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NEUROPHARMACOLOGY

N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) and Structurally Related Compounds: Novel Allosteric Enhancers of -Aminobutyric Acid_B Receptor Function

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland

Received April 14, 2003; accepted June 11, 2003.

	Abstract

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N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) and structurally related compounds are described as novel allosteric enhancers of GABA_B receptor function. They potentiate GABA-stimulated guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) binding to membranes from a GABA_B(1b/2)-expressing Chinese hamster ovary cell line at low micromolar concentrations, but do not stimulate [³⁵S]GTPS binding by themselves. Similar effects of GS39783 are seen on native GABA_B receptors in rat brain membranes. Concentration-response curves with GABA in the presence of different fixed concentrations of GS39783 reveal an increase of both the potency and maximal efficacy of GABA at the GABA_B(1b/2) heterodimer. In radioligand binding experiments, GS39783 reduces the kinetic rate constants of the association and dissociation of [³H]3-aminopropylphosphinic acid, resulting in a net increase in affinity for the agonist radioligand. In equilibrium binding experiments (displacement of the antagonist ligand [³H]CGP62349), GS39783 increases agonist affinities. Agonist displacement curves are biphasic, probably reflecting the G protein-coupled and uncoupled states of the receptor. The proportion of the high-affinity component is increased by GS39783, suggesting that the G protein coupling of the receptor is also promoted by the positive modulator. We also show that GS39783 has modulatory effects in cellular assays such as GABA_B receptor-mediated activation of inwardly rectifying potassium channels in Xenopus oocytes and Ca²⁺ signaling in human embryonic kidney 293 cells. In a more physiological context, GS39783 is shown to suppress paired pulse inhibition in rat hippocampal slices. This effect is reversed by the competitive GABA_B receptor antagonist CGP55845A and is produced most likely by enhancing the effect of synaptically released GABA at presynaptic GABA_B receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Chemical structures of GS39783 (center) and some of its analogs.

	Materials and Methods

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Culture and the preparation of membranes of CHO cell clones stably expressing human GABA_B(1b)/rat GABA_B(2) were done as described in detail previously (Urwyler et al., 2001). The preparation of rat brain cortical membranes for native receptor assays was also performed as described previously (Olpe et al., 1990).

[³⁵S]GTPS Assay. The composition of the assay mixtures (in a final volume of 250 µl in 96-well clear-bottom microtiter plates, Isoplates; PerkinElmer Wallac, Gaithersburg, MD) was as follows: 50 mM Tris-HCl buffer, pH 7.7, 10 mM MgCl₂, 0.2 mM EGTA, 2 mM CaCl₂, 100 mM NaCl, 10 µM guanosine 5'-diphosphate (30 µM with rat cortical membranes; Sigma-Aldrich, St. Louis, MO), 50 µl of the membrane suspension (approximately 10–20 µg of protein), 1.5 mg wheat germ agglutinin-coated SPA beads (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK), 0.3 nM [³⁵S]GTPS (ca. 1,000 Ci/mmol, stabilized solution; Amersham Biosciences UK, Ltd.), and the test compounds at the appropriate concentrations. Nonspecific binding was measured in the presence of unlabeled GTPS (10 µM; Sigma Chemical, Buchs, Switzerland). The samples were incubated at room temperature for 60 min, before the SPA beads were sedimented by centrifugation at 2,600 rpm for 10 min. The plates were then counted in a 1450 Microbeta liquid scintillation counter (PerkinElmer Wallac). 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 Inc., San Diego, CA) was used for all data calculations.

Radioligand Binding Experiments. The procedure to measure the binding of [³H]CGP62349 to rat cortical membranes was based on that described by Bittiger et al. (1996); it was, however, conducted in the SPA format. The assay mixture in a final volume of 250 µl contained 20 mM Tris-HCl buffer, pH 7.4, 118 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 5 mM D-glucose, 1 nM [³H]CGP62349, the test compounds at the desired concentrations, rat cortical membranes (ca. 15 µg of protein), and 1.5 mg of wheat germ agglutinin-coated SPA beads (Amersham Biosciences UK, Ltd.). Nonspecific binding was assessed in the presence of 5 µM CGP56999A. The samples were incubated for 90 min at room temperature, before being counted in a 1450 Microbeta liquid scintillation counter.

