Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

Laura Friis Eghorn; Kirsten Høstgaard-Jensen; Kenneth Thermann Kongstad; Tina Bay; David Higgins; Bente Frølund; Petrine Wellendorph

doi:10.1016/j.ejphar.2014.06.028

Positive allosteric modulation of the GHB high-affinity binding site by the GABA_A receptor modulator monastrol and the flavonoid catechin

Laura Friis Eghorn, Kirsten Høstgaard-Jensen, Kenneth Thermann Kongstad, Tina Bay, David Higgins, Bente Frølund, Petrine Wellendorph^*

^*Corresponding author for this work

17 Citations (Scopus)

Abstract

γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABA_A receptors as possible high-affinity GHB targets. GABA_A receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABA_A receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [³H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABA_A receptors, and the naturally occurring flavonoid catechin. These compounds increased [³H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [³H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [³H]GHB and the GHB high-affinity site-specific radioligands [¹²⁵I]BnOPh-GHB and [³H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABA_A receptors.

Original language	English
Journal	European Journal of Pharmacology
Volume	740
Pages (from-to)	570-577
Number of pages	8
ISSN	0014-2999
DOIs	https://doi.org/10.1016/j.ejphar.2014.06.028
Publication status	Published - 5 Oct 2014

Keywords

γ-Hydroxybutyric acid
Dihydropyrimidinone
Flavonoid
GHB receptor
NCS-382
Probe dependence

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10.1016/j.ejphar.2014.06.028

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Positive allosteric modulation of the GHB high-affinity binding site by the GABA_A receptor modulator monastrol and the flavonoid catechin. / Eghorn, Laura Friis; Høstgaard-Jensen, Kirsten; Kongstad, Kenneth Thermann et al.

In: European Journal of Pharmacology, Vol. 740, 05.10.2014, p. 570-577.

Research output: Contribution to journal › Journal article › Research › peer-review

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title = "Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin",

abstract = "γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.",

keywords = "γ-Hydroxybutyric acid, Dihydropyrimidinone, Flavonoid, GHB receptor, NCS-382, Probe dependence",

author = "Eghorn, {Laura Friis} and Kirsten H{\o}stgaard-Jensen and Kongstad, {Kenneth Thermann} and Tina Bay and David Higgins and Bente Fr{\o}lund and Petrine Wellendorph",

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T1 - Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

AU - Eghorn, Laura Friis

AU - Høstgaard-Jensen, Kirsten

AU - Kongstad, Kenneth Thermann

AU - Bay, Tina

AU - Higgins, David

AU - Frølund, Bente

AU - Wellendorph, Petrine

PY - 2014/10/5

Y1 - 2014/10/5

N2 - γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

AB - γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

KW - γ-Hydroxybutyric acid

KW - Dihydropyrimidinone

KW - Flavonoid

KW - GHB receptor

KW - NCS-382

KW - Probe dependence

U2 - 10.1016/j.ejphar.2014.06.028

DO - 10.1016/j.ejphar.2014.06.028

M3 - Journal article

C2 - 24973695

SN - 0014-2999

VL - 740

SP - 570

EP - 577

JO - European Journal of Pharmacology

JF - European Journal of Pharmacology

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