# Cell-based and isoform-selective G protein-coupled receptor kinase assays for comprehensive inhibitor evaluation

## TL;DR

This paper introduces cell-based assays to evaluate GRK inhibitors, identifying two compounds that effectively inhibit specific GRK isoforms in a controlled system.

## Contribution

The study introduces a novel cell-based assay system for evaluating GRK inhibitors with isoform specificity in a quadruple GRK knockout background.

## Key findings

- Compound 8h effectively inhibits GRK2/3, while compound 18 inhibits GRK5/6 in cellular systems.
- The developed assays enable high-throughput screening of GRK-targeted drug candidates.
- The β2 adrenergic receptor phosphorylation assay provides a reliable readout for GRK inhibitor potency.

## Abstract

G protein-coupled receptor (GPCR) signaling is regulated by four ubiquitously expressed GPCR kinase isoforms (GRKs), namely GRK2, GRK3, GRK5, and GRK6. Overexpression of individual GRKs occurs in diseases like cancer and heart failure, prompting a search for potent GRK inhibitors. While various in silico and in vitro approaches exist, few methods assess inhibitor efficacy in cellular systems. To address this, we used HEK293 cell lines co-expressing the β2 adrenergic receptor (β2) and one GRK isoform on a quadruple GRK2/3/5/6 knockout background (ΔQ-GRK). We evaluated the inhibition of isoproterenol (ISO)-induced T360/S364-β2 phosphorylation using the 7TM phosphorylation assay. This combination allowed comprehensive evaluation of commercially available GRK inhibitors. We conclude that compound 8h (GRK2/3 inhibitor) and compound 18 (GRK5/6 inhibitor) are highly recommendable tools for the study of GPCR phosphorylation and function in cellular systems. Together, these cell-based GRK inhibitor assays can facilitate medium- to high-throughput screening of future GRK-targeted drug candidates.

A GRK-isoform immunoassay was used to quantify GPCR phosphorylation and evaluate the potency of GRK inhibitors in a controlled cellular system.