For kinetic experiments with the agonist radioligand [³H]APPA (Hall et al., 1995), incubation mixtures were made up in a total volume of 25 ml in the same buffer, containing rat brain cortical membranes at the same concentration, as described above for [³H]CGP62349 binding experiments. For association experiments, the samples were preincubated for 15 min at room temperature with or without 30 µM GS39783 before the addition of [³H]APPA (3 nM). Aliquots (1 ml) were then withdrawn from the incubation mixture at different times for vacuum filtration through GF/B filters (Whatman, Maidstone, UK), followed by rapid rinsing with 2 x 5 ml of ice-cold incubation buffer. For dissociation experiments, the mixtures containing 3 nM [³H]APPA (with or without 30 µM GS39783) were preincubated for 40 min at room temperature to reach equilibrium, before L-baclofen (final concentration, 10 µM) was added in 1.5 ml to initiate the dissociation of the radioligand from the receptor. Aliquots (1 ml) were withdrawn from the incubation mixture at different times after the addition of L-baclofen for separating bound and free radioligand by filtration as in the association experiments. In both types of experiments, nonspecific binding was determined in the presence of 10 µM L-baclofen and measured in separate samples.

The results from radioligand binding assays (equilibrium displacement and kinetic experiments) were analyzed using standard procedures and GraphPad Prism software.

Measurement of Change in Intracellular Calcium Concentration by Fluorometry. For the measurement of changes in intracellular calcium concentrations, HEK293 cells were transiently transfected with human GABA_B(1a/2) or GABA_B(1b/2). All transfections included G_qzic to couple GABA_B receptors to PLC (Franek et al., 1999) and were made as described in detail previously (Pagano et al., 2001). Transfected HEK293 cells were plated into poly-D-lysinecoated 96-well plates (BD Biosciences, San Jose, CA). Post-transfection (24–72 h), cells were loaded for 45 min with 2 µM fluo-4 AM (Molecular Probes, Eugene, OR) in Hanks' balanced salt solution (Invitrogen, Basel, Switzerland) containing 20 mM HEPES and 50 µM probenecid (Sigma Chemical). Plates were washed and transferred to a fluorescence imaging plate reader (FLIPR; Molecular Devices, Crawley, UK). GS39783 was added 5 min before recording. Twenty seconds after the start of the readings, 0.3 µM or 3 µM GABA was added to all wells and their fluorescence was measured for 160 s. Relative fluorescence changes over baseline (F/F) were determined. Concentration-response curves were recorded with four 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.

Oocyte Electrophysiology. Experiments were performed as described previously (Lingenhoehl et al., 1999). Ovary lobes containing oocytes were removed surgically from anesthetized (1.2 g/l MS222; Sigma, Buchs, Switzerland) female Xenopus laevis frogs. Oocytes were separated and defolliculated and injected with 10 to 50 ng of cRNA of rat GABA_B(1a) and GABA_B(2), together with inwardly rectifying potassium channel subunits Kir3.1, 3.2, and 3.4 (rat). After incubation at 18°C for 3 to 8 days, two-electrode voltage-clamp recordings were done with a GeneClamp 500 amplifier (Axon Instruments, Union City, CA) using electrodes filled with 3 M KCl. Oocytes were continuously perfused with normal frog Ringer (115 mM NaCl, 10 mM HEPES, 2.5 mM KCl, and 1.8 mM CaCl₂, pH 7.2) or high-potassium Ringer (90 mM KCl, 27.5 mM NaCl, 10 mM HEPES, and 1.8 mM CaCl₂, pH 7.2). Recordings were performed at a clamp potential of –70 mV.

Measurement of Paired Pulse Inhibition. Semisubmerged hippocampal slices were used for extracellular recordings. Male Sprague-Dawley rats, weighing 170 to 180 g, were killed by decapitation under light isoflurane anesthesia. The brains were rapidly removed and immersed in artificial cerebrospinal fluid (124 mM NaCl, 2.5 mM KCl, 1.25 mM KH₂PO₄, 2.5 mM CaCl₂, 2 mM MgSO₄, 26 mM NaHCO₃, and 10 mM glucose; 307 ± 2 mOsM) at room temperature. The hippocampi were dissected and cut transversly into 400-µm slices using a tissue chopper (Sorvall, Newton, CT). Slices were transferred to a rectangular interface chamber and kept at room temperature for 20 min; the temperature was then raised to 32°C. The artificial cerebrospinal fluid level was adjusted to just below the upper surface of the slices and the chamber was continuously supplied with a warmed, humidified 95% O₂, 5% CO₂ mixture. The slices were left to equilibrate for a minimum of 1 h before starting the recordings.

