Exploring determinants of agonist efficacy at the CB1 cannabinoid receptor: Analogues of the synthetic cannabinoid receptor agonist EG-018

Abstract

Neutral antagonists of GPCRs remain relatively rare-indeed, a large majority of GPCR antagonists are actually inverse agonists. The synthetic cannabinoid receptor agonist (SCRA) EG-018 was recently reported as a low efficacy cannabinoid receptor agonist. Here we report a comparative characterization of EG-018 and 13 analogues along with extant putative neutral antagonists of CB₁ . In HEK cells stably expressing human CB₁ , assays for inhibition of cAMP were performed by real-time BRET biosensor (CAMYEL), G protein cycling was quantified by [³⁵ S]GTPγS binding, and stimulation of pERK was characterized by AlphaLISA (PerkinElmer). Signaling outcomes for the EG-018 analogues were highly variable, ranging from moderate efficacy agonism with high potency, to marginal agonism at lower potency. As predicted by differing pathway sensitivities to differences in ligand efficacy, most EG-018-based compounds were completely inactive in pERK alone. The lowest efficacy analogue in cAMP assays, 157, had utility in antagonism assay paradigms. Developing neutral antagonists of the CB₁ receptor has been a long-standing research goal, and such compounds would have utility both as research tools and in therapeutics. Although these results emphasize again the importance of system factors in determining signaling outcomes, some compounds characterized in this study appear among the lowest efficacy agonists described to date and therefore suggest that development of neutral antagonists is an achievable goal for CB₁ .

Keywords: CB1 receptor; EG-018; cannabinoids; signal transduction; structure-activity relationships.

FIGURE 1

Inhibition of FSK‐stimulated cAMP signaling by CP55940 and EG‐018 (A), and thirteen EG‐018 analogues (B–D) in 3HA‐hCB₁ HEK cells. Data are from a representative experiment, showing mean ± SD of conditions performed in technical duplicate. Curves are normalized to basal (vehicle only, 0%) and FSK only (100%). Combined n = 3 data are reported in Table 2

FIGURE 2

Time course of phosphorylation of ERK in 3HA‐hCB₁ HEK cells following treatment with 2‐AG (A, gray, 31.6 µM), CP55940 (B, blue, 1 µM), EG‐018 (A, red, 31.6 µM) or 13 EG‐018 analogues (B–D, all 31.6 µM). Vehicle (black) is shared between graphs. Data are from a representative experiment showing mean ± SD of conditions performed in technical duplicate or quadruplicate (vehicle and CP55940—these conditions were repeated on two plates in the assay). Curves are normalized to the peak CP55940 response at 4 min (100%)

FIGURE 3

Concentration–response experiments for cAMP (A–C) and pERK (D), showing the activity profiles of reported neutral CB₁ antagonists in comparison to SR141716A (A), the activity of AM4113 in untransfected HEK WT cells (B), and the utility of analogue 157 (10 µM) to antagonize the activity of CP55940 (C, D). Data are from representative experiments showing mean ± SD of conditions performed in technical duplicate. cAMP data are normalized to basal (0%) and FSK only (100%); pERK data are not normalized