Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias

Abstract

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and β-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses.

Single nucleotide polymorphisms in the cannabinoid CB2 receptor: Molecular pharmacology and disease associations

“…Agonist stimulation of CB 2 receptors canonically activates Gα i/oβγ heterotrimers leading to inhibition of adenylate cyclase with consequent reduced cAMP production, activation of signalling pathways such as MAPKs, and recruitment of β-arrestin which facilitates desensitisation, internalisation and potentially further signal transduction (Oyagawa & Grimsey, 2021). Coupling to Gα q and Gα s have also been reported, as well as biased agonism and temporal signalling signatures, which are hypothesised to influence responses downstream of CB 2 receptor activation (Oyagawa & Grimsey, 2021;Sharma et al, 2023).…”

Endocannabinoid system and periodontitis: mechanisms and therapeutic implications

“…CB2 receptors, however, are primarily found on immune cells and in peripheral tissues that interact with Gαi proteins to modulate various immune responses and inflammatory processes 27,28 . It is reported that the activation of CB2 receptors is also capable of inhibiting adenylyl cyclase activity, reducing cAMP levels, but leading to a decrease in protein kinase A (PKA) activation, contributing to the dampening of immune cell activation and cytokine release 29 .…”

RAMP and MRAP accessory proteins have selective effects on expression and signalling of the CB1, CB2, GPR18 and GPR55 cannabinoid receptors

“…cAMP assays were performed using the CAMYEL BRET-based biosensor (ATCC MBA-277) with methodology similar to that published previously (Sharma et al, 2023;Singh et al, 2019). Cells were prepared and transfected as for receptor and accessory protein detection, except that cells were seeded into PDL-treated white plates (Corning, Corning, NY, USA), and the CAMYEL plasmid (7 fmol per well; 23.8 ng per well) was co-transfected.…”

“…Further to our earlier study (Finlay et al, 2016), we undertook pilot experiments for dissociation of Gα i3 from β 3 γ 9 (TRUPATH; n = 1) and plasma membrane translocation of hβ-arrestin-2 (bystander BRET method described in Ibsen et al, 2019;Sharma et al, 2023;n = 2).…”