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

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

  • Commun Biol. 2026 Jan 16;9(1):287. doi: 10.1038/s42003-026-09568-0.
Nina K Blum 1,  Manuela C Kiefer 1,  Angelika Decker 1,  Laura Klement 2,  Edda S F Matthees 2,  Verena Weitzel 2,  Falko Nagel 3,  Babu Joseph 4,  Julia Drube 2,  David Uehling 4,  Carsten Hoffmann 2,  Stefan Schulz 5 6
Affiliations

Affiliations

  • 1 Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Jena, Germany.
  • 2 Institut für Molekulare Zellbiologie, CMB-Center for Molecular Biomedicine, Universitätsklinikum Jena, Jena, Germany.
  • 3 7TM Antibodies GmbH, Jena, Germany.
  • 4 Drug Discovery Program, Ontario Institute for Cancer Research, Toronto, ON, Canada.
  • 5 Institut für Pharmakologie und Toxikologie, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Jena, Germany. [email protected].
  • 6 7TM Antibodies GmbH, Jena, Germany. [email protected].
PMID: 41545717 DOI: 10.1038/s42003-026-09568-0
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.

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