Loreclezole modulates [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding via a distinct site on the GABAA receptor complex

doi:10.1016/0014-2999(95)00856-X

European Journal of Pharmacology

Volume 300, Issues 1–2, 4 April 1996, Pages 125-130

https://doi.org/10.1016/0014-2999(95)00856-X Get rights and content

Abstract

The allosteric modulation of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) and [³H]flunitrazepam binding was utilized to evaluate the actions of loreclezole at the GABA_A receptor complex in the rat brain. Loreclezole was observed to allosterically inhibit the binding of [³⁵S]TBPS in a dose-dependent manner with micromolar potency (IC₅₀ = 1 μM). Loreclezole was found to have an additive effect on neuroactive steroid modulation of [³⁵S]TBPS binding, but merely potentiated the effect of Ro5-4864 (4″-chlorodiazepam) modulation of [³⁵S]TBPS binding. These observation suggest that loreclezole modulates [³⁵S]TBPS binding through a site independent of the neuroactive steroid and Ro5-4864 sites on the GABA_A receptor complex. The enhancement of [³H]flunitrazepam binding to the benzodiazepine receptor by loreclezole as well as the effect of loreclezole on CL218872/[³H]flunitrazepam dose-response curves suggest that loreclezole does not act through the benzodiazepine site on the GABA_A receptor complex, nor does it selectively modulate benzodiazepine receptor subtypes. The potency of loreclezole as an inhibitor of [³⁵S]TBPS binding in rat brain was regionally dependent and GABA-sensitive. Loreclezole modulation of [³⁵S]TBPS binding showed greater potency and GABA sensitivity in the cerebellum and thalamus when compared to other brain regions such as the cortex, hippocampus and striatum. This finding is consistent with previous reports of the selectivity of loreclezole for GABA_A receptor complex's containing β₂ and β₃ subunits. These β subunit isoforms predominate in the cerebellum and thalamus. Collectively the evidence suggests that loreclezole modulates [³⁵S]TBPS and [³H]flunitrazepam binding through a site distinct from benzodiazepine, neuroactive steroid, Ro5-4864 and GABA sites on the GABA_A receptor complex.

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Cited by (11)

