Elsevier

Biochemical Pharmacology

Volume 66, Issue 12, 15 December 2003, Pages 2397-2407
Biochemical Pharmacology

Naturally occurring 2′-hydroxyl-substituted flavonoids as high-affinity benzodiazepine site ligands

https://doi.org/10.1016/j.bcp.2003.08.016Get rights and content

Abstract

Screening of traditional medicines has proven invaluable to drug development and discovery. Utilizing activity-guided purification, we previously reported the isolation of a list of flavonoids from the medicinal herb Scutellaria baicalensis Georgi, one of which manifested an affinity for the benzodiazepine receptor (BDZR) comparable to that of the synthetic anxiolytic diazepam (Ki=6.4 nM). In the present study, this high-affinity, naturally occurring flavonoid derivative, 5,7,2′-trihydroxy-6,8-dimethoxyflavone (K36), was chosen for further functional and behavioral characterization. K36 inhibited [3H]flunitrazepam binding to native BDZR with a Ki value of 6.05 nM. In electrophysiological experiments K36 potentiated currents mediated by rat recombinant α1β2γ2 GABAA receptors expressed in Xenopus oocytes. This potentiation was characterized by a threshold (1 nM) and half-maximal stimulation (24 nM) similar to diazepam. This enhancement was demonstrated to act via the BDZR, since co-application of 1 μM of the BDZR antagonist Ro15-1788 reversed the potentiation. Oral administration of K36 produced significant BDZR-mediated anxiolysis in the mice elevated plus-maze, which was abolished upon co-administration of Ro15-1788. Sedation, myorelaxation and motor incoordination were not observed in the chosen dosage regimen. Structure–activity relationships utilizing synthetic flavonoids with different 2′ substituents on the flavone backbone supported that 2′-hydroxyl-substitution is a critical moiety on flavonoids with regard to BDZR affinities. These results further underlined the potential of flavonoids as therapeutics for the treatment of BDZR-associated syndromes.

Introduction

GABA, the major inhibitory neurotransmitter in the central nervous system (CNS), is essential for the overall balance between neuronal excitation and inhibition through interaction with specific membrane receptors. Being a member of the fast-acting transmitter-gated ion channel superfamily, GABAA receptors share structural and functional similarities with the nicotinic acetylcholine receptor, glycine receptor and 5-HT3 receptor, which include a pentameric pseudosymmetrical trans-membrane structure with a central ion pore. GABAA receptors, mainly located postsynaptically, mediate most of the inhibitory synaptic transmission in the CNS and serve as the target for many important neuroactive drugs including BDZs, barbiturates, steroids, general anesthetics and possibly alcohol [1].

The allosteric BDZR has been proposed to reside at the interface between α and γ subunits [2], [3], [4]. Upon ligand recognition, the affinity of the GABA binding site for its substrate is modified to result in the differential regulation of chloride flux through the ion channel situated at the center of the assembly. Pharmacologically, BDZs are potent anxiolytic, sedative, muscle relaxant and anticonvulsant drugs. However, untoward effects, including ethanol potentiation and amnesia, that accompany treatment with BDZs, have stimulated research into alternatives to conventional BDZs.

Apart from the putative differential pharmacology mediated by different BDZR subtypes, BDZR ligands are also known to modulate the GABAA receptor function in both directions and in different amplitudes. In this aspect, BDZR agonists and inverse agonists have been shown to increase and decrease, respectively, the affinity of GABA for its receptor, while an antagonist at the recognition site exerts negligible effect. That these ligands possess the capacity to regulate the physiological response in a spectrum of efficacies has raised hope for the development of partial allosteric modulators at the recognition site as potential therapeutics devoid of side-effects [5], [6]. In particular, BDZR partial agonists are postulated to exert potent anxiolysis without sedative and myorelaxant effects, both of which are considered to be elicited when a majority of receptor–drug complexes are activated [7], [8]. Several lines of neuropharmacological evidences are supportive of this hypothesis, showing that BDZR partial agonists potentiate the GABA-activated current in a sub-maximal manner in comparison to full agonists, and that the therapeutic window separating anxiolysis and other pharmacological effects of these ligands are much wider than conventional BDZs [9].

