Khajehali Elham, Malone Daniel T, Glass Michelle, Sexton Patrick M, Christopoulos Arthur, Leach Katie
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Melbourne, Australia (E.K., D.T.M., P.M.S., A.C., K.L.); and Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand (M.G.).
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Melbourne, Australia (E.K., D.T.M., P.M.S., A.C., K.L.); and Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand (M.G.)
Mol Pharmacol. 2015 Aug;88(2):368-79. doi: 10.1124/mol.115.099192. Epub 2015 Jun 4.
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)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2), 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)ethyl]amide] displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK1/2 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with [(3)H]SR141716A [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)H]SR141716A, 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.
CB1大麻素受体（CB1Rs）是众多中枢神经系统疾病颇具吸引力的治疗靶点。然而，大麻素配体的临床应用因不良的靶向效应而受阻。来自CB1Rs的配体偏向性信号传导以及变构调节提供了药理学方法，通过仅调节治疗上所需的CB1R信号通路，可能有助于开发改良的CB1R药物。越来越多的证据表明CB1Rs存在配体偏向性信号传导和变构现象。因此，在本研究中，我们对CB1Rs处的配体偏向性信号传导和变构调节进行了量化。大麻素激动剂在CB1Rs上表现出不同的偏向性信号传导特征。例如，2-花生四烯酸甘油酯和WIN55,212-2 [（R）-（+）-[2,3-二氢-5-甲基-3-（4-吗啉基甲基）吡咯并[1,2,3-de]-1,4-苯并恶嗪-6-基]-1-萘基甲酮]对抑制环磷酸腺苷（cAMP）和细胞外信号调节激酶1/2（pERK1/2）的磷酸化几乎没有偏好，而N-花生四烯酸乙醇胺（花生四烯酸乙醇胺）、甲基花生四烯酸乙醇胺、CP55940 [2-[(1R,2R,5R)-5-羟基-2-(3-羟丙基)环己基]-5-(2-甲基辛烷-2-基)苯酚]和HU-210 [11-羟基-Δ(8)-四氢大麻酚-二甲基庚基]则偏向于抑制cAMP。小分子变构调节剂Org27569 [5-氯-3-乙基-1H-吲哚-2-羧酸[2-(4-哌啶-1-基苯基)乙基]酰胺]通过阻断所有测试大麻素配体介导的cAMP抑制，表现出偏向性变构效应，同时对这些配体中的一部分介导的ERK1/2磷酸化几乎没有影响。Org27569与[(3)H]SR141716A [5-(4-氯苯基)-1-(2,4-二氯苯基)-4-甲基-N-(哌啶-1-基)-1H-吡唑-3-甲酰胺]也表现出负性结合协同性；然而，它对大麻素激动剂的结合影响极小。此外，我们强调需要验证内源性配体脂氧素A4和孕烯醇酮在CB1Rs上报道的变构效应。孕烯醇酮而非脂氧素A4取代了[(3)H]SR141716A，但这些配体与大麻素激动剂之间均不存在功能相互作用。本研究展示了一种验证和量化CB1Rs处配体偏向性信号传导和变构调节的方法，揭示了配体偏向性“指纹”，这最终可能有助于开发改良的CB1R靶向疗法。
