Dual Channel Kinetic assays for detecting ligand bias at GPCRs

Speaker: Anne Marie Quinn (CEO, Montana Molecular), Kevin Harlen (Principal Investigator, Montana Molecular), Carl Peters (Senior Applications Scientist, BMG LABTECH Inc.)

GPCRs are one of the largest family of drug targets. Their activity is mediated by both G proteins and β-arrestins which activate a network of distinct signaling pathways. Drugs that activate GPCRs stabilize the receptor in different conformational states to produce distinct biological responses that represent varying degrees of are bias toward either the G-protein or β-arrestin signaling pathways. Depending on the receptor and cell type, biased drugs could produce either therapeutic or adverse effects by selectively engaging some signals, while minimizing other signals mediated by the same receptor. Despite the potential impact of ligand bias on therapeutic drug development, traditional cell-based assays are poorly suited for detecting bias. Current approaches involve comparing a series of disparate assays for arrestin recruitment, GPCR second messengers and ERK phosphorylation, each using different assay conditions, modalities, and instrumentation and measured in different cell populations. 


A novel approach allows measurement of agonist bias towards either G-protein or β-arrestin using the same cell population, same assay modality and conditions as well as the same detection instrumentation. Fluorescent biosensors measuring GPCR second messengers (e.g. calcium ions – Ca2+, inositol triphosphate – IP3, diacylglycerol – DAG or cyclic AMP – cAMP) can be combined with spectrally resolved fluorescent biosensors for β-arrestin. The reversible biosensors can be measured in parallel in a single microplate well and report on how much a specific pathway is activated. A BacMam delivery system ensures expression of the sensors in each desired cell type, preferably the cell type relevant to disease. The BacMam transduction method is applicable to immortalized cell lines, as well as to primary cells or induced pluripotent stem cells (iPSCs) and cells derived thereof such as cardiomyocytes or neurons.


Expression of the biosensors takes approximately 24 h from transduction. After the baseline fluorescence intensity of both sensors is recorded, cells are challenged with the compounds of interest using onboard injectors of the microplate reader. The fluorescent changes of the sensors in response to either second messenger increase or β-arrestin increase are recorded kinetically. The kinetic read and a high temporal resolution are highly relevant: some responses to GPCR stimulation are fast (Ca2+) and some last long (β-arrestin). A kinetic read makes the simultaneous measurement of both responses possible and fast reads ensure to record the time-point of reaction.


Bias towards either G-protein or β-arrestin pathway may only become apparent in smallest changes of the fluorescent responses towards different agonists. Therefore, the acquired data need to display excellent stability and a high signal to noise ratio. As the CLARIOstar combines all requirements (speed, precision, sensitivity, onboard reagent injectors) it was employed to establish the antagonist bias assay.


The acquired data were then analyzed according to an algorithm that first calculates the rate of β-arrestin activation and of G-protein activation. Subsequently both rates are compared to result in a bias ratio reporting on the bias towards β-arrestin of a compound.


In this tutorial, Montana Molecular describes a new approach that takes advantage of the dual channel fluorescence detection capability of the BMG CLARIOstar plate reader to introduce an efficient and practical method to simultaneously measure kinetic responses of both arrestin and G-protein second messenger signaling. We will discuss results from assays using agonists to both the Angiotensin and Vasopressin Receptors to illustrate the precision with which agonists that are known to be arrestin biased can be distinguished from unbiased or partially biased agonists in a single experiment.

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