Cyclothiazide binding to the GABAA receptor

Neuroscience Letters 439 (2008) 66–69

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Cyclothiazide binding to the GABAA receptor a,∗ ´ ´ Szarics ´ ´ ´ Banka c , Eva , Agnes Simon a , Julia Visy b , Edit Simon-Trompler c , Zoltan a d e a ´ o´ Heja ´ ´ o´ Gabor ´ ´ ´ Laszl , Laszl Harsing , Gabor Blasko´ , Julianna Kardos a

Department of Neurochemistry, Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary Department of Molecular Pharmacology, Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary Department of Applied Organic Chemistry, Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary d Division of Preclinical Research, EGIS Pharmaceuticals Plc, Budapest, Hungary e Servier Research Institute for Medicinal Chemistry, Budapest, Hungary b c

a r t i c l e

i n f o

Article history: Received 21 December 2007 Received in revised form 22 April 2008 Accepted 29 April 2008 Keywords: [3 H]-Cyclothiazide Diastereomeric fractions [3 H]-Flunitrazepam [3 H]-GABA (−)[1S,9R]-Bicuculline methiodide Binding in rat brain synaptic membrane

a b s t r a c t In order to explore the molecular interaction between cyclothiazide (CTZ) and ␥-aminobutyric acidA (GABAA ) receptors, possibly underlying inhibition of GABAA receptor currents, [3 H]-CTZ was synthesized. Binding of [3 H]-CTZ to rat brain synaptic membranes could be observed only in the presence of the GABAA receptor antagonist (−)[1S,9R]-bicuculline methiodide (BMI) (EC50,BMI = 500 ± 80 ␮M). GABA decreased [3 H]-CTZ binding induced by the presence 300 ␮M and 3 mM BMI with IC50,GABA values of 300 ± 50 ␮M and 5.0 ± 0.7 mM, respectively. Binding of CTZ to [3 H]-CTZ labeled sites was characterized by IC50,CTZ values of 0.16 ± 0.03 ␮M ([BMI] = 300 ␮M) and 7.0 ± 0.5 ␮M ([BMI] = 3 mM). Binding of the diastereomeric fraction [3 H]-(3R,1 S,4 S,5 R + 3S,1 R,4 R,5 S)-CTZ induced by 3 mM BMI was quantitatively the more significant in cerebrocortical and hippocampal membranes. It was characterized by IC50,CTZ = 80 ± 15 nM and IC50,GABA = 13 ± 3 mМ. In the absence of BMI, CTZ (1 mM) significantly decreased GABA-induced enhancement of [3 H]-flunitrazepam binding. Our findings suggest that functional inhibition may occur through binding of CTZ to an allosteric site of GABAA receptors. This allosteric site is possibly emerged in the receptor conformation, stabilized by BMI binding. © 2008 Elsevier Ireland Ltd. All rights reserved.

Cyclothiazide (CTZ) enhances synaptic currents induced by glutamate and increases the duration of currents gated by ␣-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [30], showing selectivity for the flip variants of AMPA receptor subunits conferred by the norbornenyl moiety of the molecule [14]. It is suggested that allosteric CTZ binding [25] reduces affinity of both agonists and antagonists of AMPA receptors in freshly prepared rat brain membranes [17]. In addition to the impairment of AMPA receptor desensitization, CTZ has been reported to inhibit recombinant metabotropic glutamate receptors non-competitively [26] and to reversibly inhibit ␥-aminobutyric acid (GABA) induced membrane currents via reduction of the open probability of GABAA receptor channel [7]. The CTZ molecule possesses four chiral carbons (Table 1), which allows 16 separate isomers, i.e., eight racemates to be envisaged, however, due to the restriction of the methylene bridge of the norbornenyl moiety, the number of possible stereoisomers is reduced

∗ Corresponding author. Tel.: +36 1 4384141x263; fax: +36 1 4381143. ´ Szarics). ´ E-mail address: [email protected] (E. 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.04.092

