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 , Gα₁₁ and Gα₁₄ expressed in CRISPR/Cas9 Gα_q -knockout cells, but not for Gα₁₅ . 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.