Abstract
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 (kτ), a biologically-meaningful kinetic drug efficacy parameter, by fitting time course data using routine curve-fitting methods. Biased agonism was assessed by comparing kτ values for arrestin recruitment with those for Gq signaling via the AT1 receptor. The kτ 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.
Highlights
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
Biased agonism is the capacity of a ligand to selectively activate one or more of multiple signaling pathways transduced by the GPCR9
Biased agonism was assessed; the initial rate of arrestin recruitment was compared with the initial rate of downstream signaling measured using the same biosensor modality (DAG generation and Ca2+ mobilization)
Summary
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. Quantifying the kinetics of signaling with useful drug parameters would aid the development of kinetically-optimized molecules, tuned, for example, to the optimum duration of signaling, timeframe of desensitization, and residence period in signaling compartments This requires appropriately optimized kinetic assays and a data analysis platform for extracting drug parameter values from time course data. The goal of this study was to optimize and integrate the biosensor modality and the data analysis framework to create a unified platform suitable for robustly measuring and quantifying signaling kinetics and biased agonism for numerous GPCR pathways. This first required extending the framework described above to arrestin recruitment, since our biosensor and analysis technologies were developed only for G-protein signaling. This work demonstrated a novel approach for quantifying biased agonism in kinetic terms using a unified assay modality
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