Biased Agonism and Biased Allosteric Modulation at the CB1 Cannabinoid Receptor.
CB1 cannabinoid receptors (CB1Rs) are attractive therapeutic targets for numerous central nervous system disorders. However, clinical application of cannabinoid ligands has been hampered owing to their adverse on-target effects. Ligand-biased signaling from, and allosteric modulation of, CB1Rs offer pharmacological approaches that may enable the development of improved CB1R drugs, through modulation of only therapeutically desirable CB1R signaling pathways. There is growing evidence that CB1Rs are subject to ligand-biased signaling and allosterism. Therefore, in the present study, we quantified ligand-biased signaling and allosteric modulation at CB1Rs. Cannabinoid agonists displayed distinct biased signaling profiles at CB1Rs. For instance, whereas 2-arachidonylglycerol and WIN55,212-2 (R)-()-2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo1,2,3-de-1,4-benzoxazin-6-yl-1-napthalenylmethanone showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 12 (pERK12), N-arachidonoylethanolamine (anandamide), methanandamide, CP55940 2-(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl-5-(2-methyloctan-2-yl)phenol, and HU-210 11-hydroxy-Δ(8)-THC-dimethylheptyl were biased toward cAMP inhibition. The small-molecule allosteric modulator Org27569 5-chloro-3-ethyl-1H-indole-2-carboxylic acid 2-(4-piperidin-1-yl-phenyl)ethylamide displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK12 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with (3)HSR141716A 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide however, it had minimal effects on binding of cannabinoid agonists. Furthermore, we highlight the need to validate the reported allosteric effects of the endogenous ligands lipoxin A4 and pregnenolone at CB1Rs. Pregnenolone but not lipoxin A4 displaced (3)HSR141716A, but there was no functional interaction between either of these ligands and cannabinoid agonists. This study demonstrates an approach to validating and quantifying ligand-biased signaling and allosteric modulation at CB1Rs, revealing ligand-biased fingerprints that may ultimately allow the development of improved CB1R-targeted therapies.