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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Published on: February 20, 2018
Kinetic multiplex assay to assess biased signaling of clinical GPCR agonists
Jara Bouma1, Elisa J M de Koning1, Cas van der Horst1
1Division of Medicinal Chemistry, LACDR, Leiden University & Oncode Institute, the Netherlands.
Abstract:
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 (kon) 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.
Insights
A new multiplex assay simultaneously measures G protein-coupled receptor (GPCR) signaling kinetics, aiding drug discovery. This kinetic analysis of cannabinoid receptor 2 (CB2R) agonists reveals time-dependent effects and potential for predicting in vivo responses.
Area of Science:
- Pharmacology and Drug Discovery
- Molecular and Cellular Biology
- Biochemistry
Background:
- Biased signaling of G protein-coupled receptors (GPCRs) offers therapeutic potential but is challenging to study accurately.
- Existing methods risk system or observation bias, hindering the precise characterization of ligand-specific signaling pathways.
- Understanding receptor kinetics is crucial for predicting in vivo drug efficacy and safety.
Purpose of the Study:
- To develop and validate a novel multiplex assay for simultaneous, kinetic detection of cAMP production and ß-arrestin-2 recruitment.
- To investigate the kinetic signaling profiles of seventeen cannabinoid receptor 2 (CB2R) agonists.
- To assess the utility of kinetic analysis in characterizing GPCR agonist bias and predicting pharmacological effects.
Main Methods:
- Development of a multiplex assay enabling simultaneous kinetic measurement of cAMP and ß-arrestin-2 signaling in the same well.
- Profiling of seventeen clinically tested CB2R agonists using the developed kinetic multiplex assay.
- Kinetic and semi-kinetic analysis to determine potency, efficacy, and signaling rate constants (k1, k2, kon).
Main Results:
- Agonist-mediated CB2R activation and signaling kinetics were time-dependent and agonist-specific.
- Kinetic analysis provided additional signaling rate constants, revealing correlations between fast engagement (kon) and increased potency.
- No significant biased signaling was observed for the tested CB2R agonists; however, the assay successfully detected bias in a CB1R validation experiment.
Conclusions:
- The developed kinetic multiplex assay provides a comprehensive preclinical profile of GPCR agonists.
- Kinetic parameters offer deeper insights into ligand-receptor interactions and signaling dynamics than traditional methods.
- This approach has the potential to advance drug discovery by improving the prediction of in vivo pharmacological effects for GPCR-targeted therapeutics.
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