Cell‐permeable high‐affinity tracers for G(q) proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

BACKGROUND AND PURPOSE: G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The G(q) protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up‐regulated in cancer and inflammatory diseases. G(q) inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for G(q) proteins are lacking. EXPERIMENTAL APPROACH: We have now developed G(q)‐specific, cell‐permeable (3)H‐labelled high‐affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM‐254890 (YM). The tracers served to specifically label and quantify G(q) proteins in their native conformation in cells and tissues with high accuracy. KEY RESULTS: FR and YM displayed low nanomolar affinity for Gα(q), Gα(11) and Gα(14) expressed in CRISPR/Cas9 Gα(q)‐knockout cells, but not for Gα(15). The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a “dowel” effect of the pseudoirreversibly binding FR. A high‐throughput binding assay led to the discovery of novel G(q) inhibitors, which inhibited G(q) signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation. CONCLUSIONS AND IMPLICATIONS: The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.