In recent years, drug screening from traditional medicinal herbs has attracted much attention in the hope to identify novel therapeutics for the treatment of various diseases. The discovery of chrysin, one of the first flavonoids shown to possess in vivo activity through interaction with the BDZR [10], marked the search for such natural anxiolytics. A number of flavonoids have been found to possess partial allosteric modulatory action at the GABAA receptor complex, and play a role in the modulation of anxiety [11], [12], [13]. They therefore constitute a promising class of naturally occurring compounds for the treatment of anxiety.

Naturally occurring flavonoids often bind to the BDZR with only moderate affinities. However, through synthesis of chemical libraries and molecular modeling of the flavonoid binding to the BDZR pharmacophore, several groups have been able to generate synthetic derivatives with higher affinities for the BDZR [14], [15], [16], [17], [18].

As part of our effort in identifying potent BDZR ligands from natural resources, a range of flavonoids was isolated from the medicinal herb Scutellaria baicalensis Georgi guided by radioreceptor binding assay for the BDZR [19], [20]. One of these naturally occurring flavonoids, K36, exhibited the highest affinity for the BDZR, comparable to that of diazepam. In the present study, K36 was further investigated with respect to its functional and behavioral properties. Using electrophysiological techniques, its role as a GABAA receptor function modulator was examined. Neuropharmacological studies employing animal models routinely adopted for BDZ evaluations were carried out. In addition, affinities of a series of flavonoid derivatives for the BDZR were examined in order to better understand the structural basis of the high-affinity of K36 for the BDZR.

Section snippets

Chemicals

Radioactive [3H]flunitrazepam (N-methyl-[3H], 88.0 Ci mmol−1) was purchased from Amersham. [3H]Ro15-1788 (N-methyl-[3H], 78.6 Ci mmol−1) was from NEN Life Science Products. Diazepam was from Sigma Chemical Co. Anexate (Ro15-1788, 0.1 mg mL−1 ampoules) was purchased from Hoffmann-La Roche Ltd. 5,7,2′-Trihydroxy-6,8-dimethoxyflavone, 5,7,2′-trihydroxy-6-methoxyflavone, 5,7-dihydroxy-6-methoxyflavone, 5,7-dihydroxy-6,8-dimethoxyflavone, 5,7-dihydroxy-8-methoxyflavone and 5,6,7-trihydroxyflavone were

Determination of Kd and Bmax of [3H]flunitrazepam binding to the BDZR

From representative Rosenthal (Scatchard) plot analysis of [3H]flunitrazepam saturation binding experiments, the dissociation constant (Kd) and the maximal binding density (Bmax) of the high and low affinity binding sites were determined to be 1.23±0.08 nM and 2.17±0.17 pmol mg−1, and 18.90±1.63 nM and 2.30±0.16 pmol mg−1 membrane preparation, respectively. From competitive binding experiments carried out in the presence of 1 nM of [3H]flunitrazepam, Ki values were calculated from ic50 of test

Discussion

Flavone and its derivatives have been shown to possess affinity for the BDZR [19], [32], [33] and a number of them have been demonstrated to be active in vivo[34], [35]. However, these naturally occurring flavonoids characterized so far exhibited affinities for the BDZR only in the micromolar range, substantially lower than that of conventional BDZs. Until the protein structure of the BDZR is elucidated, facilitating rational drug design to generate potent ligands for the recognition site,

Acknowledgements

This work was supported by the Research Grant Council and the Innovation and Technology Fund of Hong Kong (Grant No. UIM40), and by the Swiss National Science Foundation Grant 3100-064789.01/1.

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