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

N,N -Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6diamine (GS39783) and structurally related compounds are described as novel allosteric enhancers of GABAB receptor function. They potentiate GABA-stimulated guanosine 5 -O-(3-[S]thio)triphosphate ([S]GTP S) binding to membranes from a GABAB(1b/2)-expressing Chinese hamster ovary cell line at low micromolar concentrations, but do not stimulate [S]GTP S binding by themselves. Similar effects of GS39783 are seen on native GABAB receptors in rat brain membranes. Concentrationresponse 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 GABAB(1b/2) heterodimer. In radioligand binding experiments, GS39783 reduces the kinetic rate constants of the association and dissociation of [H]3aminopropylphosphinic acid, resulting in a net increase in affinity for the agonist radioligand. In equilibrium binding experiments (displacement of the antagonist ligand [H]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 GABAB receptor-mediated activation of inwardly rectifying potassium channels in Xenopus oocytes and Ca 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 GABAB receptor antagonist CGP55845A and is produced most likely by enhancing the effect of synaptically released GABA at presynaptic GABAB receptors. The receptors for the major inhibitory neurotransmitter in the central nervous system, GABA, are subdivided into ionotropic GABAA receptors and metabotropic GABAB receptors. Whereas GABAA receptors form a chloride-permeable ion channel that elicits short-lasting inhibitory postsynaptic potentials, GABAB receptors are G-protein coupled receptors (GPCRs) that inhibit cyclic AMP formation and modulate the activity of inwardly rectifying potassium channels and voltage-sensitive calcium channels. By these mechanisms, they act postand presynaptically to inhibit neuronal excitability (by producing a late, long-lasting component of inhibitory postsynaptic potentials) and neurotransmitter release, respectively. Whereas benzodiazepines are well established positive allosteric modulators of GABAA receptor function, the first examples of such allosteric enhancers for GABAB receptors have only recently been described (Urwyler et al., 2001). As a therapeutic principle, positive modulators are expected to have several advantages over compounds acting as agonists, because they are only effective in the presence of the endogenous ligand and therefore act in line with physiological neurotransmission in its temporal and spatial organization. Agonists, on the other hand, activate receptors independently of synaptic activity, possibly leading to unwanted side effects. For these reasons, the search for posArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. 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; [S]GTP S, guanosine 5 -O-(3-[S]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-hydroxy3-[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)-[[3cyclohexylmethyl)hydroxyphosphinyl]-2-(S)-hydroxypropyl]amino]ethyl]-benzoic acid, monolithium salt. 0022-3565/03/3071-322–330$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 307, No. 1 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 53074/1090867 JPET 307:322–330, 2003 Printed in U.S.A. 322 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from itive modulators not only for ionotropic receptors but also for GPCRs has recently attracted considerable interest (Pin et al., 2001; Christopoulos, 2002; Christopoulos and Kenakin, 2002). In this study, we present a novel class of allosteric enhancers of GABAB receptors, GS39783 and some of its analogs (Fig. 1). Furthermore, we also provide some new insights into the molecular and physiological mechanisms of allosteric GABAB receptor modulation. Materials and Methods Culture and the preparation of membranes of CHO cell clones stably expressing human GABAB(1b)/rat GABAB(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). [S]GTP S Assay. The composition of the assay mixtures (in a final volume of 250 l 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 M guanosine 5 -diphosphate (30 M with rat cortical membranes; Sigma-Aldrich, St. Louis, MO), 50 l of the membrane suspension (approximately 10–20 g of protein), 1.5 mg wheat germ agglutinin-coated SPA beads (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK), 0.3 nM [S]GTP S (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 GTP S (10 M; 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 [H]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 l 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 Dglucose, 1 nM [H]CGP62349, the test compounds at the desired concentrations, rat cortical membranes (ca. 15 g 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 M CGP56999A. The samples were incubated for 90 min at room temperature, before being counted in a 1450 Microbeta liquid scin-