Foyzun, T.; Connor, M.; Zaman, H.; Kassiou, M.; Kallinen, A.; Santiago, M.

Introduction: Cannabinoid receptor-2 (CB2) is an emerging therapeutic target for chronic and inflammatory pain, cancer, and neurological disorders. Understanding the efficacy of CB2 ligands is crucial for future drug design and development. Aims: We aimed to establish a simple and robust system to control CB2 expression using a tetracycline-regulated mammalian expression system (T-REx), to enable application of the Black and Leff operational model to measure the operational efficacy ({tau}) of CB2 ligands. Methods: Ligand-induced hyperpolarisation of AtT20 cells transfected with T-REx and human CB2 was measured by FLIPR membrane potential assay. Maximal and submaximal responses of the CB2 ligands were produced by regulating CB2 expression using tetracycline. Data were fitted to the operational model of receptor depletion to quantify the efficacy of seven ligands. Additionally, the maximal initial rate of signalling (IRmax-), another putative measure of ligand efficacy, was determined. Results: AK-F-064, CP55940 and 2-AG exhibited similar efficacy with a {tau} values of 11.4, 11 and 10.4 respectively, while anandamide (AEA) had the lowest efficacy ({tau}=1.07) among the tested agonists. The rank order of operational efficacy and IRmax- was similar and was estimated as: AK-F-064 = CP55940 = 2-AG > 5F-AB-PICA = WIN55212-2 > HU-308 = AEA. Conclusion: This inducible expression system provides a reliable platform for quantifying and comparing CB2 ligand efficacy using the operational model. This approach may facilitate more precise CB2-targeted drug development and can be readily extended to other GPCR targets.