Radioligand binding studies of caloporoside and novel congeners with contrasting effects upon [35S] TBPS binding to the mammalian GABA<inf>A</inf> receptor
2005, Biochemical Pharmacology
Citation Excerpt :
The positive modulatory effect of octyl-β-d-glucoside was occluded in the presence of GABA, in a similar fashion to benzodiazepines, indicating that the modulatory action of octyl-glucoside is related to the conformational state of the chloride channel [25]. GABA sensitivity is shared by a number of other GABAA receptor modulators, as well as benzodiazepines, including loreclezole, propofol and lactose [6,25,26]. The lack of inhibitory action of octyl-β-d-glucoside at high concentrations is a property shared by diazepam, but not loreclezole or propofol.
Caloporoside is a natural active fungal metabolite, which was isolated from Caloporous dichrous and was described to exhibit antibacterial, antifungal and phospholipase C inhibitory activity. We have previously reported evidence that related β-linked compounds, lactose and octyl-β-d-mannoside, bind and functionally modulate rodent GABA_A receptors, respectively. We have characterized the binding pharmacology of synthetic caloporoside and two further congeners, 2-hydroxy-6-{[(16R)-(β-d-mannopyranosyloxy)heptadecyl]} benzoic acid and octyl-β-d-glucoside on GABA_A receptors using a [³⁵S]-t-butylbicyclophosphoorothionate (TBPS) radioligand binding assay. Caloporoside and 2-hydroxy-6-{[(16R)-(β-d-mannopyranosyloxy)heptadecyl]} benzoic acid produced concentration-dependent complete inhibition of specific [³⁵S] TBPS binding with overall apparent IC₅₀ values of 14.7 ± 0.1 and 14.2 ± 0.1 μM, respectively. In contrast, octyl-β-d-glucoside elicited a concentration-dependent stimulation of specific [³⁵S] TBPS binding (E_max = 144 ± 4%; EC₅₀ = 39.2 ± 22.7 nM). The level of stimulation was similar to that elicited by diazepam (E_max = 147 ± 6%; EC₅₀ = 0.8 ± 0.1 nM), and was occluded by GABA (0.3 μM). However, the three test compounds failed to elicit any significant effect (positive or negative) upon [³H] flunitrazepam or [³H] muscimol binding, indicating that they did not bind directly, or allosterically couple, to the benzodiazepine or agonist binding site of the GABA_A receptor, respectively. The constituent monosaccharide, glucose, and both the closely related congeners octyl-β-d-glucoside or hexyl-β-d-glucoside have no significant effect upon [³⁵S] TBPS binding. These data, together, provide strong evidence that a β-glycosidic linkage and chain length are crucial for the positive modulation of [³⁵S] TBPS binding to the GABA_A receptor by this novel chemical class.
Modulation of [3H] TBOB binding to the rodent GABA<inf>A</inf> receptor by simple disaccharides
2003, Biochemical Pharmacology
We have recently reported evidence that a simple β-linked alkylated mannose reversibly increased the magnitude of GABA_A receptor currents evoked in cultured rat pyramidal neurons whilst concomitantly reducing the incidence of spontaneous synaptic activity. In this present study, the effects of the simple β-linked disaccharide, lactose was investigated using a [ $^{3} H$ ] TBOB (t-[ $^{3} H$ ] butylbicycloorthobenzoate) binding assay in adult rat forebrain and cerebellum membranes. Lactose elicited a significant potentiation of [ $^{3} H$ ] TBOB binding to well-washed forebrain and cerebellar membranes (mean E_max values=367 and 287%; mean ec₅₀ values=1.5 and 30 μM, respectively, N=4). The α-linked disaccharides, maltose and sucrose also potentiated [ $^{3} H$ ] TBOB binding, but with 100–600-fold higher ec₅₀ values than lactose. The lactose-mediated potentiation of [ $^{3} H$ ] TBOB in the forebrain and cerebellum was completely abolished in the presence of 0.3 μM GABA. Over the concentration range in which significant potentiation of [ $^{3} H$ ] TBOB binding was detected, lactose elicited no significant effect upon [ $^{3} H$ ] flunitrazepam binding. This study demonstrated that lactose can modulate the GABA_A receptor channel, allosterically coupled to the agonist site, but independent of the benzodiazepine site. Furthermore, lactose displayed differential effects upon forebrain and cerebellar GABA_A receptors indicating that it may be a novel subtype selective agent.
Use of bicuculline, a GABA antagonist, as a template for the development of a new class of ligands showing positive allosteric modulation of the GABA(A) receptor
2000, Bioorganic and Medicinal Chemistry Letters
Analogues of bicuculline devoid of the benzo ring fused to the lactone moiety were prepared by reacting 2-(tert-butyldimethylsiloxy)furans with 3,4-dihydroisoquinolinium salts. Some of these compounds (e.g., ROD185, 8) acted as modulators of the GABA_A receptor, displacing ligands of the benzodiazepine binding site. They also strongly stimulated GABA currents mediated by recombinant GABA_A receptors expressed in Xenopus oocytes.
Recent Advances in Synthesis and Anticancer Potential of Triazole-Containing Scaffolds
2022, Anti-Cancer Agents in Medicinal Chemistry
A multifaceted GABA<inf>A</inf> receptor modulator: Functional properties and mechanism of action of the sedative-hypnotic and recreational drug methaqualone (Quaalude)
2015, Molecular Pharmacology
Characterization of [ 35S]t-butylbicyclophosphorothionate ([ 35S]TBPS) binding to GABA <inf>A</inf> receptors in postmortem human brain
2007, British Journal of Pharmacology

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European Journal of Pharmacology

Abstract

References (22)

In vivo studies on the mechanism of action of the broad spectrum anticonvulsant lorereclezole

Epilepsy Res.

