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

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 new 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 signaling (kτ), a biologicallymeaningful 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. Receptor excess over arrestin

Analyzing kinetic signaling data for G-protein-coupled receptors

“…The association exponential profile was observed in direct receptor-transducer interaction assays, including receptor-G-protein interaction and receptor-arrestin interaction ( Supplementary Fig. S2a,b) 22,46 . This was expected; mechanistically, the response is likely resultant from a bimolecular interaction and the association exponential equation is a general form of the familiar bimolecular association kinetics equation.…”

“…4c 61 . Other receptors giving this profile include the BB1 receptor 62 and AT 1 receptor 22 . It is important to note that other time course shapes are observed for cytoplasmic Ca 2+ mobilization, including the rise-and-fall to steady-state curve (considered separately below), and calcium oscillations and waves 63,64 .…”

“…4) The initial rate can be determined regardless of the shape of the time course, enabling comparison of a ligand's efficacy between responses with different temporal profiles (see below). We recently applied the initial rate approach to arrestin-receptor interaction, a special case in which there was no signal transduction 22 . Here it is applied universally to GPCR signaling responses.…”

“…For the CB 1 receptor, the potency for internalization increased over time 21 . For AT 1 angiotensin receptor-mediated arrestin recruitment the potency increased over time, as did the E max of partial agonists 22 .…”

“…A data analysis framework for time course data of GPCR signaling would enable the quantification of efficacy in kinetic terms. One way to do this is to measure the initial rate of signaling, analogous to the initial rate of enzyme activity [22][23][24][25][26][27] , an approach recently applied to receptor receptor-arrestin interaction 22 . Kinetic analysis could also enable measurement of regulation of signaling parameters, such as the rate of receptor desensitization.…”