flow (16.21 ± 1.71 mL/min to 21.94 ± 1.38 mL/min). SUL-121 and SUL-150 competitively inhibited PIRA contraction responses to phenylephrine, while other 6-chromanols were without effect. SUL-150 similarly inhibited phenylephrine-induced calcium influx and effectively displaced [7-Methoxy-3H]-prazosin in CHO cells. Docking simulation to the α1-AR revealed shared binding characteristics between prazosin and SUL-150. SUL-150 is a novel α1-AR antagonist with the potential to improve renal graft perfusion after hypothermic storage. In combination with previously reported protective effects, SUL-150 emerges as a novel protectant in organ transplantation.

Cannabinoid receptor 1 (CB 1 R) and cannabinoid receptor 2 (CB 2 R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G protein pathway and the recruitment of β-arrestins. Differences in the activation level of these pathways lead to biased signaling. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB 1 R and CB 2 R using the PathHunter ® assay. This is a cellular assay that uses a β-galactosidase complementation system which has a chemiluminescent read-out and can be performed in 384-well plates. We have successfully used this assay to characterize a set of reference ligands (both agonists, antagonists, and an inverse agonist) on human CB 1 R and CB 2 R, of which some examples will be presented here.