Kinetic multiplex assay to assess biased signaling of clinical GPCR agonists

Studying biased signaling of G protein-coupled receptors (GPCRs) holds promise for the identification of ligands with a better therapeutic window. However, proper examination of biased signaling remains challenging by risking introduction of system or observation bias. Therefore, we developed a novel multiplex assay that simultaneously and kinetically detects cAMP production and ß-arrestin-2 recruitment in the same well. To investigate the applicability of the kinetic multiplex assay, we profiled seventeen clinically tested agonists for the cannabinoid CB₂ receptor (CB₂R), a promising GPCR for treating tissue injury and inflammation. Agonist-mediated CB₂R activation and signaling was time sensitive, dependent on the agonist. Similar potency and efficacy parameters were obtained from semi-kinetic and kinetic analysis, while the latter provided additional signaling rate constants for signaling onset (k₁) and decline (k₂). Fast CB₂R engagement (k_on) of agonists resulted in increased affinity and potency. Slow dissociation of agonists extended the interaction between CB₂R and ß-arrestin-2. Moreover, superagonists Tedalinab, Olorinab, PRS-211375 and ART-27.13 were characterized by fast k₁ values. No significant biased signaling was observed for the investigated CB₂R agonists. To validate the assay, the reported biased compound JWH145 was tested in a multiplex assay developed for CB₁R and confirmed that our new method is able to detect bias. Altogether, this study accentuates the potential of multiplexing functional responses and performing kinetic analyses to provide an extensive preclinical profile for agonists that may better predict their in vivo pharmacological effects. Ultimately, providing full kinetic context for binding and signaling of GPCR agonists could advance drug discovery efforts.