Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on Gα Subunit

Marjorie Damian,Christian Gespach,Jean-Louis Banères,Olivier Lavergne,Mohammed Akli Ayoub,Grégoire Pierre Prévost,Olivier De Wever,Eric Ferrandis,Jean-Philippe Pin

doi:10.1074/jbc.m109.042333

Abstract

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

Highlights

The simultaneous activation of many distinct G proteincoupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions
Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the G␣ subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation
We have reported in vivo inhibition of the GPCR signaling pathway by two closely related imidazopirazine containing small molecules, displaying potent antiproliferative activity (BIM-46174) [17] and potent pain relief activity (BIM46187) [18]

Summary

EXPERIMENTAL PROCEDURES

Reagents, and Plasmids—The cDNA of PAR1, PAR2, LPAR1, and ␤2-adrenergic receptors in pcDNA3.1 were purchased from cDNA Resources Center (Rolla, MO). The assay was incubated for 1 h at 4 °C, and Europium Cryptate fluorescence and the time resolved FRET signal at 620 and 665 nm were measured 50 ␮s after excitation at 337 nm using a RubyStar instrument (BMG Labtechnologies, Champigny-sur-Marne, France). The data represent cAMP levels in pmol/well calculated from the calibration curve generated with the increasing concentrations of cAMP added in the assay according to the manufacturer’s instructions. The cells were lysed by adding the HTRF௡ assay reagents, the Europium Cryptate-labeled anti-IP1 antibody, and the d2-labeled IP1 analog, previously diluted in a the lysis buffer containing 1% Triton X-100. The assay was incubated for 1 h at room temperature, and Europium Cryptate fluorescence and the time resolved FRET signal were measured 50 ␮s after excitation at 337, 620, and 665 nm, respectively, using a RubyStar instrument (BMG Labtechnologies, Champigny, France). Absorbance values in the 1 range were used to avoid any saturation of the photomultiplicator

RESULTS

99 Ϯ 17 79 Ϯ 16 20 Ϯ 6

DISCUSSION