A Cell-Permeable Inhibitor to Trap Gαq Proteins in the Empty Pocket Conformation

Abstract

SUMMARY In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date. Our data show that Gα proteins may be “frozen” pharmacologically in an intermediate conformation along their activation pathway and propose a pharmacological strategy to specifically silence Gα subclasses with cell-permeable inhibitors.

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

“…Next, we studied whether binding of the G q tracer [ 3 H]PSB‐15900 would be modulated by metal cations, nucleotides, phospholipids, agonists of G q protein‐coupled receptors or the known G protein inhibitors BIM‐46187 (BIM dimer) and BIM‐46174 (BIM monomer; Ayoub et al, ; Schmitz et al, ). Effects of sodium, lithium, magnesium and calcium chloride (10 and 100 mM) on the binding of [ 3 H]PSB‐15900 (5 nM) to human platelet membranes were investigated.…”

“…Ebselen was synthesized following reported procedures (Küppers et al, ; Pietka‐Ottlik, Potaczek, Piasecki, & Mlochowski, ). BIM‐46174 and BIM‐46178 were synthesized as described (Schmitz et al, ). GPCR agonists were purchased from Tocris Bioscience, Bristol, UK.…”

“…BIM-46174 and BIM-46178 were synthesized as described (Schmitz et al, 2014). GPCR agonists were purchased from Tocris Bioscience, Bristol, UK.…”

Recent achievements in developing selective Gq inhibitors

“…The chemical structures were determined by 1 H‐ and 13 C‐NMR, mass spectrometry, IR, and melting point. B, Acquired from Schmitz et al [Color figure can be viewed at wileyonlinelibrary.com]…”

“…There is not a clear correlation between the inhibition and the level of expression of G q protein, thus, the mechanistic basis underlying cellular context‐dependent inhibition needs to be further investigated. By a combination of the radioligand binding assays, BRET assays, docking, MD simulations, and in vitro GDP dissociation studies, the mode of action of 58 was determined; 58 traps G q in an empty pocket conformation permitting GDP exit, but preventing GTP entry, hereby inhibiting receptor‐catalyzed activation of G q proteins (Figure B).…”