Stephan Urwyler

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

Survival signaling and selective neuroprotection through glutamatergic transmission.

In the brain, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate glutamatergic neurotransmission and, when intensely activated, can induce excitotoxic cell death. In addition to their ionotropic properties, however, AMPA receptors have been functionally coupled to a variety of signal transduction events involving Src-family kinases, G-proteins, and the mitogen-activated protein kinase (MAPK). In the present study, we tested whether AMPA receptors are linked to appropriate signaling events in order to prevent neuronal injury and/or enhance recovery. AMPA stimulation in hippocampal slice cultures caused the selective activation of MAPK through the upstream activator MAPK kinase (MEK). Inhibition of either component of the AMPA receptor--MAPK pathway potentiated cellular damage due to serum deprivation, suggesting that this pathway facilitates compensatory signals in response to injury. Correspondingly, positive modulation of AMPA receptors with the Ampakine 1-(quinoxalin-6-ylcarbonyl)piperidine (CX516) enhanced MAPK activation and reduced the extent of synaptic and neuronal degeneration resulting from excitotoxic episodes. CX516 was neuroprotective when infused into slices either before or after the insult. The Ampakine derivative also elicited neuroprotection in an in vivo model of excitotoxicity as evidenced by reduction in lesion size and preservation of two different types of neurons. Interestingly, the AMPA receptor--MAPK pathway selectively protects against excitotoxicity since enhancing the pathway did not protect against the nonexcitotoxic, slow pathology initiated by lysosomal dysfunction. The results indicate that glutamatergic communication is important for cellular maintenance and that AMPA receptors activate survival signals to counterpoise their own excitotoxic potential.

Receptor activation involving positive allosteric modulation, unlike full agonism, does not result in GABAB receptor desensitization.

Allosteric modulators act more physiologically than orthosteric ligands, targeting only endogenously activated receptors and not their whole population, which is why they are expected to produce less side effects and tolerance. To inspect the role of the positive allosteric modulator GS39783 in GABAB receptor desensitization, we examined receptor function and cell surface expression in a recombinant GABAB cell line and in primary neuronal cultures upon persistent treatments with GABAB agonists, and combinations of agonists and GS39783. The potency of GABA to inhibit 7beta-forskolin-induced cAMP formation in recombinant cells decreased after the exposure to a saturating GABA concentration, but not after a combination of a low GABA concentration and GS39783, that activated the receptor to the same extent. Concordantly, a significant decrease of cell surface receptors was found after GABA-induced desensitization, unlike after the combined treatment with GABA and GS39783. Similar observations regarding receptor function were found in primary neurons for baclofen-induced inhibition of spontaneous Ca2+ oscillations. However, the cell surface receptor density remained unaffected upon baclofen-induced desensitization in the primary neurons, possibly due to different mechanisms of desensitization in the neurons and the recombinant cell line. These findings indicate that the degree of occupancy of the orthosteric site determines desensitization rather than the degree of receptor activation. In summary, our results conform to predictions that positive allosteric modulators have less propensity for the development of tolerance due to receptor desensitization than classical agonists.

Roles of GABAB receptor subtypes in presynaptic auto- and heteroreceptor function regulating GABA and glutamate release

AbstractΓ-Aminobutyric acid B (GABAB) receptors are heterodimers composed of two subunits GABAB(1) and GABAB(2), the former existing in two isoforms GABAB(1a) and GABAB(1b). The contributions of individual receptor subunits and isoforms to GABAB auto- and heteroreceptor functions were investigated, using release experiments in cortical slice preparations from corresponding knockout mice. Presynaptic GABAB autoreceptors are located on GABAergic terminals and inhibit GABA release, whereas presynaptic GABAB heteroreceptors control the release of other neurotransmitters (e.g. glutamate). Neither baclofen nor the selective antagonist CGP55845 at maximally active concentrations affected [3H]GABA release in slices from GABAB(1)−/− mice. The amount of [3H]GABA released per pulse was unaffected by the stimulation frequency in slices from GABAB(1)−/− and GABAB(2)−/− demonstrating a loss of GABAB autoreceptor function in these knockout animals. The GABAB receptor agonist baclofen was ineffective in modulating glutamate release in cortical slices from GABAB(2)−/− mice, showing that heteroreceptor function was abolished as well. Next we investigated knockout mice for the two predominant GABAB(1) isoforms expressed in brain, GABAB(1a) and GABAB(1b). In cortical, hippocampal and striatal slices from both GABAB(1a)−/− and GABAB(1b)−/− mice, the frequency dependence of [3H]GABA released per pulse was maintained, suggesting that both isoforms participate or can substitute for each other in GABAB autoreceptor function. By contrast, the efficacy of baclofen to inhibit glutamate release was substantially reduced in GABAB(1a)−/−, but essentially unaltered in GABAB(1b)−/− mice. Our data suggest that functional GABAB heteroreceptors regulating glutamate release are predominantly, but not exclusively composed of GABAB(1a) and GABAB(2) subunits.

