Cannabinoid Receptor 2 Signalling Bias Elicited by 2,4,6-Trisubstituted 1,3,5-Triazines

Abstract

Cannabinoid receptor 2 (CB2) is predominantly distributed in immune tissues and cells and is a promising therapeutic target for modulating inflammation. In this study we designed and synthesised a series of 2,4,6-trisubstituted 1,3,5-triazines with piperazinylalkyl or 1,2-diethoxyethane (PEG2) chains as CB2 agonists, all of which were predicted to be considerably more polar than typical cannabinoid ligands. In this series, we found that triazines containing an adamantanyl group were conducive to CB2 binding whereas those with a cyclopentyl group were not. Although the covalent attachment of a PEG2 linker to the adamantyl triazines resulted in a decrease in binding affinity, some of the ligands produced very interesting hCB2 signalling profiles. Six compounds with notable hCB2 orthosteric binding were functionally characterised in three pathways; internalisation, cyclic adenosine monophosphate (cAMP) and ERK phosphorylation (pERK). These were predominantly confirmed to be hCB2 agonists, and upon comparison to a reference ligand (CP 55,940), four compounds exhibited signalling bias. Triazines 14 (UOSD017) and 15 were biased towards internalisation over cAMP and pERK, and 7 was biased away from pERK activation relative to cAMP and internalisation. Intriguingly, the triazine with an amino-PEG2-piperazinyl linker (13 [UOSD008]) was identified to be a mixed agonist/inverse agonist, exhibiting apparent neutral antagonism in the internalisation pathway, transient inverse agonism in the cAMP pathway and weak partial agonism in the pERK pathway. Both the cAMP and pERK signalling were pertussis toxin (PTX) sensitive, implying that 13 is acting as both a weak agonist and inverse agonist at CB2 via Gαi/o. Compound 10 (UOSD015) acted as a balanced high intrinsic efficacy agonist with the potential to produce greater hCB2-mediated efficacy than reference ligand CP 55,940. As 10 includes a Boc-protected PEG2 moiety it is also a promising candidate for further modification, for example with a secondary reporter or fluorophore. The highest affinity compound in this set of relatively polar hCB2 ligands was compound 16, which acted as a slightly partial balanced agonist in comparison with CP 55,940. The ligands characterised here may therefore exhibit unique functional properties in vivo and have the potential to be valuable in the future development of CB2-directed therapeutics.

Cannabinoid Receptor 2 (CB2) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes

“…This was followed by a steady reduction in cAMP concentration, reaching a maximum extent by 30 min (to 60.3 ± 2.8% of forskolin-stimulated), with cAMP subsequently returning back to the forskolin-induced level by 60 min. Prior studies in cells overexpressing CB 2 describe an immediate onset and earlier peak cAMP inhibition . Given that we cannot readily measure inhibition of cAMP production without forskolin being present, we hypothesized that low basal cAMP and/or slow response to forskolin in the primary leukocytes might mask the ability to detect inhibition at early time-points and hence produce the observed delay.…”

Cannabinoid Receptor 2 (CB2) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes

“…This was followed by a steady reduction in cAMP concentration, reaching a maximum extent by 30 min (to 60.3 ± 2.8% of forskolin-stimulated), with cAMP subsequently returning back to the forskolin-induced level by 60 min. Prior studies in cells overexpressing CB2 describe an immediate onset and earlier peak cAMP inhibition 37 .…”

“…A time-course with PBMC stimulated by 1µM HU308(Fig. 5A) revealed a wave of CREB phosphorylation which was maximal around[30][31][32][33][34][35][36][37][38][39][40] min and resolved by 60 min. We applied G protein inhibitors NF023, NF449, gallein and PTX (Fig.…”

Cannabis and cannabinoids as an alternative remedy in metabolic syndrome

“…Activation of CB2 receptors by natural or synthetic ligands, in general, affects similar signaling pathways: links to G proteins, inhibition of cAMP via adenylate cyclase and activation of ERK1/2, Akt, and MAPK cascade (stimulating cell survival, migration, and growth), stimulation of ceramide synthesis and potential activation of arrestin-specific signaling, decreased PKA activity and stimulated MAPK pathways. These pathways result in the positive regulation of many genes, by activation of a pathway, inhibition/ downregulation of a pathway or through of combination of all of them (Oyagawa et al, 2018). Activation of MAP kinase, probably mediated by PKC, has also been reported.…”