Extracellular population spikes were recorded in the CA1 pyramidal cell layer. Drugs were administered by addition to the superfusion medium at a rate of 1 ml/min and were applied for a sufficient period (10–20 min) to give their full effect before the start of the experiment. The recordings were made using glass microelectrodes (5–7 M) filled with 4 M NaCl. Synaptic responses were evoked by stimulating the Schaffer collateral-commissural fibers in the stratum radiatum (orthodromic stimulation) using a bipolar stimulation electrode (twisted Pt/iridium wire; 25 µm in diameter) and constant current stimuli (1.5–4.0 µA; 100-µs pulse width). GABAergic inhibitory circuits were studied using the paired-pulse inhibition paradigm at a fixed interstimulus interval of 20 ms delivered every 15 s. The amount of paired-pulse inhibition was determined by calculating the ratio of the amplitude of the population spike in response to the second stimulus (test pulse, U_test) to the amplitude of the population spike in response to the first stimulus (conditioning pulse, U_cond). Test and conditioning stimuli were of the same strength and adjusted so that the size of U_cond was 90% maximal under control conditions. Records were captured and averaged (average of four responses) using an oscilloscope (LeCroy 9430; Le Croy SA, Geneva, Switzerland) and plotted on a Hewlett Packard plotter (HP 7475). Slices were accepted for experiments if the evoked maximum orthodromic population spike during the control period was at least 6 mV and free from multiple spikes.

Chemicals. GS39783 and its analogs were synthesized in house (Fischer, 1972, 1974, 1975). Stock solutions were usually prepared in dimethyl sulfoxide and subsequently diluted in the respective assay buffers. The final dimethyl sulfoxide concentrations usually did not exceed 0.3% and did not interfere with the measured parameters. [³H]APPA (40 Ci/mmol) and [³H]CGP62349 (85 Ci/mmol) were from American Radiolabeled Chemicals (St. Louis, MO). The sources of other chemicals used are given above.

	Results

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GS39783 Enhances the Stimulation of [³⁵S]GTPS Binding by Agonists Activating Native and Recombinant GABA_B Receptors. As shown in Fig. 2, GABA stimulated the binding of [³⁵S]GTPS to membranes from CHO cells stably expressing GABA_B(1b/2) receptors by close to a 3-fold of basal activity at a maximally active concentration (100 µM). At 1 µM GABA, a stimulation above baseline corresponding to about 20% of the maximal GABA effect was obtained, which was strongly amplified by GS39783 (1 and 10 µM; Fig. 2). In the presence of 10 µM GS39783, the stimulation of [³⁵S]GTPS binding produced by 1 µM GABA exceeded the effect observed with GABA alone at a saturating concentration (100 µM). Similar amplifications of [³⁵S]GTPS binding were observed when APPA or L-baclofen were used as agonists instead of GABA (Fig. 2). On the other hand, in the absence of an agonist, GS39783 (1 and 10 µM) did not produce any stimulation of [³⁵S]GTPS binding above baseline levels (Fig. 2). Also, in the presence or in the absence of GS39783, no increase above basal levels was observed when the activation produced by GABA (1 µM) was blocked by the competitive GABA_B receptor antagonist CGP56999A (10 µM; Fig. 2). In membranes from CHO cells stably expressing metabotropic glutamate receptor 2 instead of GABA_B receptors, GS39783 did not enhance the stimulation of [³⁵S]GTPS binding produced by glutamate (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 2. Effects of GS39783 and its analogs (Fig. 1) on the stimulation of [35S]GTPS binding via recombinant GABAB(1b/2) receptors. A, potentiation of the effects of low concentrations of the agonists GABA (1 µM), APPA (0.1 µM), and L-baclofen (1 µM) by GS39783, as well as the absence of effect with GS39783 alone or in combination with GABA and the competitive antagonist CGP56999A is visible from the bars labeled accordingly. The basal level and the stimulation obtained with GABA alone at a maximally active concentration (100 µM) are also shown for reference. Horizontal dotted lines indicate baseline values and the degree of stimulation with agonists alone (controls), respectively. The data shown are means ± S.E.M. from a typical experiment performed in triplicates. B, effects of 1 µM GABA in combination with the compounds shown in Fig. 1 are expressed in percentage of the effect of GABA alone at a saturating concentration (100 µM). All compounds were tested at 1 µM (hatched columns) and 10 µM (black columns). The control group represents the stimulation of [35S]GTPS binding (above basal levels) by 1 µM GABA alone.