Studies on the subcellular localization of monoamine oxidase types A and B and its importance for the deamination of dopamine in the rat brain.

Abstract The distribution of the MAO-forms A and B between intra- and extrasynaptosomal rat brain mitochondria was studied with the aid of their known substrate and inhibitor specificities. The activities with the selective substrates serotonin, PEA and benzylamine indicated that intrasynaptosomal mitochondria have about a 3.4-fold higher MAO A:MAO B ratio than extrasynaptosomal mitochondria. However, PEA was found to be a selective substrate for MAO B only at low concentrations (such as 5 × 10 −6 M), whereas at higher concentrations (such as 10 −3 M) it was a substrate for both forms of MAO. The different ratios of the two enzyme forms in the two mitochondrial populations were confirmed when the selective inhibitors clorgyline and deprenyl were used with dopamine or 10 −3 M PEA. With these two amines, the ratios of MAO A: MAO B activities were 3–4.5 times higher in intrasynaptosomal than in extrasynaptosomal mitochondria. In particular, when the activity with dopamine was measured in intact synaptosomes, deamination being preceded by a specific uptake into these particles, the inhibitor sensitivities clearly showed that MAO activity was almost exclusively attributable to the A-form of the enzyme. Thus, mitochondria in the terminals of dopaminergic neurones have an even more pronounced enrichment in MAO A than the mitochondria obtained by osmotic lysis of a total brain synaptosomal preparation. It was also found that clorgyline and deprenyl have an inhibitory effect on the uptake of dopamine into nerve endings with IC 50 values in the range of 10 −5 to 10 −4 M. These results are discussed in terms of possible physiological significancies of the properties and distribution of the two forms of MAO.

Discovery of NMDA Glycine Site Inhibitors from the Chemical Universe Database GDB

Drug development faces the ever-increasing challenge of discovering new bioactive small molecules that have not already been investigated in the more than 150-year history of medicinal chemistry. [1] This problem is particularly critical for drug targets that require very small organic ligands, for which the range of potential structures is limited. One possible way to overcome this difficulty would be to search through the entire chemical space of these small molecules using virtual screening tools and to identify promising ligands for synthesis and testing. Herein we report the first example of such an approach for ligands of the N-methyl-d-aspartic acid (NMDA) receptor glycine site. This receptor is an important drug target implicated in synaptic plasticity, neuronal development, learning, and memory. Inhibiting the NMDA receptor may help prevent neuronal cell death caused by glutamate excitotoxicity in acute and chronic neurodegenerative disorders such as stroke, epilepsy, Huntington’s, and Alzheimer’s disease. [2] Starting with our recently reported chemical universe database (GDB) that lists all compounds of C, N, O, and F up to 11 atoms obeying simple stability and synthetic feasibility rules, [3] we show that virtual screening followed by synthesis and testing leads to several new NMDA glycine site ligands. Both ligand binding assays and functional investigations reveal that the identified ligands interact with the glycine binding site of the NDMA receptors, inhibiting receptor function by direct competition with glycine. The crystal structure of the NMDA receptor glycine site was recently reported. [4] Glycine is bound through a series of hydrogen bonds at the bottom of a narrow channel, leaving limited free space. The known NMDA glycine site ligands are indeed very small analogues of glycine, such as d-alanine and dserine, [5] d-cycloserine, [6] and small cyclic amino acids, [7] sug

The positive allosteric modulator GS39783 enhances GABAB receptor-mediated inhibition of cyclic AMP formation in rat striatum in vivo

