Reversal of Ethanol-induced Intoxication by a Novel Modulator of Gβγ Protein Potentiation of the Glycine Receptor

Abstract

The acute intoxicating effects of ethanol in the central nervous system result from the modulation of several molecular targets. It is widely accepted that ethanol enhances the activity of the glycine receptor (GlyR), thus enhancing inhibitory neurotransmission, leading to motor effects, sedation, and respiratory depression. We previously reported that small peptides interfered with the binding of Gβγ to the GlyR and consequently inhibited the ethanol-induced potentiation of the receptor. Now, using virtual screening, we identified a subset of small molecules capable of interacting with the binding site of Gβγ. One of these compounds, M554, inhibited the ethanol potentiation of the GlyR in both evoked currents and synaptic transmission in vitro When this compound was tested in vivo in mice treated with ethanol (1-3.5 g/kg), it was found to induce a faster recovery of motor incoordination in rotarod experiments and a shorter sedative effect in loss of righting reflex assays. This study describes a novel molecule that might be relevant for the design of useful therapeutic compounds in the treatment of acute alcohol intoxication.

Keywords: G protein; Gβγ; alcohol; glycine receptor; molecular pharmacology; small molecule.

FIGURE 1.

Molecules identified by virtual screening and inhibition of the ethanol potentiation of GlyR. A, structures of six molecules identified as a result of the virtual screening protocol showing the obvious importance of amine groups. B, patch clamp experiments were performed in HEK cells overexpressing the GlyR α1 subunit. Traces show chloride current evoked by co-application of glycine with ethanol with intracellular diffusion of different small molecules (200 μm). The percentage of potentiation was obtained from the glycine plus ethanol glycinergic currents at minute 15 in relation to glycine alone. C, the percentage of ethanol potentiation of chloride current evoked as described in the presence of different molecules (200 μm) selected from the virtual screening. Molecules were allowed to diffuse intracellularly. D, the percentage of ethanol potentiation of chloride current evoked as described in the presence of the extracellularly applied molecules M554 and M890 (200 μm, incubated for 45 min). The values represent the mean ± S.E. of the percentage of potentiation from at least six cells. **, p < 0.01; *, p < 0.05.

FIGURE 2.

Concentration-response curve of the inhibitory effects of small molecules on ethanol potentiation of the GlyR. A, concentration-response curves of ethanol potentiation of the GlyR in the presence of molecules M554, M890, M554-1, and M554-2, applied intracellularly at concentrations of 1, 10, 30, 100, 200, and 1000 μm in HEK cells overexpressing the GlyR. B, values of half-maximal concentration and maximal inhibition were determined from the concentration-response curves for each molecule evaluated.

FIGURE 3.

Binding mode and molecular interaction between selected small molecules and Gβγ surface. A, predicted position of molecules M554 and M890 interacting with Gβγ. The Gβ amino acids interacting with both compounds are the same. Carbon atoms are shown in cyan or magenta, nitrogen in blue, and oxygen in red. B, modulation of PLCβ activated by Gβγ. HL60 cells expressing the fMLP receptor stimulated with fMLP activates PLCβ through Gβγ (fMLP column). Compounds M554, M119, and M890 were evaluated to determine any modulatory activity on this transduction system. C, modulation of GIRK channels activated by Gβγ. HEK cells overexpressing GIRK subunits and the GABA_B receptor were stimulated with baclofen in the presence or absence of molecules M554, M890, and M119. The values represent the mean ± S.E. from at least six cells. *, p < 0.05.

FIGURE 4.

Inhibition of ethanol synaptic effects by small molecules. A, representative traces of pharmacologically isolated glycinergic synaptic activity of cultured spinal cord neurons. For each condition (no ethanol, ethanol, +M554, and +M890), the cumulative probability of interevent interval (B) and amplitude (C) were obtained. D, isolated glycinergic miniature inhibitory postsynaptic currents from synaptic activity of cultured spinal neurons recorded in the absence (black traces) and presence of ethanol (100 mm, gray traces) plus M554 or M890 (200 μm each, applied intracellularly). E, decay time constant variation of glycinergic events registered in the presence or absence of ethanol and molecules as indicated. The values represent the mean ± S.E. **, p < 0.01.

FIGURE 5.

Effect of molecules M554 and M890 in open field assays in the presence of ethanol in C57BL/6J mice. Mice were tested in an open field assay after 10 min of an intraperitoneal injection of saline or ethanol (1.0 g/kg) in the presence or absence of M554 and M890 (100 mg/kg). A, sample trajectories of saline, ethanol, ethanol plus M554, and ethanol plus M890-injected mice in the open field test. B, total distance traveled during the 5-min test period revealed an effect of ethanol in mice that was not affected by the presence of small molecules. The values represent the mean ± S.E. *, p < 0.05; **, p < 0.01.

FIGURE 6.

Molecule M554 attenuated the sedative effect of ethanol in C57BL/6J mice tested on the accelerating rotarod. A, the latency to fall was determined before and after injection of ethanol (2 g/kg, ●), molecule M554 (100 mg/kg, ▵), ethanol plus M554 (♢), ethanol plus M890 (○) and saline solution (■). Five trials before and four trials after the respective injections were performed. Differences in the recovery of the group injected with both ethanol and molecule M554 were statistically significant. B, the duration of ethanol-induced LORR (3.5 g/kg) was quantified in the absence and presence of M554 at different concentrations in C57BL/6J mice. Statistical differences were detected in groups injected with 100 and 200 mg/kg in relation to the control condition. C, effect of M554 on duration of LORR in GlyRα1 KI mice. No statistical differences were detected. The values represent the mean ± S.E. *, p < 0.05.