Figure 2, bottom, also shows similar modulating effects of a number of analogs of GS39783 (Fig. 1) in the [³⁵S]GTPS assay. None of these compounds stimulated [³⁵S]GTPS binding in the absence of GABA (data not shown). Because GS39783 showed the most marked effects of them all, further characterization was done with this compound only.

Figure 3 shows concentration-response curves for the enhancing effect of GS39783 at two different fixed concentrations of GABA (1 and 20 µM). It can be seen that the amplifying effect of GS39783 is observed with both recombinant (top) and native (bottom) GABA_B receptor preparations, with very similar EC₅₀ values in the low micromolar range (Fig. 3; Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Concentration-response curves for the amplification by GS39783 of GABA-stimulated [35S]GTPS binding to membranes from CHO cells expressing GABAB receptors (A) or to rat cortical membranes (B). The amplifying effect of GS39783 was measured at two different fixed concentrations of GABA, 1 µM () and 20 µM (); the corresponding control levels, measured in the presence of GABA alone, are indicated by horizontal lines (, 1 µM GABA; , 20 µM GABA). The upper broken line () represents the level of maximal stimulation obtained by a saturating concentration (100 µM) of GABA alone. The data are means ± S.E.M. from triplicate determinations in a typical experiment. A summary of the results obtained from three independent experiments is given in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Characteristics of the potentiation of the effect of GABA on [35S]GTPS binding by GS39783 Concentration-response curves for GS39783 were measured with recombinant (expressed in CHO cells) or native receptor preparations in the presence of a lower (1 µM) and higher (20 µM) concentration of GABA, as shown in Fig. 3. The data shown are means ± S.E.M. from three independent experiments. Maximal effects are expressed in percentage of the effect produced by a saturating concentration of GABA in the absence of GS39783. Hill coefficients were not significantly different from one in all cases.

GS39783 Enhances Both Agonist Affinity and Efficacy at Recombinant GABA_B(1b/2) Receptors in the [³⁵S]GTPS Assay. Concentration-response curves for GABA at different fixed concentrations of GS39783 reveal that a dual mechanism of GABA_B receptor modulation results in an increase of both the potency (maximally about an 8-fold increase) as well as the maximal intrinsic efficacy (about a 2.2-fold increase) of GABA. These effects are dependent on the concentration of the positive modulator GS39783 (Fig. 4; Table 2). The 8-fold increase in the potency of GABA produced by GS39783 represents the effect of a maximally active concentration of the modulator, compared with a control obtained with GABA alone. On the other hand, it is not possible to measure a potency of GS39783 in the absence of GABA, because the compound has no effect on its own. Therefore, the effects of GS39783 on GABA potency (Table 2) cannot be compared with the reciprocal effects of GABA on the potency of the modulator, obtained at two nonsaturating GABA concentrations (Table 1).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Concentration-response curves for GABA in the [35S]GTPS binding assay in the absence () and in the presence of GS39783 (, 0.3 µM; , 1 µM; , 3 µM; , 10 µM; , 30 µM). GABA responses were measured at recombinant GABAB receptors using membranes from stably transfected CHO cells as described under Materials and Methods. The data shown are means ± S.E.M. from triplicate determinations in a typical experiment. See Table 2 for a summary of the results from several independent experiments.

View this table: [in this window] [in a new window]   TABLE 2 Effects of GS39783 on the potency and efficacy of GABA to stimulate [35S]GTPS binding to membranes from CHO cells expressing recombinant GABAB receptors Concentration-response curves for GABA were measured in membranes from CHO cells expressing recombinant GABAB(1b/2) receptors in the absence and in the presence of different fixed concentrations of GS39783. The data shown are means ± S.E.M. from n independent experiments. A typical experiment with the data expressed in cpm values is shown in Fig. 4.