to eight, i.e., four diastereomeric racemates [21,6]. The separation of diastereomers of CTZ showed different AMPA receptor potentiating efficacy in Xenopus oocytes [6]. In order to explore the molecular interaction between CTZ and GABAA receptor possibly underlying inhibition of GABA currents, [3 H]-CTZ was synthesized. Specific binding of [3 H]-CTZ as well as the effect of CTZ on [3 H]-GABA and [3 H]-flunitrazepam binding in rat brain synaptic membrane fractions were explored. In addition, the four diastereomeric fractions of [3 H]-CTZ (A: 3S,1 S,4 S,5 R + 3R,1 R,4 R,5 S; B: 3R,1 S,4 S,5 R + 3S,1 R,4 R,5 S; C: 3R,1 S,4 S,5 S + 3S,1 R,4 R,5 R; and D: 3S,1 S,4 S,5 S + 3R,1 R,4 R,5 R) were separated and tested. Male Wistar rats (4–6 weeks old) were purchased from Toxicoop. They were kept and used in accordance with the European Council Directive of 24 November 1986 (86/609/EEC) and the Hungarian Animal Act 1998 and associated guidelines. The following compounds were used in the experiments: saccharose; tris-(hydroxy-methyl)-amino-methane (TRIS); dimethylsulfoxide (DMSO); ethanol; NaOH (Reanal); GABA; 7-chloro-1methyl-5-phenyl-3H-1,4-benzodiazepine-2(1H)one (diazepam); 5-ethyl-5-(1-methylbutyl)-2,4,6-trioxohexahydro-pyrimidine (pentobarbital); picrotoxin (Sigma); 6-chloro-3,4-dihydro-3-(2-

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67

Table 1 Comparison of specific binding of [3 H]-CTZ and its diastereomeric pairs to synaptic membranes isolated from the rat brain cortex or hippocampus in the presence of 3 mM BMI and their effects on AMPA-elicited currents in Xenopus oocytes

[3 H]-Ligand

Specific bindinga , cortex (%)

CTZ A-CTZ (3S,1 S,4 S,5 R + 3R,1 R,4 R,5 S) B-CTZ (3R,1 S,4 S,5 R + 3S,1 R,4 R,5 S) C-CTZ (3R,1 S,4 S,5 S + 3S,1 R,4 R,5 R) D-CTZ (3S,1 S,4 S,5 S + 3R,1 R,4 R,5 R)

50 44 68 20 48

a b

± ± ± ± ±

7 (N = 18) 5 (N = 2) 6 (N = 16) 2 (N = 2) 2 (N = 2)

Specific bindinga , hippocampus (%)

48 38 67 27 50

± ± ± ± ±

1% (N = 2) 2% (N = 2) 4% (N = 2) 2% (N = 2) 1% (N = 2)

AMPA receptor efficacyb (␮M)

2 3 20 10 1.8

In the presence or 3 mM BMI. Concentration causing a two-fold increase in the current elicited by application of 30 ␮M AMPA in Xenopus oocytes [6].

norbornen-5-yl)2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1-dioxide (CTZ); 2,3-dioxo-6-nitro-1,2,3,4-tetrahydro-benzo[f]quinoxaline-7-sulfonamide (NBQX); 1,2,3,6-tetrahydro-4-pyridinecarboxylic acid hydrochloride (isoguvacine); (−)[1S,9R]-bicuculline methiodide (BMI); 6-imino-3-4-mehoxyphenyl-1(6H)-pyridazine butanoic acid (SR 95531) (Tocris); n-heptane; isopropyl alcohol (Merck); MicroSil Silica 7.5 ␮m, 300 mm × 5 mm I.D. chromatographic column (Micrometrica); Optiphase HiSafe scintillation mixture (LKB-Wallac); [3 H]-GABA (87 Ci/mmol); [3 H]-flunitrazepam (93 Ci/mmol) (Amersham Biosciences). Stock solution of diazepam (2 mM) was made in DMSO, with the final DMSO concentration in the [3 H]-flunitrazepam binding assay being 0.5%. CTZ (100 mM) was dissolved in 1 M NaOH solution, the pH value in the binding assay being 7.50. Stock solutions of all other compounds were made in double-distilled water. [3 H]-CTZ (15 Ci/mmol) was synthesized according to the method described by Whitehead et al., 1961 [29]. Separation of diastereomeric fractions was carried out according to the method described in Nusser et al. [21]. The ISCO HPLC-system (Model 2360 Gradient Programmer, Model 2350 HPLC-Pump, V4 Variable Wavelength Absorbance Detector; Lincoln, Nebraska, USA) was used under the following conditions: the detector was set at 225 nm and the Valco injector was used with a 50 ␮l loop. The stationary phase used was a MicroSil Silica (7.5 ␮m, 300 mm × 5.0 mm I.D., Micromeritics, Norcross, Georgia, USA) column. The mobile phase was composed of 90% n-heptane and 10% isopropyl alcohol, and the flow rate was 1 ml/min. The four diastereomeric fractions were prepared, with the separation of peaks having retention times of 25, 28, 33 and 43 min (in this order). The products of the four runs were then combined, evaporated to dryness and dissolved in ethanol. The four diastereomeric fractions of [3 H]-CTZ corresponding to those of 1, 2, 3 and 4 described by Cordi et al. [6] were separated with a purity of 92% ([3 H]-A-CTZ), 90% ([3 H]-B-CTZ), 97% ([3 H]-C-CTZ) and 97% ([3 H]-D-CTZ) and a yield of 8.5% ([3 H]-A-CTZ), 8% ([3 H]-B-CTZ), 39% ([3 H]-C-CTZ) and 44.5% ([3 H]-D-CTZ) was obtained. Synaptic membrane fragments containing A type GABA receptors were prepared as described earlier [11]. The final protein concentration in binding assays was 0.35 ± 0.03 mg/ml [19]. Aliquots (600 ␮l) of synaptosomal membrane fragments containing A type GABA receptors were incubated with 600 ␮l of 0.05 M TRIS–HCl (pH 7.1 at 4 ◦ C) containing [3 H]-CTZ or one of its diastereomeric fractions (with a final concentration of 1.5 nM, 0.023 ␮Ci/ml) in the absence or (to define non-specific binding) presence of 500 ␮M of CTZ for 60 min at 4.0 ± 0.1 ◦ C. Samples of 500 ␮l were filtered through glass microfiber filters (Whatman

