Evaluation of the profiles of CB1 cannabinoid receptor signalling bias using joint kinetic modelling

Background and Purpose Biased agonism describes the ability of ligands to differentially regulate multiple signalling pathways when coupled to a single receptor. Signalling is affected by rapid agonist-induced receptor internalisation. Hence, the conventional use of equilibrium models may not be optimal, because (i) receptor numbers vary with time and, in addition, (ii) some pathways may show non-monotonic profiles over time. Experimental Approach Data were available from internalisation, cAMP inhibition and phosphorylation of ERK (pERK) of the cannabinoid-1 (CB1) receptor using a concentration series of six CB1 ligands (CP55,940, WIN55,212-2, anandamide, 2-arachidonylglycerol, Delta(9)-tetrahydrocannabinol and BAY59,3074). The joint kinetic model of CB1 signalling was developed to simultaneously describe the time-dependent activities in three signalling pathways. Based on the insights from the kinetic model, fingerprint profiles of CB1 ligand bias were constructed and visualised. Key Results A joint kinetic model was able to capture the signalling profiles across all pathways for the CB1 receptor simultaneously for a system that was not at equilibrium. WIN55,212-2 had a similar pattern as 2-arachidonylglycerol (reference). The other agonists displayed bias towards internalisation compared to cAMP inhibition. However, only Delta(9)-tetrahydrocannabinol and BAY59,3074 demonstrated bias in the pERK-cAMP pathway comparison. Furthermore, all the agonists exhibited little preference between internalisation and pERK. Conclusion and Implications This is the first joint kinetic assessment of biased agonism at a GPCR (e.g. CB1 receptor) under non-equilibrium conditions. Kinetic modelling is a natural method to handle time-varying data when traditional equilibria are not present and enables quantification of ligand bias.