Effects of GS39783 on Agonist Affinities and Kinetic Rate Constants in Radioligand Binding Assays. To determine the effects of GS39783 on the affinities of different agonists for native GABA_B receptors, binding experiments with [³H]CGP62349 (a competitive antagonist radioligand) using membranes from rat brain cortex were performed. As shown in Fig. 5, displacement curves obtained with GABA, L-baclofen, and APPA revealed enhanced affinities for all three agonists in the presence of 30 µM GS39783. In all cases, a two-site model yielded a significantly better curve fit than a one-site model. As shown in Table 3, the affinities of both the high- and low-affinity components of agonist binding were increased by 30 µM GS39783. (It should be noted that the pK_i value found for the high-affinity component of APPA was considerably higher than expected from that found earlier in saturation experiments (Urwyler et al., 2001). However, this is due to different assay conditions (the earlier experiments were conducted in the presence of 50 µM GDP, which was omitted in the experiments shown here in Table 3 and Fig. 5.) Furthermore, the relative proportion of high affinity sites was slightly, but consistently increased in all experiments in the presence of the modulator. If the percentages of high-affinity site values for all three agonists were pooled (assuming that high- and low-affinity receptor states are the same regardless of the agonist used), the means were 48 ± 0.9% in the absence and 55 ± 1.5% in the presence of 30 µM GS39783 (p < 0.01, n = 12 in each group).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Displacement of [3H]CGP62349 from native GABAB receptors in rat cortical membranes by GABA (squares), L-baclofen (circles), and APPA (triangles) in the absence (open symbols) and in the presence (filled symbols) of 30 µM GS39783. The assay was performed as described under Materials and Methods. The results shown are from a single typical experiment; the data points represent means ± S.E.M. from triplicate determinations. A summary of the relevant parameters from several such experiments is given in Table 3.

In these experiments, the control specific binding of 1 nM [³H]CGP62349 was somewhat lower in the presence of 30 µM GS39783 than in its absence (not shown); however, this inhibition was obviously not of a competitive nature. In fact, saturation experiments with the antagonist radioligand [³H]CGP62349 revealed a slight increase in the K_d value (0.54 nM in the absence and 0.92 nM in the presence of 30 µM GS39783), without any change in the total number of binding sites produced by 30 µM GS39783 (curves not shown). This change in affinity could be calculated to fully account for the lower control binding of the radioligand in the presence of the modulator and was taken into account for the calculation of the K_i values given in Table 3. Thus, it follows that GS39783 does not bind to the orthosteric ligand binding site and does not act by changing the total number of available GABA_B receptors.

The high agonist-affinity state of the GABA_B receptor can be directly labeled by the agonist radioligand [³H]APPA. We made use of this possibility to determine the effects of GS39783 on the rates of association and dissociation of this agonist at native GABA_B receptors (Fig. 6). In association experiments, as expected, the level of binding once equilibrium was reached was higher in the presence of the modulator than in its absence (top, inset), due to the increased affinity for APPA. However, somewhat unexpectedly, the rate of association was slower in the presence of 30 µM GS39783. This effect was overcompensated by an even greater decrease in the rate of dissociation produced by the modulator, the net effect thus resulting in an increased binding affinity of [³H]APPA, as is reflected in the K_d values, which can be calculated from the different rate constants given in Table 4.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Effects of GS39783 on the rates of association (A) and dissociation (B) of [3H]APPA binding to native GABAB receptors in rat cortical membranes. The kinetic experiments were performed in the absence () and in the presence () of 30 µM GS39783 as described under Materials and Methods. Because of the increased affinity of the agonist radioligand, equilibrium binding is higher in the presence of GS39783. To make the effect of GS39783 on the rate of association more visible, the data shown in the top are normalized to the level of equilibrium; the actual cpm values are given in the inset. Because aliquots were drawn for filtration at different times from a single incubation mixture, only single data points are available for each time. The kinetic constants derived from three independent experiments are given in Table 4.

