Cell-permeable high-affinity tracers for G 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 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 inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for G proteins are lacking.

EXPERIMENTAL APPROACH: We have now developed G -specific, cell-permeable 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 proteins in their native conformation in cells and tissues with high accuracy.

KEY RESULTS: FR and YM displayed low nanomolar affinity for Gα , Gα and Gα expressed in CRISPR/Cas9 Gα -knockout cells, but not for Gα . 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 inhibitors, which inhibited G 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.

© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.

PMID: 31881095