GRK Assays and Inhibitors

G protein-coupled receptors (GPCRs) are among the most important drug targets in modern medicine. Their activity is tightly regulated by GPCR kinases (GRKs), enzymes that are often overactive in diseases such as cancer and heart failure. Despite strong interest in GRK inhibitors, reliable tools to test their effectiveness directly in living cells have been limited. We have developed a powerful cell-based assay platform that enables precise and isoform-specific analysis of GRK inhibition. Using engineered human cells that express a single GRK alongside a clinically relevant GPCR, our system allows accurate measurement of receptor phosphorylation under physiologically relevant conditions. This makes it possible to directly compare and validate GRK inhibitors in a cellular context. With this approach, we identified highly effective inhibitors for GRK2/3 and GRK5/6, providing validated tools for GPCR research and drug discovery. Our scalable assay platform supports medium- to high-throughput screening and offers a robust solution for companies developing next-generation GRK-targeted therapeutics.

Identification and functional testing of potent and selective GRK inhibitors. Inhibition of agonist-induced β2-adrenoceptor phosphorylation by GRK inhibitors. HEK293 cell lines stably expressing β2 and a GRK isoform (from left to right: ΔQ+GRK2, ΔQ+GRK3, ΔQ+GRK5, ΔQ+GRK6) were preincubated with increasing concentrations of compound 8h or compound 18 (30 min, 37°C) before being treated with 10 µM isoproterenol (ISO) (30 min, 37°C). Receptor phosphorylation was determined using the 7TM phosphorylation assay, and data were normalized to 10 µM ISO without inhibitor. The pan-inhibitor LDC9728 (dashed line in gray) serves as a reference in each graph. Data points represent n=5 independent experiments performed in duplicate ± SEM.

Effects of GRK inhibition on phosphorylation of various GPCRs. HEK293 cells stably expressing the β2-adrenergic receptor (β2), the µ-opioid receptor (MOP), the sphingosine-1-phosphate receptor 1 (S1P1),  were were pretreated for 30 min with 30 µM GRK2/3 inhibitor Compound 8h, 30 µM GRK5/6 inhibitor Compound 18, as well as a combination of both compounds or vehicle (V). Cells were then either exposed to 10 µM isoproterenol (ISO), 10 µM (DAMGO), 10 µM fingolimod (FTY) or not exposed. Receptor phosphorylation was analyzed using phosphosite-specific antibodies in a Western blot analysis. Total receptor levels were verified using anti-HA antibodies.

Effects of GRK inhibition on internalization of various GPCRs. HEK293 cells stably expressing the β2-adrenergic receptor (β2), the µ-opioid receptor (MOP), the sphingosine-1-phosphate receptor 1 (S1P1),  were were pretreated for 30 min with 30 µM GRK2/3 inhibitor Compound 8h, 30 µM GRK5/6 inhibitor Compound 18, as well as a combination of both compounds or vehicle (V). Cells were then either exposed to 10 µM isoproterenol (ISO), 10 µM (DAMGO), 10 µM fingolimod (FTY) or not exposed. receptor localization was examined using an anti-HA antibody.