Positive Allosteric Modulation of Native and Recombinant -Aminobutyric AcidB Receptors by 2,6-Di-tert-butyl-4-(3- hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and its Aldehyde Analog CGP13501

The compounds CGP7930 [2,6-Di-tert-butyl-4-(3-hydroxy-2,2dimethyl-propyl)-phenol] and its close analog CGP13501 were identified as positive modulators of -aminobutyric acidB (GABAB) receptor function. They potentiate GABA-stimulated guanosine 5 -O-(3-[S]thiotriphosphate) (GTP [S]) binding to membranes from a GABAB(1b/2) expressing Chinese hamster ovary (CHO) cell line at low micromolar concentrations and are ineffective in the absence of GABA. The structurally related compounds propofol and malonoben are inactive. Similar effects of CGP7930 are seen in a GTP [S] binding assay using a native GABAB receptor preparation (rat brain membranes). Receptor selectivity is demonstrated because no modulation of glutamate-induced GTP [S] binding is seen in a CHO cell line expressing the metabotropic glutamate receptor subtype 2. Dose-response curves with GABA in the presence of different fixed concentrations of CGP7930 reveal an increase of both the potency and maximal efficacy of GABA at the GABAB(1b/2) heteromer. Radioligand binding studies show that CGP7930 increases the affinity of agonists but acts at a site different from the agonist binding site. Agonist affinity is not modulated by CGP7930 at homomeric GABAB(1b) receptors. In addition to GTP [S] binding, we show that CGP7930 also has modulatory effects in cellular assays such as GABAB receptor-mediated activation of inwardly rectifying potassium channels in Xenopus laevis oocytes and Ca signaling in human embryonic kidney 293 cells. Furthermore, we show that CGP7930 enhances the inhibitory effect of L-baclofen on the oscillatory activity of cultured cortical neurons. This first demonstration of positive allosteric modulation at GABAB receptors may represent a novel means of therapeutic interference with the GABAergic system. -Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. It activates two classes of receptors: ionotropic, chloride-permeable GABAA receptors and metabotropic GABAB receptors. The structure and function of GABAB receptors have been reviewed extensively (Bettler et al., 1998; Marshall et al., 1999; Bowery and Enna, 2000; Couve et al., 2000; Jones et al., 2000; Kuriyama et al., 2000; Marshall, 2000). The GABAB receptor is a member of the “family 3” G-protein-coupled receptors (GPCRs) (reviewed in Couve et al., 2000), which also comprises metabotropic glutamate receptors (mGluRs), the calciumsensing receptor, and a group of mammalian vomeronasal and candidate taste receptors (Hoon et al., 1999). Like the other members of this family, the GABAB receptor has a molecular structure that is characterized by its seven transmembrane-spanning domains and a large extracellular N-terminal ligand binding domain related to periplasmic bacterial amino acid binding proteins. GABAB receptors modulate the activity of inwardly rectifying potassium channels and high voltage-activated calcium channels. Furthermore, they also inhibit adenylate cyclase activity in native and recombinant systems. By these mechanisms, they act postand presynaptically to inhibit neuronal excitability and neurotransmitter release, respectively. A thorough molecular investigation of GABAB receptors was initiated by the cloning of a first receptor protein GABAB(1), which exists in two N-terminal splice variants, 1a and 1b (Kaupmann et al., 1997). Unexpectedly, however, heterologous expression of GABAB(1) receptor protein has not made possible the measurement of robust funcABBREVIATIONS: GABA, -aminobutyric acid; GPCR, G-protein-coupled receptor; mGluR, metabotropic glutamate receptor; CHO, Chinese hamster ovary; GTP S, guanosine 5 -O-(3-thiotriphosphate); SPA, scintillation proximity assay; CGP7930, 2,6-di-tert-butyl-4-(3-hydroxy-2,2dimethyl-propyl)-phenol; APPA, 3-aminopropylphosphinic acid; HEK, human embryonic kidney; HBSS, Hanks’ balanced salt solution; FLIPR, fluorescence imaging plate reader. 