GF/B), soaked with 4 ml of ice-cold 0.05 M TRIS–HCl buffer (pH 7.1 at 4 ◦ C) and washed three times with 4 ml of the same buffer. [3 H]-GABA binding experiments were performed as described earlier [11]. Final concentration of [3 H]-GABA was 3 nM (0.25 ␮Ci/ml). Non-specific binding was determined in the presence of 1 mM isoguvacine for 60 min at 4.0 ± 0.1 ◦ C. The non-specific to total ratio for high-affinity [3 H]-GABA binding was 0.12 ± 0.02. [3 H]-flunitrazepam binding experiments were performed as described [27]. Final concentration [3 H]-flunitrazepam was 1 nM (0.087 ␮Ci/ml). Non-specific binding was determined in the presence of 10 ␮M diazepam. The non-specific to total ratio for [3 H]-flunitrazepam binding was 0.05 ± 0.01. Comparison of BMI and enantiomers of CTZ diastereomeric fractions were performed using the Surflex-Sim module of the SYBYL program package (SYBYL 7.3, Tripos Inc., St. Louis, MO, USA). Surflex-Sim optimizes the pose of a query molecule to maximize 3D similarity to the object molecule [5]. The resulting positions are ranked according to decreasing order of morphological similarity, which is defined on a scale of 0–1 and is quantified by a “score” value. A score value greater than 0.70 is generally significant in terms of the similarity it indicates in the functional relationship of the molecules. Results are reported as mean ± S.E.M. Statistical differences were determined by paired t-test or by one-way ANOVA (analysis of variance). A p-value < 0.05 was considered significant. In order to explain the GABAA inhibitory action of CTZ observed by Deng and Chen [7], enantiomers of B-CTZ and D-CTZ were aligned to the fixed, experimentally determined [11] threedimensional structure of BMI which was shown to effectively inhibit the binding of [3 H]-GABA to the GABAA receptors [11]. None of the 10 alignments suggested by Surflex-Sim reached the threshold indicating significant similarity between CTZ and BMI (score values between 0.48 and 0.53). As a control, BMI itself was flexibly aligned to the fixed BMI structure, resulting in a score value of 0.99 in the best position. Molecular mechanics data, therefore, indicate that CTZ and BMI do not share their binding sites on the GABAA receptor. Preliminary experiments showed that the binding of BMI, an antagonist of the GABAA receptor [11,20] to rat brain synaptic membranes induced binding of [3 H]-CTZ and its diastereomers. In the presence of saturating concentration of BMI (3 mM) the specific [3 H]-CTZ binding was 50 ± 7% of the total binding (Table 1). Antagonists other than BMI, such as the orthosteric site antagonist SR 95531 (30 ␮M); [28] or the channel blocking picrotoxin (1 mM);

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Fig. 1. Effect of BMI (triangle), CTZ (square) and GABA (circle) on [3 H]-CTZ binding in the presence of 0.3 mM (filled symbols) and 3 mM (open symbols) BMI.