GS39783 Augments GABA_B Receptor-Mediated Calcium Signaling in a Cellular Assay. In HEK293 cells transiently transfected with human GABA_B(1a/2) or GABA_B(1b/2) and cotransfected with G_qzic to couple GABA_B receptors to PLC, GABA elicited a transient calcium signal (Fig. 7, inset). Thus, as observed previously with CGP7930 in Xenopus oocytes (Urwyler et al., 2001), this effect of GS39783 was not dependent on the GABA_B(1) receptor isoform. At a low (0.3 µM) and at a higher (3 µM) concentration of GABA, the signal was potentiated in a concentration-dependent manner by GS39783 (Fig. 7). Like in the [³⁵S]GTPS assay, the signal measured in the presence of GS39783 clearly exceeded the maximal effect produced by GABA on its own. Table 5 gives the corresponding EC₅₀ values and maximal effects of GS39783 under the different conditions used. GS39783, when applied in the absence of GABA, did not produce any calcium signal on its own (Fig. 7).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Positive modulation of GABAB responses by GS39783 in a cellular assay [intracellular calcium signal in HEK293 cells transiently cotransfected with human GABAB(1a/2) and Gqzic]. The graph shows representative concentration-response curves for GS39783 either without GABA (), 0.3 µM GABA (), or 3 µM GABA (). The data points are normalized to the maximal effect of GABA alone and represent averages (± S.E.M.) of the readings from four wells. The lines are Hill equations fitted to the data points. The inset shows fluorescence responses to stimulation with 3 µM GABA alone or to 3 µM GABA in combination with 1, 3, or 10 µM GS39783 (from bottom to top). The calibration bars in the inset correspond to time (horizontal) and fluorescence intensity (FLIPR counts, arbitrary units, vertical), respectively. Similar results were obtained in cells expressing GABAB(1b/2) (Table 5).

GS39783 Enhances the Effects of GABA on Inwardly Rectifying Potassium Channels in Xenopus Oocytes. The effects of GS39783 on the regulation of inwardly rectifying potassium channels via GABA_B receptors in Xenopus oocytes is shown in Fig. 8. Exposure of the oocytes to a high potassium (90 mM) Ringer solution elicited an inward current that was reversibly amplified in the presence of GABA. The effect of a low concentration (0.3 µM, EC₂₀ value) of GABA was increased in the presence of GS39783 (Fig. 8, top). Upon adding 10 µM GS39783 to ascending concentrations of GABA, a left-shift in the GABA concentration-response curve [EC₅₀ = 1 µM (95% confidence interval: 0.58–1.74 µM), compared with 1.63 µM (95% confidence interval: 1.34–1.98 µM) for the control curve] and an increase in the maximal GABA response to 127 ± 5 (S.E.M.) percentage of the control value were observed (Fig. 8, bottom). No intrinsic agonistic activity of GS39783 was seen in oocytes (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 8. Positive modulation of the effects of GABA on inwardly rectifying potassium channels in X. laevis oocytes by GS39783. Top, typical current record of an oocyte expressing GABAB(1a/2) receptors in the presence of high potassium (90 mM) Ringer solution. Control responses to GABA are shown at two different concentrations of GABA [0.3 µM (EC20) and 5.4 µM (EC80), black columns). In the presence of 3 µM GS39783 (gray column), the response to 0.3 µM GABA is enhanced. Bottom, concentration-response curves for GABA in the absence () and in the presence of 10 µM GS39783 (). GS39783 caused a left shift of the GABA concentration-response curve and an increase in the maximal response amplitude. All values were normalized to a 100 µM GABA response (Io) that was measured on every oocyte. The values shown are means ± S.E.M. from at least three different oocytes.

GS39783, Like L-Baclofen, Reduces Paired-Pulse Inhibition in Hippocampal Slices. In a hippocampal slice preparation, the application of two consecutive stimuli to afferent pathways results in inhibition of the second population response. In our experiments, addition of L-baclofen (1 µM) completely suppressed paired pulse inhibition (U_test/U_cond. = 0.36 ± 0.08 for controls and 1.07 ± 0.09 in the presence of baclofen; mean ± S.E.M., n = 8, p < 0.01; recordings not shown). This effect of L-baclofen was reversed in the presence of the competitive antagonist CGP55845A (3 µM; data not shown). A similar reduction of paired pulse inhibition was produced by 10 µM GS39783 (Fig. 9); in fact, the ratio U_test/U_cond. was 0.36 ± 0.03 in the absence and 0.90 ± 0.1 in the presence of 10 µM GS39783 (n = 4; p = 0.01). This effect was also antagonized by 3 µM CGP55845A (Fig. 9).

View larger version (16K): [in this window] [in a new window]   Fig. 9. Effect of the GABAB receptor modulator GS39783 on synaptic inhibition of extracellularly recorded pyramidal cell population spikes and its reversal by the GABAB receptor antagonist CGP55845A. The traces show typical records of pairs of evoked conditioning (Ucond) and test (Utest) potentials at an interstimulus interval of 20 ms. Shown are control responses in artificial cerebrospinal fluid (top trace) and responses under the influence of 10 µM GS39783 alone (middle trace) or in combination with 3 µM CGP55845A (bottom trace). The stimulus strength was identical for all records. Asterisks mark the time of stimulation. The calibration pulse has, as indicated, an amplitude of 5 mV and a duration of 10 ms.

