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.
authors: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, 2100 Copenhagen, Denmark.
authors: Laura F
authors: LF
authors: Eghorn
authors: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, 2100 Copenhagen, Denmark.
authors: Kirsten
authors: K
authors: Hoestgaard-Jensen
authors: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, 2100 Copenhagen, Denmark.
authors: Kenneth T
authors: KT
authors: Kongstad
authors: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, 2100 Copenhagen, Denmark.
authors: Tina
authors: T
authors: Bay
authors: Department of Histopathology, University of Dublin, Trinity College, Dublin, Ireland.
authors: David
authors: D
authors: Higgins
authors: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, 2100 Copenhagen, Denmark.
authors: Bente
authors: B
authors: Frølund
authors: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, 2100 Copenhagen, Denmark. Electronic address: pw@sund.ku.dk.
authors: Petrine
authors: P
authors: Wellendorph
mesh_terms: D000818:Animals; D001567:Benzocycloheptenes / Q000494:pharmacology; D001665:Binding Sites; D002392:Catechin / Q000494:pharmacology*; D005260:Female; D006885:Hydroxybutyrates / Q000494:pharmacology*; D009865:Oocytes / Q000502:physiology; D011743:Pyrimidines / Q000494:pharmacology*; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D013871:Thiones / Q000494:pharmacology*; D014982:Xenopus laevis
chemical_list: D001567:Benzocycloheptenes; D006885:Hydroxybutyrates; D011743:Pyrimidines; D011963:Receptors, GABA-A; D013871:Thiones; C066471:NCS 382; C111420:4-hydroxybutyric acid; C400223:monastrol; D002392:Catechin
keywords: Dihydropyrimidinone; Flavonoid; GHB receptor; NCS-382; Probe dependence; γ-Hydroxybutyric acid