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. 2009 Oct 16;284(42):29136-45.
doi: 10.1074/jbc.M109.042333. Epub 2009 Jul 31.

Inhibition of heterotrimeric G protein signaling by a small molecule acting on Galpha subunit

Mohammed Akli Ayoub  1 , Marjorie Damian, Christian Gespach, Eric Ferrandis, Olivier Lavergne, Olivier De Wever, Jean-Louis Banères, Jean-Philippe Pin, Grégoire Pierre Prévost
Affiliations

Inhibition of heterotrimeric G protein signaling by a small molecule acting on Galpha subunit

Mohammed Akli Ayoub et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
BIM-46187 inhibits several GPCR downstream signaling pathways. A, chemical structure of BIM-46187. B and C, BIM-46187 inhibits cAMP accumulation (B) or IP1 (C) induced by activation of the indicated GPCR transiently expressed in COS-7 cells. Agonist concentrations used were: AVP (1 μm for V2 vasopressin), isoproterenol (10 μm for β2-adrenergic), serotonin (10 μm for 5HT4a), forskolin (10 μm on mock cells), thrombin (5 units/ml for PAR1), LPA (10 μm for LPA1), and GABA (100 μm for GABAb coexpressed with Gαq/i9). The data are the means ± S.E. from triplicate determinations from a representative experiment of three independent experiments. D, SRE-Luc gene reporter assay on cells transiently expressing PAR1 and stimulated by thrombin (5 units/ml) for 6 h after pretreatment overnight with Pertussis toxin (200 ng/ml) or 2 h with U73122 (10 μm), Y27632 (10 μm), or BIM-46187 (10 μm). E, SRE-Luc gene reporter assay on cells co-expressing SRE-Luc gene reporter without or with PAR1 and stimulated with thrombin (5 units/ml) or serum (10%) for 6 h after their pretreatment for 2 h with BIM-46187 (10 μm). The data are the means ± S.E. from triplicate determinations from a representative experiment of three independent experiments.
FIGURE 2.
FIGURE 2.
BIM-46187 specifically inhibits heterotrimeric G protein downstream pathways. A, BIM-46187 inhibits the cyclic AMP accumulation in the MCF-7 cells treated with increasing concentrations of the G protein α subunit activator cholera toxin. B, BIM-46187 specifically inhibits the endothelin-induced calcium release in the A2058 cells without effects on the ionophore induced calcium release. C, BIM-46174 was ineffective to down-regulate the activation status of Rac1-GTP in human colon cancer cells HCT8/S11 cultured for 1 or 20 h in the presence of the G protein inhibitor. Similar data were obtained with BIM-46187 (1 mm, 10 min of incubation) in HCT8/S11 cells cultured under standard conditions. The data are representative of three separate experiments.
FIGURE 3.
FIGURE 3.
BIM-46187 inhibits agonists mediated effect on receptor-G protein complexes in living cells. A, for BRET experiments, G proteins and receptors were fused to energy donor (Rluc) and energy acceptor (YFP), respectively. B–F, BRET experiments were performed in COS-7 cells transiently co-expressing PAR1-YFP and Gαi1-Rluc (B), Gαo-Rluc (C), Gα12-Rluc (D), and Rluc-β-arrestin 1 (F) or V2-YFP and Gαs-Rluc (E). The cells were first pretreated with BIM-46187 and then stimulated with PBS (□), Thrombin 5 units/ml (▲), or AVP 10 μm (●). The data are the means ± S.E. from triplicate determinations from a representative experiment of at least three independent experiments. DMSO, dimethyl sulfoxide.
FIGURE 4.
FIGURE 4.
BIM-46187 inhibits the physical interaction between GPCR and the heterotrimeric G protein and the GDP/GTP exchange. A, fluorescence emission spectra of the purified Alexa-488-labeled BLT1 receptor mixed with the G protein trimer Gαi2β1γ2 where the Gαi2 subunit is labeled with Alexa-568 in the presence of LTB4 (1 μm) and in the absence or presence of BIM-46187. The absence of nonspecific FRET was assessed by using Gαsβ1γ2 instead of Gαi2β1γ2. B, changes in the intensity of the 603-nm band as a function of BIM-46187 concentration (■). Open triangles (△) correspond to the changes when using Alexa-568-labeled β-arrestin 1 instead of Gαi2β1γ2. C, inhibition of BLT1-catalyzed GTPγS binding on the purified Gαi2β1γ by increasing concentrations in BIM-46187. D, effects of BIM-46187 on GTP binding induced by the GPCR peptidomimetic Mastoparan-7. The data are the means ± S.E. of three independent experiments. E, effects of BIM-46187 on FUB132-induced GTP binding to isolated Gαi2. The data are normalized to the maximal effect observed in the absence of BIM-46187. F, effects of BIM-46187 on AlF4−-induced fluorescence changes of Gαi2. The data are normalized to the maximal effect observed in the absence of BIM-46187. The data are the means ± S.E. of three independent experiments. DMSO, dimethyl sulfoxide.
FIGURE 5.
FIGURE 5.
BIM-46187 does not prevent Gα-Gβγ subunit association, but it blocks their molecular rearrangements upon agonist activation. A, the interaction between Gα, Gβ1, and Gγ2 protein subunits was studied in living cells by BRET approach. For this, COS-7 cells were transiently cotransfected with Gαi1-Rluc or Gαo-Rluc and either YFP-Gβ1 (B) or Venus-Gγ2 (C) subunits, as indicated. The cells were then pretreated (■) or not (□) with BIM-46187 (100 μm) for 2 h at 37 °C, and BRET signal was measured. D, difference near-UV circular dichroism spectra obtained by subtracting from the spectra of a Gαi2-BIM-46187 or Gβ1γ2-BIM-46187 mixture the contribution of the individual components (Gαi2 and BIM-46187 or Gβ1γ2 and BIM-46187, respectively). E and F, for the activation of G proteins, the cells were transiently cotransfected with Gαo-Rluc and either YFP-Gβ1 (E) or Venus-Gγ2 (F) subunits in the presence of various GPCRs as indicated. The cells were pretreated (■) or not (□) with BIM-46187 for 2 h and then stimulated with 5 units/ml thrombin (for PAR1), 10 μm PAR2-Amide peptide (for PAR2), 100 μm GABA (for GABAb), and 10 μm serotonin (for 5-HT4a), and BRET was immediately measured. G, dose response of BIM-46187 on PAR1-promoted BRET changes between either Gαi1-Rluc (□) or Gαo-Rluc (▲) and YFP-Gβ1 after thrombin stimulation. The data are the means ± S.E. of three independent experiments. DMSO, dimethyl sulfoxide.
FIGURE 6.
FIGURE 6.
Conformational changes of Gαi2 following BIM-46187 binding. A, near-UV circular-dichroism spectra of Gαi2 in the absence (closed circles) or the presence (open circles) of BIM-46187. B, variations in the molar ellipticity at 280 nm as a function of BIM-46187 concentration. [θ] is the ellipticity at a given BIM-46187 concentration, [θ]max the ellipticity in the absence of BIM-46187.
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