Functional Selectivity of CB2 Cannabinoid Receptor Ligands at a Canonical and Noncanonical Pathway

Abstract

The CB2 cannabinoid receptor (CB2) remains a tantalizing, but unrealized therapeutic target. CB2 receptor ligands belong to varied structural classes and display extreme functional selectivity. Here, we have screened diverse CB2 receptor ligands at canonical (inhibition of adenylyl cyclase) and noncanonical (arrestin recruitment) pathways. The nonclassic cannabinoid (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940) was the most potent agonist for both pathways, while the classic cannabinoid ligand (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran JWH133) was the most efficacious agonist among all the ligands profiled in cyclase assays. In the cyclase assay, other classic cannabinoids showed little [(-)-trans-Δ(9)-tetrahydrocannabinol and (-)-(6aR,7,10,10aR)-tetrahydro-6,6,9-trimethyl-3-(1-methyl-1-phenylethyl)-6H-dibenzo[b,d]pyran-1-ol] (KM233) to no efficacy [(6aR,10aR)-1-methoxy-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromene(L759633) and (6aR,10aR)-3-(1,1-dimethylheptyl)-6a,7,8,9,10,10a-hexahydro-1-methoxy-6,6-dimethyl-9-methylene-6H-dibenzo[b,d]pyran]L759656. Most aminoalkylindoles, including [(3R)-​2,​3-​dihydro-​5-​methyl-​3-​(4-​morpholinylmethyl)pyrrolo[1,​2,​3-​de]-​1,​4-​benzoxazin-​6-​yl]-​1-​naphthalenyl-​methanone,​ monomethanesulfonate (WIN55212-2), were moderate efficacy agonists. The cannabilactone 3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c)chromen-6-one (AM1710) was equiefficacious to CP55940 to inhibit adenylyl cyclase, albeit with lower potency. In the arrestin recruitment assays, all classic cannabinoid ligands failed to recruit arrestins, indicating a bias toward G-protein coupling for this class of compound. All aminoalkylindoles tested, except for WIN55212-2 and (1-​pentyl-​1H-​indol-​3-​yl)(2,​2,​3,​3-​tetramethylcyclopropyl)-​methanone (UR144), failed to recruit arrestin. WIN55212-2 was a low efficacy agonist for arrestin recruitment, while UR144 was arrestin biased with no significant inhibition of cyclase. Endocannabinoids were G-protein biased with no arrestin recruitment. The diarylpyrazole antagonist 5-​(4-​chloro-​3-​methylphenyl)-​1-​[(4-​methylphenyl)methyl]-​N-​[(1S,​2S,​4R)-​1,​3,​3-​trimethylbicyclo[2.2.1]hept-​2-​yl]-​1H-​pyrazole-​3-​carboxamide (SR144258) was an inverse agonist in cyclase and arrestin recruitment assays while the aminoalkylindole 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630) and carboxamide N-(1,3-benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-(pentyloxy)-3-quinolinecarboxamide (JTE907) were inverse agonists in cyclase but low efficacy agonists in arrestin recruitment assays. Thus, CB2 receptor ligands display strong and varied functional selectivity at both pathways. Therefore, extreme care must be exercised when using these compounds to infer the role of CB2 receptors in vivo.

CB 1 and CB 2 Receptor Pharmacology

“…Example studies have provided predictive clues regarding pathway-preferring ligands for both CB 1 receptors (Baillie et al, 2013; Delgado-Peraza et al, 2016) and CB 2 receptors (Dhopeshwarkar & Mackie, 2016). …”

Biased Agonism of Three Different Cannabinoid Receptor Agonists in Mouse Brain Cortex

“…For instance, for the CB 1 receptor has been reported that, whereas 2-AG and WIN55,212 have little preference for inhibition of cAMP and phosphorylation of ERK1/2, anandamide and CP55940 were biased toward cAMP inhibition (Khajehali et al, 2015). Moreover, in a recent study Dhopeshwarkar and Mackie (2016) demonstrated that CB 2 receptor ligands display strong and varied functional selectivity at canonical (inhibition of adenylyl cyclase) and non-canonical (arrestin recruitment) pathways. Moreover, the intracellular signaling activated by a receptor depends on the cellular system where it is expressed, which may vary across different neuronal environments.…”

Presynaptic cannabinoid CB2 receptors modulate [3H]‐Glutamate release at subthalamo‐nigral terminals of the rat

“…First, different isoforms of CB2r have been described among species (Liu et al, ; Zhang, Kolaj et al, ); thus, these agonists could possess different affinities and/or potency in the different receptor subtypes. Second, the different functional selectivity that has been described could be responsible for the different responses observed by the agonists (Atwood, Straiker, et al, ; Dhopeshwarkar & Mackie, ). Because GW833972A exhibited the highest potency and an IC 50 of 126 nM, that is comparable with 50% of the Effective Concentration (EC 50 ) of 28 and 90 nM reported for cAMP inhibition and β‐arrestin recruitment assays (Dhopeshwarkar & Mackie, ), it was feasible to suppose that it is a good agonist for the response studied; thus, it was utilized throughout our study.…”

“…Second, the different functional selectivity that has been described could be responsible for the different responses observed by the agonists (Atwood, Straiker, et al, ; Dhopeshwarkar & Mackie, ). Because GW833972A exhibited the highest potency and an IC 50 of 126 nM, that is comparable with 50% of the Effective Concentration (EC 50 ) of 28 and 90 nM reported for cAMP inhibition and β‐arrestin recruitment assays (Dhopeshwarkar & Mackie, ), it was feasible to suppose that it is a good agonist for the response studied; thus, it was utilized throughout our study. Moreover, the effect of GW833972A is prevented by AM630 (200 nM) and JTE907 (100 nM), two selective antagonists/inverse agonists that, at the concentration employed, do not affect CB1r, indicating specificity (Iwamura et al, ; Ross et al, ) (Table ).…”