	Discussion

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Only recently, the first allosteric enhancers of GABA_B receptor function, CGP7930 and CGP13501, have been described (Urwyler et al., 2001). Three arylalkylamine derivatives have meanwhile also been reported to enhance GABA_B receptor-mediated responses in rat cortical slices (Kerr et al., 2002). However, in that report molecular mechanisms of action were not addressed. Two of these compounds were found to be totally inactive in our biochemical assays (S. Urwyler, T. Gjoni, K. Kaupmann, M. F. Pozza, and J. Mosbacher, manuscript in preparation). In the present study, we introduce for the first time a novel structure, GS39783 (Fig. 1), with the pharmacological characteristics of an allosteric GABA_B receptor modulator. Some analogs of this compound (Fig. 1) have also been found to amplify GABA_B receptor-mediated responses; however, GS39783 was the most efficacious modulator in this series (Fig. 2).

The actions of GS39783 are very similar to those of the previously described compound CGP7930 (Urwyler et al., 2001) in [³⁵S]GTPS binding and cellular assays (intracellular calcium release, effects on Kir3 channels in Xenopus oocytes). In these experiments, GS39783 had no effect without the concomitant activation of the orthosteric ligand binding site by an agonist, but potentiated the effects of agonists, and the maximal effects of GABA in the presence of the modulator exceeded those of GABA at a saturating concentration alone (Figs. 3, 4, 7, and 8). However, although the potencies of the two compounds (in terms of EC₅₀ values; Tables 1 and 5) are very similar, GS39783 seems to be more efficacious than CGP7930 in enhancing GABA_B receptor-mediated responses. In fact, the maximal stimulation of [³⁵S]GTPS binding by GABA was increased to about 200% of control at maximally active concentrations of GS39783 (Table 2), whereas with CGP7930 only about a 40% increase was obtained (Urwyler et al., 2001). On the other hand, at the highest concentration tested, GS39783 increased the potency of GABA by an 8-fold, whereas a maximally 6-fold increase was obtained previously with CGP7930. Thus, like CGP7930, GS39783 acts through a dual mechanism, by enhancing at the same time the affinity and the maximal efficacy of GABA. This mode of action seems unusual compared with that of previous examples of allosteric modulators at other GPCRs, which affect only agonist potencies but not the maximal responses. However, recently at least two other examples of allosteric enhancers have become known, which increase the affinities as well as the intrinsic efficacies of agonists at glutamate metabotropic glutamate receptor 1 (Knoflach et al., 2001) and adenosine A3 receptors (Gao et al., 2002). This mechanism of allosteric modulation is accounted for by a recently developed theoretical model (Hall, 2000).

The allosteric effects of GS39783 on native and recombinant GABA_B receptors were further corroborated in radioligand binding experiments. The effects of the previously described GABA_B receptor modulator CGP7930 on the displacement of [³H]CGP62349 by GABA in recombinant receptor preparations were found to be complex due to the lack of sensitivity of the overexpressed GABA_B(1) monomer to the allosteric effects of this compound (Urwyler et al., 2001). For this reason, we have chosen to use native receptors for this type of experiment in this study. Agonist displacement curves were biphasic (Fig. 5), presumably due to the presence of GABA_B receptors coupled to and uncoupled from their G proteins. In fact, it is well known that GABA_B receptors, like most GPCRs, have different agonist affinities in these two different states (Hill et al., 1984; Parmentier et al., 2002). Interestingly, the affinities of both receptor forms for agonists were significantly enhanced by GS39783 (Table 3; Fig. 5). Moreover, the relative proportion of the high-affinity state was increased to some extent by GS39783, strongly suggesting that the coupling of the receptor to its G proteins was also promoted by this compound. This mechanism might well contribute to the finding that the maximal efficacy of GABA_B receptor activation was enhanced by the positive modulator.

