A kinetic method for measuring agonist efficacy and ligand bias using high resolution biosensors and a kinetic data...

The kinetics/dynamics of signaling are of increasing value for G-protein-coupled receptor therapeutic development, including spatiotemporal signaling and the kinetic context of biased agonism. Effective application of signaling kinetics to developing new therapeutics requires reliable kinetic assays and an analysis framework to extract kinetic pharmacological parameters. Here we describe a platform for measuring arrestin recruitment kinetics to GPCRs using a high quantum yield, genetically encoded fluorescent biosensor, and a data analysis framework to quantify the recruitment kinetics. The sensor enabled high temporal resolution measurement of arrestin recruitment to the angiotensin AT₁ and vasopressin V₂ receptors. The analysis quantified the initial rate of arrestin recruitment (k_τ), a biologically-meaningful kinetic drug efficacy parameter, by fitting time course data using routine curve-fitting methods. Biased agonism was assessed by comparing k_τ values for arrestin recruitment with those for Gq signaling via the AT₁ receptor. The k_τ ratio values were in good agreement with bias estimates from existing methods. This platform potentially improves and simplifies assessment of biased agonism because the same assay modality is used to compare pathways (potentially in the same cells), the analysis method is parsimonious and intuitive, and kinetic context is factored into the bias measurement.