0026-895X/01/6005-963–971$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 5 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 1107/942258 Mol Pharmacol 60:963–971, 2001 Printed in U.S.A. 963 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from tional responses. This finding has remained unexplained until the discovery that the formation of heterodimeric assemblies between GABAB(1) and a novel GABAB(2) protein is a prerequisite to form functional GABAB receptors (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999). Allosteric modulation of GABAB and some mGluR receptors by calcium has been described previously (Kubo et al., 1998; Saunders et al., 1998; Wise et al., 1999; Galvez et al., 2000a). The calcium sensing receptor is, in turn, allosterically activated by amino acids (Conigrave et al., 2000). Furthermore, noncompetitive inhibitors of “group I” mGluRs acting at a site distinct from the agonist binding site have also been found (for reviews, see Pin et al., 1999; Spooren et al., 2001). However, no allosteric modulation of GABAB receptor activity by low-molecular-weight organic compounds has been observed to date. This study describes two molecules with such effects, CGP13501 and CGP7930 (Fig. 1). Positive allosteric modulators act synergistically with an agonist, but have no intrinsic efficacy on their own. Thus, they act only when and where the endogenous agonist is present and thus have more physiological effects than pure agonists, which activate receptors independently of synaptic activity. Therefore, positive allosteric modulators are expected to have a better side effect profile than conventional agonists and thus are of considerable therapeutic interest. Materials and Methods Stable Transfection and Culture of CHO Cell Clones. Chinese hamster ovary K1 (CHO-K1) cells were stably transfected with GABAB(1b) and GABAB(2) cDNAs. Human GABAB(1b) [in pcDNA3.1, (Invitrogen, Carlsbad, CA)] and rat GABAB(2) [in pC1-neo (Promega, Madison, WI)] constructs were cotransfected (1:1 ratio of plasmids) using the Superfect transfection system from QIAGEN AG (Basel, Switzerland). Stably transfected cell clones were selected and cultured in Dulbecco’s modified eagle medium (glutamine-free Dulbecco’s modified Eagle’s medium; Invitrogen) supplemented with 10% fetal calf serum, 20 g/ml L-proline, 400 g/ml L-glutamine, 1 mg/ml geneticin, 250 g/ml zeocin. The cells were grown to 80 to 90% confluence in 14-cm cell culture dishes. For some specificity experiments, a CHO cell line stably expressing the mGluR2 metabotropic glutamate receptor (Flor et al., 1995) was also used. Preparation of Membranes from CHO Cells. The culture dishes were washed twice with ice-cold HEPES buffer, pH 7.4. Buffer was added and the cells were scraped off. Crude membranes from several dishes were collected in a 50-ml tube and centrifuged at 4°C for 20 min at 15,000 rpm in an SS34 rotor (Sorvall, Newton, CT). The pellet was resuspended in buffer and homogenized using a glassglass homogenizer (10 strokes). Afterward, the suspension was centrifuged (18,000 rpm, 30 min, 4°C), and the pellet was resuspended in a small volume of buffer and homogenized again (20 strokes). Aliquots were frozen in liquid nitrogen and stored at 80°C. On the day of the experiment, the frozen membranes were thawed and then centrifuged for 10 min at 15,000 rpm and 4°C. The pellet was resuspended in 1 ml of ice-cold distilled water and incubated for 1 h on ice. After a further centrifugation as before, the final pellet was resuspended in the appropriate amount of assay buffer (see below). Preparation of Rat Brain Membranes for Native Receptor Assays. Membranes from rat brain cortex were prepared as described in detail earlier (Olpe et al., 1990). GTP [S] Assay. The composition of the assay mixtures [in a final volume of 250 l in 96-well, clear-bottomed microtiter Isoplates (PerkinElmer Wallac, Turku, Finland)] was as follows: 50 mM TrisHCl buffer, pH 7.7, 10 mM MgCl2, 0.2 mM EGTA, 2 mM CaCl2, 100 mM NaCl, 10 M guanosine 5 -diphosphate (30 M with rat cortical membranes; Sigma Chemical, Buchs, Switzerland), 50 l of the membrane suspension described above (approximately 10–20 g of protein), 1.5 mg of wheat germ agglutinin-coated SPA beads (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK), 0.3 nM [S]GTP S ( 1000 Ci/mmol, stabilized solution; Amersham Pharmacia Biotech), and the test compounds (agonists and/or modulators) at the appropriate concentrations. Nonspecific binding was measured in the presence of unlabeled GTP S (Sigma) in excess (10 M). The samples were incubated at room temperature for 60 min before the SPA beads were sedimented by centrifugation at 2600 rpm for 10 min. The plates were then counted in a Wallac 1450 MicroBeta liquid scintillation counter. For data analysis, nonspecific binding was subtracted from all the other values; the effects of GABA and modulators were expressed relative to basal activity, measured in the absence of agonist. Concentration-response curves were analyzed by nonlinear regression. Prism 3.0 software (GraphPad