[18], did not induce specific [3 H]-CTZ binding. Pentobarbital was also ineffective at both 200 ␮M and 1 mM, the later concentration having previously been shown to antagonize GABAA receptor function [4]. GABA (100 ␮M), diazepam (10 ␮M) and the AMPA receptor antagonist NBQX (2 ␮M) did not induce specific [3 H]-B-CTZ binding. BMI (3 mM) induced specific binding of [3 H]-CTZ to rat brain synaptic membranes with a half-maximal enhancement value of EC50,BMI = 500 ± 80 ␮M (Fig. 1). Apparently, CTZ and GABA displaced [3 H]-CTZ bound to sites in rat brain synaptic membranes (Fig. 1). In the presence of saturating concentration (3 mM) of BMI, CTZ displaced bound [3 H]-CTZ binding with a half-maximal inhibition value of IC50,CTZ = 7.0 ± 0.5 ␮M. In the presence of 0.3 mM BMI, however, the IC50, CTZ value was reduced to 0.16 ± 0.03 ␮M (Fig. 1). Inhibition of [3 H]-CTZ binding by GABA also varied with the concentration of BMI. The IC50 values obtained for GABA were 5.0 ± 0.7 mM and 0.30 ± 0.05 mM in the presence of 3 mM and 0.3 mM BMI, respectively. [3 H]-CTZ diastereomers displayed equal specific binding in cerebrocortical and hippocampal membranes (Table 1) with [3 H]-B-CTZ showing the highest specific binding in both brain regions in the presence of 3 mM BMI. Under the same condition, CTZ and GABA displaced [3 H]-B-CTZ from specific binding sites with an IC50 value of 80 ± 15 nM and 16 ± 3 mM, respectively. The effect of CTZ on GABAA receptor has been evaluated in combination with its effect on GABA-induced [3 H]-flunitrazepam binding (‘GABAshift’). Enhancements of [3 H]-flunitrazepam binding by GABA were characterized by an EC50 value of 0.50 ± 0.02 ␮M. CTZ (100 ␮M) significantly decreased [3 H]-flunitrazepam binding when measured in the presence of 10 ␮M GABA (163 ± 3% vs. 128 ± 1%; P < 0.05, oneway ANOVA). It is noteworthy that slight but significant reduction of high-affinity [3 H]-GABA binding by 1 mM CTZ was also observed (84 ± 2%; P < 0.05, paired t-test). CTZ was reported to inhibit evoked and spontaneous inhibitory postsynaptic currents (IPSCs) and GABA-induced membrane currents [7], challenging the specificity of this widely used AMPA receptor antagonist. IPSCs were abolished (98 ± 0.8% reduction) by application of 500 ␮M CTZ. The molecular interaction between CTZ and GABAA receptors, however, remained undisclosed. The major finding of the present study is that the GABAA receptor antagonist BMI (0.3 mM) specifically induces binding of CTZ to rat brain synaptic sites with submicromolar affinity (with an estimated Kd of 400 ± 50 nM). In addition, inhibition of GABA-induced flunitrazepam binding by CTZ was also observed. CTZ was shown to inhibit [3 H]-S-fluoro-willardiine and [3 H]NBQX binding in freshly prepared rat brain membranes [17]. In