We studied the effects of the positive allosteric modulator GS39783 on GABAB receptors at a biochemical level in vivo. Changes in extracellular levels of cyclic AMP following GABAB receptor activation were monitored in the striatum of freely moving rats using microdialysis. Locally applied GABAB agonist R(–)‐baclofen inhibited cyclic AMP formation stimulated by a water‐soluble forskolin analogue in a concentration‐dependent manner (EC50 7.3 µm, maximal inhibition 40%). The selective GABAB antagonist CGP56999 reversed R(–)‐baclofen‐induced cyclic AMP inhibition to control levels, but not higher. Orally applied GS39783 lacked effects on its own but, together with a threshold concentration of R(–)‐baclofen (1 µm), significantly decreased cyclic AMP formation in a dose‐dependent fashion. Effects of GS39783 were revoked with CGP56999, showing dependence on GABAB receptor activation and suggesting allosteric modulation as a mechanism of action in vivo. Administered with a maximally active dose of R(–)‐baclofen, GS39783 failed to further inhibit cyclic AMP formation. The data obtained with CGP56999 and the lack of effect of GS39783 alone suggest that there is no detectable endogenous activation of GABAB receptors controlling cyclic AMP formation in rat striatum. To our knowledge, these results provide the first biochemical demonstration of in vivo activity of a G protein‐coupled receptor‐positive allosteric modulator.

Quinazolinedione sulfonamides: A novel class of competitive AMPA receptor antagonists with oral activity

Quinazoline-2,4-diones with a sulfonamide group attached to the N(3) ring atom constitute a novel class of competitive AMPA receptor antagonists. One of the synthesized compounds, 28, shows nanomolar receptor affinity, whereas other examples of the series display oral anticonvulsant activity in animal models.

Novel N-methyl-d-aspartate receptor antagonists: a review of compounds patented since 2006

Importance of the field: The NMDA receptor is a complex ligand gated, voltage-dependent ion channel. It has been a drug target for > 25 years for neurological and psychiatric indications. Whereas the initial optimism to turn preclinically active compounds rapidly into drugs for human use was dampened, new insights into cellular receptor localization, role of subunits and receptor operation have kept the interest alive to modulate this receptor for therapeutic intervention. Areas covered in this review: The article describes the NMDA receptor antagonists patented since 2006. Also included are novel NMDA receptor ligands potentially useful for positron emission tomography imaging. What the reader will gain: The first section summarizes the current status of NMDA receptor pharmacology. This serves as a base for the next sections discussing the patented compounds with respect to their mode of action, potency and, in some cases, drugability. Take home message: The most important recent strategies aiming for inhibition of NMDA receptor-mediated neurotransmission avoid for safety reasons full receptor blockade but allow a low degree of normal receptor function. Approaches pursued by the latest patents comprise blocking the channel with compounds of low affinity, antagonizing receptor activity by highly potent NR2B ligands, partial agonism at the glutamate or glycine-binding site and improvement of pharmacokinetic properties of well established, safe antagonists by deuteration.

Identification of Dopamine “D3′’ and “D4′’ Binding Sites, Labelled with [3H]2-Amino-6,7-Dihydroxy- 1,2,3,4- Tetrahydronaphthalene, as High Agonist Affinity States of the D1 and D2 Dopamine Receptors, Respectively

Abstract: On the basis of affinity differences for spiperone, two binding sites for [3H](±)‐2‐amino‐6,7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene ([3H]ADTN) in the rat brain could be distinguished: “D3′’ with a low and “D4′’ with a high affinity for spiperone. Evidence is provided that D3 and D4 sites are related to high agonist affinity states of the D1 and D2 dopamine receptors, respectively. Various well‐known selective D1 and D2 agonists and antagonists showed potencies at these sites in agreement with this hypothesis. A comparison of the Bmax values for [3H]ADTN binding to D3 and D4 sites with the numbers of D1 receptors (labelled by [3H]SCH 23390) and of D2 receptors (labelled by [3H]spiperone), both in the striatum and in the mesolimbic system, indicated that under the conditions used for 3H‐agonist binding experiments, both populations of D1 and D2 receptors were converted to their high agonist affinity states to a considerable, although different extent. In fact, when competition experiments with [3H]spiperone were performed under the conditions otherwise used for [3H]ADTN binding experiments (instead of the conditions usually used for antagonist binding), substantial shifts of the displacement curves of 3,4‐dihydroxyphenylethylamine (dopamine) and ADTN toward higher affinities were observed. A comparison of the effects of various agonists and antagonists in the [3H]ADTN binding experiments and in functional tests revealed a significant correlation between their potencies at D4 binding sites and at D2 receptors modulating the release of [3H]acetylcholine from striatal slices. However, in the situation of the D1/D3 pair, when the measurement of adenylate cyclase activity was taken as a functional test for D1 receptors, agonists were more active in the binding than in the functional test, whereas for many antagonists the opposite was found. The results are discussed with regard to the classification and functional aspects of brain dopamine receptors.