Software, San Diego, CA) was used for all data calculations. Radioligand Binding Experiments. The protocols for measuring the binding of the radioligands [H]CGP62349 (a competitive antagonist) and [H]APPA ([H]CGP27492, an agonist ligand) were based essentially on methods described previously (Olpe et al., 1990; Hall et al., 1995; Bittiger et al., 1996). The [H]CGP62349 binding assay was performed in the SPA format; in the [H]APPA binding assay, bound and free radioligand were separated by centrifugation. Saturation and displacement curves were analyzed by nonlinear curve fitting to the appropriate models and using Prism 3.0 software. Measurement of Change in Intracellular Calcium Concentration by Fluorometry. For measurement of changes in intracellular calcium concentrations, HEK293 cells were transiently transfected with GABAB(1b/2a). All transfections included G qo5 to couple GABAB receptors to phospholipase C (Franek et al., 1999) and were made as described in detail previously (Pagano et al., 2001). Transfected HEK293 cells were plated into poly-D-lysine coated 96-well plates (BD Biosciences, San Jose, CA). Twenty-four to seventy-two hours after transfection, cells were loaded for 45 min with 2 M fluo-4 AM (Molecular Probes, Eugene, OR) in HBSS (Invitrogen) containing 50 M probenecid (Sigma). Plates were washed twice in the incubation buffer (HBSS) and transferred to a fluorescence imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA). Fluorescence was measured at room temperature for 3 min after the Fig. 1. Chemical structures of compounds used. 964 Urwyler et al. at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from addition of CGP7930 to check for agonistic effects of the compound. A second recording period of 3 min was initiated 10 min after the start of the first measurement. CGP7930 was present from the start, and 1 M GABA in HBSS was added at 20 s after the start of the second reading. Relative fluorescence changes over baseline ( F/F) were determined. Concentration-response curves were recorded with three to eight wells per concentration and experiment; the data were pooled and fitted using Igor Pro (Wavemetrics, Lake Oswego, OR) with a logistic equation using weighted nonlinear regression. FLIPR Experiments on Neuronal Networks. Primary cultures of cortical neurons were prepared from embryonic day 16 to 18 Sprague-Dawley rats (Wang and Gruenstein, 1997). Dissociated cells were plated on poly-L-lysine coated plates and incubated at 37°C in 5% CO2 for 7 to 10 days. About 15 min before experiments, the culture medium was removed and cells were loaded with 2 M fluo-4 AM in HBSS supplemented with 10 mM HEPES, pH adjusted to 7.4. After loading, cells were washed twice in the incubation medium (HBSS without Mg ) and then transferred to the fluorescence reader. Fluorescence was measured at room temperature and at a sampling rate of 0.5 Hz. Drugs were dissolved in HBSS without Mg and added to the cultures during recording. Oscillations were analyzed using IgorPro by peak detection and calculation of the ratio of peak frequencies before and after compound addition. Oocyte Electrophysiology. Experiments were performed as described earlier (Lingenhoehl et al., 1999). Briefly, lobes of oocytes were removed surgically from anesthetized (1.2 g/l MS222) female Xenopus laevis frogs. Oocytes were separated and defolliculated and injected with 10 to 50 ng of rat GABAB(1a) (or GABAB(1b)) together with GABAB(2) and rat Kir3.1, 3.2, and 3.4 coding mRNAs and incubated at 18°C for 3 to 8 days. Two-electrode voltage clamp recordings were done with electrodes filled with 3 M KCl. Oocytes were continuously perfused with normal frog Ringer solution (115 mM NaCl, 10 mM HEPES, 2.5 mM KCl, 1.8 mM CaCl2, pH 7.2) or high-potassium Ringer solution (90 mM KCl, 27.5 mM NaCl, 10 mM HEPES, 1.8 mM CaCl2, pH 7.2). Recordings were performed at a clamp potential of 70 mV. To test the positive modulatory activity of CGP7930, the compound was applied with ascending concentrations and a fixed GABA concentration. Chemicals. CGP7930 and CGP13501 were synthesized in house. Propofol and malonoben were from Tocris Cookson Ltd. (Bristol, UK). Stock solutions of these compounds were prepared in dimethyl sulfoxide and subsequently diluted in the respective assay buffers. The final concentrations of dimethyl sulfoxide in the various assays usually did not exceed 0.3% and did not interfere with the measured parameters. [H]APPA ([H]CGP27429, 50 Ci/mMol) and [H]CGP62349 (85 Ci/mMol) were obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO).