Abstract
The endocannabinoid system plays essential roles in normal physiology and a variety of disease states. A component of this system is the type-2 cannabinoid receptor (CB2); a G protein-coupled receptor predominantly expressed in peripheral tissues, primarily in the immune system. Thus, CB2 is a promising candidate therapeutic target for treating inflammation, diabetes, cancer, pain, and other diseases. However, CB2-selective drug candidates thus far have failed in clinical trials. A contributor to this problem is difficulties in developing CB2-selective compounds. As a result, novel therapeutic strategies for modulating CB2 are currently under study, with allosteric modulation being proposed as a potentially important approach for targeting CB2 receptors. A positive allosteric modulator would potentiate the functional response to an orthosteric agonist, allowing affinity modulation and/or efficacy modulation. However, proper spaciotemporal regulation of signalling would theoretically remain under physiological control due to the allosteric compound being dependent on an endogenous orthosteric ligand. The compounds C2 and A5 are among the first small synthetic CB2 positive allosteric modulators (PAMs) reported. This project aimed to characterise the molecular pharmacology of these allosteric modulators together with a series of proposed novel allosteric modulators (which comprise analogues of C2 and A5, and novel compounds from a in silico virtual screen) at CB2 using G protein dissociation (TRUPATH) and β- arrestin-2 translocation assays; both real-time bioluminescence resonance energy transfer (BRET) assays. Assays were conducted in human embryonic kidney (HEK) cells, where compounds were tested alone, and in the presence of the orthosteric agonist CP55,940. Results showed that neither C2 or A5 could modulate CP55,940-dependent G protein dissociation or CP55,940-dependent β- arrestin-2 translocation at CB2, contradicting the published evidence describing these compounds as PAMs. However, C2 was active as an agonist alone. Surprisingly, C2 alone also induced G protein dissociation at the CB1 receptor, and increased CP55,940-dependent G protein dissociation through CB1, suggesting that this compound is both an agonist and a positive allosteric modulator at CB1 (i.e., an ago-PAM). The remaining set of proposed novel allosteric modulators (C2 and A5 analogues and “hits” from the in silico virtual screen) did not able appear to modulate CP55,940- dependent G protein dissociation or CP55,940-dependent β-arrestin-2 translocation at CB2. The findings from this study add to the body of work on allosteric modulators of CB2 and highlight discrepancies from published literature, which present C2 and A5 as PAMs. Therefore further investigation requires more rigour to address these differences.