Lack of effect of flumazenil and CGS 8216 on the anxiolytic-like properties of loreclezole

Eur. J. Pharmacol.

The neuroactive steroid 3α-hydroxy-5β-pregnan-20-one is a two site modulator of [³⁵S]TBPS and [³H]flunitrazepam binding to the GABA_A receptor

Eur. J. Pharmacol. Mol. Pharmacol.

Resolution of two biochemically and pharmacologically distinct benzodiazepine receptors

Pharmacol. Biochem. Behav.

Protein measurement with the Folin phenol reagents

J. Biol. Chem.

A novel allosteric modulatiory site on the GABA_A receptor β subunit

Neuron

Heterotropic cooperativity between putative recognition sites for progesterone metabolites and the atypical benzodiazepine Ro5-4864

J. Neurochem.

Statistical estimation in pharmacokinetics

J. Pharmacokinet. Biopharmacol.

Phenylquinolines PK 8165 and PK 9084 allosterically modulate [³⁵S]-t-butylbicyclophosphorothionate binding to a chloride ionophore in rat brain via a novel Ro5-4864 binding site

J. Pharmacol. Exp. Ther.

Benzodiazepine receptor heterogeneity: a consequence of multiple conformational states of a single receptor or multiple populations of structurally distinct macromolecules?

Modulation of the chloride ionophore by benaodiazepine receptor ligands: Influence of GABA and ligand efficacy

Mol. Pharmacol.

Cited by (11)

Radioligand binding studies of caloporoside and novel congeners with contrasting effects upon [<sup>35</sup>S] TBPS binding to the mammalian GABA<inf>A</inf> receptor

Modulation of [<sup>3</sup>H] TBOB binding to the rodent GABA<inf>A</inf> receptor by simple disaccharides

Use of bicuculline, a GABA antagonist, as a template for the development of a new class of ligands showing positive allosteric modulation of the GABA(A) receptor

Recent Advances in Synthesis and Anticancer Potential of Triazole-Containing Scaffolds

A multifaceted GABA<inf>A</inf> receptor modulator: Functional properties and mechanism of action of the sedative-hypnotic and recreational drug methaqualone (Quaalude)

Characterization of [ <sup>35</sup>S]t-butylbicyclophosphorothionate ([ <sup>35</sup>S]TBPS) binding to GABA <inf>A</inf> receptors in postmortem human brain

Loreclezole modulates [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding via a distinct site on the GABAA receptor complex

Abstract

Epilepsy Res.

Eur. J. Pharmacol.

Eur. J. Pharmacol. Mol. Pharmacol.

Pharmacol. Biochem. Behav.

J. Biol. Chem.

Neuron

Heterotropic cooperativity between putative recognition sites for progesterone metabolites and the atypical benzodiazepine Ro5-4864

J. Neurochem.

Statistical estimation in pharmacokinetics

J. Pharmacokinet. Biopharmacol.

Phenylquinolines PK 8165 and PK 9084 allosterically modulate [35S]-t-butylbicyclophosphorothionate binding to a chloride ionophore in rat brain via a novel Ro5-4864 binding site

J. Pharmacol. Exp. Ther.

Benzodiazepine receptor heterogeneity: a consequence of multiple conformational states of a single receptor or multiple populations of structurally distinct macromolecules?

Modulation of the chloride ionophore by benaodiazepine receptor ligands: Influence of GABA and ligand efficacy

Mol. Pharmacol.

Loreclezole modulates [³⁵S]t-butylbicyclophosphorothionate and [³H]flunitrazepam binding via a distinct site on the GABA_A receptor complex

Phenylquinolines PK 8165 and PK 9084 allosterically modulate [³⁵S]-t-butylbicyclophosphorothionate binding to a chloride ionophore in rat brain via a novel Ro5-4864 binding site