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

Abstract

Background and Purpose G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The Gq 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. Gq inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for Gq proteins are lacking. Experimental Approach We have now developed Gq‐specific, cell‐permeable 3H‐labelled high‐affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM‐254890 (YM). The tracers served to specifically label and quantify Gq proteins in their native conformation in cells and tissues with high accuracy. Key Results FR and YM displayed low nanomolar affinity for Gαq, Gα11 and Gα14 expressed in CRISPR/Cas9 Gαq‐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 Gq inhibitors, which inhibited Gq 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.

Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359

“…33 , Supplementary Table 6 ). In addition, competition binding assays with 1 and 2 against the radiolabeled FR-derivative [³H]PSB-15900 19 , performed with human platelet membrane preparations, revealed a 207-fold decrease in binding affinity for 2 compared to 1 . Here, the measured p IC 50 -value decreased from 7.88 ± 0.09 for 1 to 5.56 ± 0.04 for 2 (Fig.…”

“…Therefore, inhibition of Gq, one of the four major G protein families, by FR is a very efficient strategy to simultaneously shut down the intracellular signaling of multiple GPCRs 16 , 17 . FR1-4 and YM inhibit Gq with similar potency and selectivity 14 , although YM is slightly less active and has altered binding kinetics characterized by a significantly shorter residence time 19 .

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G protein-coupled receptor signaling regulates ER-mitochondria contacts

“…Another question we sought out was: Is Gaq activation, then, required for isoproterenol-induced coupling? To test this, we treated HEK293T cells with a recently identified Gq inhibitor, ebselen, which binds to Gaq and inhibits Ca 2+ increase triggered by receptor activation (58). Ebselen eliminated the positive effect of isoproterenol ( Fig 6C), indicating that Gaq activation is indeed required for isoproterenol-induced ER-Mito coupling.…”

Sensitive LC-MS/MS Method for the Quantification of Macrocyclic Gαq Protein Inhibitors in Biological Samples

“…Binding of YM and FR to the Gα q protein subunit is believed to prevent nucleotide exchange of GDP for GTP which keeps the heterotrimeric G protein arrested in its inactive state (Nishimura et al, 2010 ; Schrage et al, 2015 ). Inhibition of G q signaling has been investigated in several disease models, and FR in particular, due to its long residence time (Kuschak et al, 2020 ), was found to be a promising drug candidate for the treatment of complex diseases such as asthma (Matthey et al, 2017 ), hypertension (Meleka et al, 2019 ), metabolic syndrome (Klepac et al, 2016 ), and cancers (Onken et al, 2018 ; Annala et al, 2019 ; Lapadula et al, 2019 ).…”