this study, the binding interaction of [3 H]-CTZ with some GABAA receptor sites was explored in a frozen-thawed synaptic membrane preparation. It could not be observed in the absence of BMI, nor could it be seen in the presence of agonist AMPA or antagonist NBQX indicating the lack of interaction between [3 H]-CTZ and AMPA receptors. Reportedly, interaction between CTZ and [3 H]-AMPA or [3 H]-S-fluoro-willardiine binding in frozen-thawed membranes was absent or reduced if the chaotropic agent KSCN was not present [10], which explains the absence of binding of [3 H]-CTZ to AMPA receptors observed in this study. Several molecular interactions can be addressed to explain the inhibition of IPSCs by CTZ. These include the indirect effect of CTZ on GABA receptor currents and the binding of CTZ at orthosteric or allosteric GABAA binding sites. Besides inhibiting GABAA receptor binding (IC50 = 15 ␮M) [11], bicuculline isomers were shown to inhibit GABA uptake, (−)[1S,9R]bicuculline being the less active [13]. In addition, BMI was reported to inhibit slow conductance potassium (SK) channels in rat preoptic neurons (IC50 = 15 ␮M) [8] and Xenopus oocytes (IC50 = 1 ␮M) [15], which can imply some interaction between CTZ and SK channels. However, indirect effect of CTZ on GABAA receptors is questioned by both electrophysiological and binding results. CTZ instantaneously inhibited GABAA receptor mediated currents, implying a direct interaction with GABAA receptors [7]. This statement is further supported by the inhibition of GABA-induced [3 H]-flunitrazepam binding by CTZ (this work). Moreover, BMI-induced [3 H]-CTZ binding was reduced by GABA. These data together demonstrate that CTZ interacts with GABAA receptors at the molecular level. BMI induced binding of CTZ at the orthosteric GABAA receptor site is also unlikely because IC50 value for inhibition of [3 H]-CTZ binding by GABA was found to be 500 ± 80 ␮M, order of magnitudes higher than IC50 value for replacement of [3 H]-GABA by GABA (15 ␮M) [11]. Therefore CTZ is expected to interact with an allosteric binding site of the GABAA receptor. In a previous study, we demonstrated that a genuine allosteric interaction may appear as competitive [17], an observation supported by the present findings as well. The enhancement of CTZ binding by saturating BMI is reversed by saturating GABA. By lowering the BMI concentration to 0.3 mM, a similar reduction in the IC50 value of GABA was also observed. Failure to align CTZ and BMI suggests that these structures are unlikely to occupy similar recognition sites despite a partial overlap in their structures. This can explain the binding of CTZ in the presence of BMI, although both are reported to be antagonists of the A type GABA receptors. Deng and Chen [7] showed that the EC50 value of GABA did not change in the presence of 100 ␮M CTZ (EC50,GABA = 19.0 ␮M, EC50,GABA+CTZ = 22.2 ␮M), indicating that CTZ does not influence GABA binding. In accordance, high-affinity [3 H]-GABA binding was only slightly inhibited by CTZ in this study. Therefore, the binding site of CTZ is likely to be different from the binding sites of GABA and BMI. Binding of CTZ to GABAA receptors could only be observed in the presence of BMI, suggesting that conformation of BMIbound GABAA receptor is preferred for CTZ binding. In resting conditions, GABAA receptors are in equilibrium between closed and open states. The open state at resting is determined by the low-micromolar [22] extracellular concentration of GABA. GABAA receptor models assuming two different receptor sites for GABA binding has been proposed in several studies [20,9,23]. Dependence of receptor activation on GABA concentration indicates that channel opening is mediated by two types of GABA binding sites [3], i.e. one for channel opening and the other for receptor desensitization. Binding of GABA to one of these sites leads to channel opening [3]. This site can be blocked by the antagonist SR 95531. Activation of the other binding site of GABA, however, results in receptor

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desensitization [3]. BMI is able to bind to both GABA sites as well as to the barbiturate and the steroid binding sites [28,2]. These interactions of BMI are not shared by SR 95531 [28], which may explain the inability of SR 95531 to induce CTZ binding. Desensitization of GABAA receptors may occur with only one ligand molecule bound [3,12] and it is regulated through phosphorylation of the receptor subunits [24]. Occupation of the site for desensitization by BMI keeps the receptor in the closed state [1], suggesting that BMI-bound and closed states are indistinguishable. Conclusively, these findings suggest that CTZ binds to an allosteric site of the GABAA receptor which is emerged in a closed GABAA R conformation. Correlation of the BMI-sensitive specific binding of [3 H]CTZ diastereomeric fractions in cerebrocortical and hippocampal synaptic membranes suggests that the presumed CTZ binding site of GABAA receptors may possibly be located either on subunits present in both brain areas, i.e. ␣1, ␣2, ␤1, ␤3, ␥2 [16] or CTZ lacks selectivity to GABAA receptor subunits. If one assumes that the rank order of diastereomeric CTZ racemates to potentiate AMPA receptor function (D-CTZ > ACTZ > C-CTZ > B-CTZ; [6]) reflects binding affinities, the different rank order of BMI-sensitive GABAA receptor binding (B-CTZ > DCTZ > A-CTZ > C-CTZ; this study) suggests that CTZ diastereomeric pairs differentiate between GABAA and AMPA receptors. In conclusion, our data provides evidence that functional inhibition of A type GABA receptors may occur through allosteric binding of CTZ to the closed conformation of A type GABA receptors.

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Acknowledgements

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This work was supported by grants Bolyai BO 00226/03, MediChem2 1/A/005/2004 NKFP (Hungary), and EGIS Pharmaceu´ ´ is greatfully ticals. The skilful assistance of Erzsebet Fekete Kuti acknowledged. The authors thank Ulrike Uhrig and Antal Lopata (Tripos Inc.) for their advice relating to the molecular similarity search process and Karun Singh Arora for proofreading the manuscript.

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