A reliable and widely used method to demonstrate and quantify allosteric effects is kinetic (nonequilibrium) radioligand binding experiments (Christopoulos and Kenakin, 2002). The rate constants of association and dissociation of an orthosteric ligand are very sensitive to changes in receptor conformation induced by an allosteric ligand. In particular, whereas the rate of association can also be decreased by conventional competitive agents, the dissociation is a zero-order reaction and is most indicative of such a conformational change induced by another agent acting at a distant site. It is the balance of the changes in kinetic association and/or dissociation rate constants that makes up the effects of allosteric modulators on orthosteric ligand affinity at equilibrium. Thus, a positive modulator could enhance agonist affinity by enhancing the rate of association or reducing the rate of dissociation of an agonist, or both. In our experiments, however, GS39783 reduced the rate of association of [³H]APPA to native GABA_B receptors, but this effect was overcompensated by an even greater reduction in its rate of dissociation, resulting in a net increase of affinity for this agonist radio-ligand. It is of interest in this context that, although an increase of association rate could theoretically be the underlying mechanism of action of a positive allosteric modulator, to date no such mechanism seems to have been conclusively shown for any GPCR (Holzgrabe and Mohr, 1998; Christopoulos and Kenakin, 2002).

In a hippocampal slice preparation, the application of two consecutive stimuli to afferent pathways results in inhibition of the second population response, due to the activation of local inhibitory GABAergic interneurons (Alger and Nicoll, 1982). Therefore, paired-pulse inhibition provides a reliable measure of GABA receptor-mediated synaptic inhibitory function. Activation of presynaptic GABA_B receptors leads to a suppression of this paired pulse synaptic inhibition, most likely through inhibition of GABA release from interneurons (Deisz and Prince, 1989; Thompson and Gaehwiler, 1989). Thus, in our experiments, 1 µM L-baclofen completely suppressed paired pulse inhibition; upon coapplication of the competitive GABA_B receptor antagonist CGP55845A (Davies et al., 1993), this effect of baclofen was completely reversed (data not shown). The positive modulator GS39783, on its own, had an effect similar to that of the agonist baclofen (Fig. 9). However, because our other assays have shown that the compound does not directly activate the GABA_B receptor on its own, its suppression of paired pulse inhibition is most likely due to modulation of the effects of endogenous GABA. The finding that the competitive antagonist CGP55845A reversed the effect of GS39783 is in line with this interpretation. In fact, a competitive antagonist would not be expected to inhibit GABA_B receptor activation by a compound acting at a different site than GABA, if any agonistic activity by GS39783 were to be present.

In conclusion, GS39783 has been shown to act as a positive allosteric modulator at native and recombinant GABA_B receptors in different in vitro assay systems. A key question that remains to be answered is certainly that of whether the two structurally different positive modulators CGP7930 and GS39783 exert their effects through the same or through distinct allosteric sites on the GABA_B receptor. Parmentier et al. (2002) have recently suggested that allosteric modulators of family 3 GPCRs act through their seven transmembrane domains; however, in the case of the GABA_B receptor this hypothesis awaits experimental confirmation.

	Acknowledgements

 
We thank Dr. Delphine Dupuis for comments on the manuscript, and R. Brom, J. Heid, M. Horvath, D. Monna, and C. Schöffel for expert technical assistance.

	Footnotes

 
DOI: 10.1124/jpet.103.053074.

ABBREVIATIONS: GPCR, G protein-coupled receptor; GS39783, N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine; CHO, Chinese hamster ovary; [³⁵S]GTPS, guanosine 5'-O-(3-[³⁵S]thio)triphosphate; SPA, scintillation proximity assay; APPA, 3-aminopropylphosphinic acid; HEK, human embryonic kidney; PLC, phospholipase C; FLIPR, fluorescence imaging plate reader; CGP62349, [3-[1-(R)-[[(2S)-2-hydroxy-3-[hydroxyl[4-methoxyphenyl)methyl]phosphinyl]propyl]amino]ethyl]-benzoic acid; CGP55845A, [3-[[1-(S)-(3,4-dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]phenylmethyl-phosphinic acid hydrochloride; MS222, ethyl 3-aminobenzoate methanesulfonate; CGP56999A, [3-[1-(R)-[[3-cyclohexylmethyl)hydroxyphosphinyl]-2-(S)-hydroxypropyl]amino]ethyl]-benzoic acid, monolithium salt.

Address correspondence to: Dr. Stephan Urwyler, Novartis Institutes for BioMedical Research, Novartis Pharma AG, WKL-125.11.03, CH-4002 Basel, Switzerland. E-mail: stephan.urwyler{at}pharma.novartis.com

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