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Cell‐permeable high‐affinity tracers for G q proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors
Author(s) -
Kuschak Markus,
Namasivayam Vigneshwaran,
Rafehi Muhammad,
Voss Jan H.,
Garg Jaspal,
Schlegel Jonathan G.,
Abdelrahman Aliaa,
Kehraus Stefan,
Reher Raphael,
Küppers Jim,
Sylvester Katharina,
Hinz Sonja,
Matthey Michaela,
Wenzel Daniela,
Fleischmann Bernd K.,
Pfeifer Alexander,
Inoue Asuka,
Gütschow Michael,
König Gabriele M.,
Müller Christa E.
Publication year - 2020
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.14960
Subject(s) - intracellular , g protein coupled receptor , drug discovery , small molecule , extracellular , biology , biochemistry , chemistry , receptor
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.