Fig. 2: ABM300 increases agonist binding but inhibits CB₁R orthosteric agonist signaling through arrestin recruitment, ERK phosphorylation and cAMP signaling.

a ABM300 increases [³H]CP55,940 binding to hCB₁R CHO cell membranes. b ABM300 concentration-dependently decreases CP55,940 (10 nM)-mediated arrestin recruitment, with the PathHunter® β-arrestin assay. c ABM300 concentration-dependently decreases ERK phosphorylation at the EC₈₀ concentration of CP55,940 (40 nM), using the AlphaScreen® SureFire® ERK1/2 phosphorylation kit in hCB₁R CHO cells. d ABM300 has no effect alone, but decreases the E_max for CP55,940-stimulated ERK phosphorylation in a concentration-dependent manner in hCB₁R CHO cells. e ABM300 decreases the E_max of AEA-stimulated ERK1/2 phosphorylation in a concentration-dependent manner in hCB₁R CHO cells. f ABM300 concentration-dependently inhibits CP55,940-(EC₈₀ of 40 nM) mediated inhibition of forskolin-stimulated cAMP signaling in hCB₁R CHO cells. Data shown as mean ± SEM from 3–5 independent experiments conducted in triplicate. g BRET CAMYEL real-time cAMP signaling data in hCB₁R HEK cells, showing ABM300 concentration-dependently inhibiting the reduction in cAMP level induced by 5 µM forskolin and 1 µM CP55,940; the time-dependent activity of ABM300 is particularly apparent at moderate concentrations (1 µM and 100 nM), in which the onset of the ABM300 effect is delayed (representative experiment). h Area-under-the-curve analysis of g, showing that ABM300 concentration-dependently inhibits CP55,940-mediated cAMP reductions in HEK cells. At high concentrations of ABM300, cAMP levels are increased above forskolin alone (100%).