From NMDA receptor antagonists to discovery of selective σ2 receptor ligands

Following previous studies focused on the search for new molecules targeting GluN2B-containing NMDA, a small series of 1-(1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone derivatives has been synthesized by using Microwave Assisted Organic Synthesis (MAOS). Given that GluN2B ligands frequently exert off-target effects we also tested their affinity towards sigma receptors. Binding assay revealed that only the 1-(5-hydroxy-1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone (7a) retained GluN2B affinity. Interestingly, the 5-methoxyindoles 5a and 6a were efficient and selective ligands toward σ₂ receptor (K_i values of 10 nM and 20 nM, respectively). Thus, in this case the discovery of new σ₂ receptor selective ligands was an unexpected result emerging from the screening of cross-activity against other CNS receptors.     

Activation of NMDA receptor partly involved in beta-bungarotoxin-induced neurotoxicity in cultured primary neurons

In this study, we demonstrated that a snake presynaptic toxin, beta-bungarotoxin (beta-BuTX), was capable of binding to NMDA receptors of the cultured primary neurons (cerebellar granule neurons, CGNs). We labeled beta-BuTX with fluorescent FITC (FITC-beta-BuTX) and showed that the binding of FITC-beta-BuTX was inhibited by unlabeled beta-BuTX and MK801 (an NMDA receptor antagonist). Meanwhile, the binding of [3H]-MK801 was also reduced by unlabeled MK801 and beta-BuTX. In addition, beta-BuTX produced a very potent neurotoxic effect on mature CGNs with the EC(50) of 3ng/ml (equivalent to 144pM), but was less effective in immature CGNs. We explored the signaling pathway of neuronal death and found that it was apparently due to the excessive production of reactive oxygen species (ROS) induced by beta-BuTX. MK801 and antioxidants (Vitamin C, N-acetylcysteine (NAC), melatonin, epigallocatechin gallate (EGCG), superoxide dismutase (SOD) and catalase) attenuated not only ROS production but also beta-BuTX-neurotoxicity. The downstream signaling of ROS was identified as the activation of caspase-3. Caspase inhibitor (z-DEVD-fmk) and antioxidants depressed both caspase-3 activation and neurotoxicity. Based on these findings and our previous reports, we conclude that the binding and activation of NMDA receptors by beta-BuTX was crucial step to produce the potent neurotoxic effect. The binding of NMDA receptors resulted in excessive Ca(2+) influx, followed by ROS production and activation of caspase-3. This snake toxin is considered not only to be a useful tool for exploring the death-signaling pathway of neurotoxicity, but also provides a model for searching neuroprotective agents.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

A novel series of benzimidazole NR2B-selective NMDA receptor antagonists

A series of novel benzimidazoles are discussed as NR2B-selective N-methyl-d-aspartate (NMDA) receptor antagonists. High throughput screening (HTS) efforts identified a number of potent and selective NR2B antagonists such as 1. Exploration of the substituents around the core of this template identified a number of compounds with high potency for NR2B (pIC(50) >7) and good selectivity against the NR2A subunit (pIC(50) <4.3) as defined by FLIPR-Ca(2+) and radioligand binding studies. These agents offer potential for the development of therapeutics for a range of nervous system disorders including chronic pain, neurodegeneration, migraine and major depression.     

In vitro characterization of novel NR2B selective NMDA receptor antagonists

N-Methyl-D-aspartate (NMDA) subunit specific receptor antagonism has potential therapeutic application for multiple CNS pathologies. MERCK 1, MERCK 2, and MERCK 3 are novel NR2B subtype selective NMDA receptor antagonists. The affinity and the kinetic mechanism of inhibition by these antagonists and ifenprodil were investigated using the whole-cell configuration of the patch clamp technique, calcium flux, and radioligand binding on a mouse cell line L(tk-) expressing recombinant human heteromeric NMDA receptors consisting of NR1a/NR2B subunit combinations. The rank order of potency, as determined by electrophysiology, was ifenprodil相似文献   

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the 'developmental switch' of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol-treated cells than in control cells. Cross-linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell-surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol-treated cells, as determined by NMDA/glycine-induced increases in intracellular Ca(2+). The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.     

Glycine-insensitive desensitization of NMDA responses in cultured mouse embryonic neurons

The influence of glycine on the desensitization of NMDA-induced currents was studied using cultured embryonic mouse neurons. Although glycine often appeared to reduce desensitization in the whole-cell mode, it had no effect on desensitization in outside-out patches. Various interpretations can be proposed for this discrepancy, such as the presence in intact cells of an intracellular factor regulating desensitization, or the masking of desensitization in intact cells by restricted diffusion of the agonist in the extracellular space. The fact that glycine potentiates the NMDA responses under conditions where it does not regulate desensitization indicates that the potentiation cannot be explained by a reduction of desensitization.     

Differentiated human NT2-N neurons possess a high intracellular content of myo-inositol

myo-Inositol plays a key role in signal transduction and osmotic regulation events in the CNS. Despite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol homeostasis was studied in NT2-N cells, a unique cell culture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2-N neurons by means of retinoic acid treatment resulted in an increase in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2-N cells, respectively. The high concentrations of inositol in NT2-N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2-N and NT2 cells, respectively) and (2) a decreased efflux of inositol (1.7%/h for NT2-N neurons vs. 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The observation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS.     

Discovery of N-aryl-2-acylindole human glucagon receptor antagonists

A novel class of N-aryl-2-acylindole human glucagon receptor (hGCGR) antagonists is reported. These compounds demonstrate good pharmacokinetic profiles in multiple preclinical species. One compound from this series, indole 33, is orally active in a transgenic murine pharmacodynamic model. Furthermore, a 1mg/kg oral dose of indole 33 lowers ambient glucose levels in an ob/ob/hGCGR transgenic murine diabetes model. This compound was deemed suitable for preclinical safety studies and was found to be well tolerated in an 8-day experimental rodent tolerability study. The combination of preclinical efficacy and safety observed with compound 33 highlights the potential of this class as a treatment for type 2 diabetes.     

Corticotropin-releasing factor requires CRF binding protein to potentiate NMDA receptors via CRF receptor 2 in dopamine neurons

Stress increases addictive behaviors and is a common cause of relapse. Corticotropin-releasing factor (CRF) plays a key role in the modulation of drug taking by stress. However, the mechanism by which CRF modulates neuronal activity in circuits involved in drug addiction is poorly understood. Here we show that CRF induces a potentiation of NMDAR (N-methyl-D-aspartate receptor)-mediated synaptic transmission in dopamine neurons of the ventral tegmental area (VTA). This effect involves CRF receptor 2 (CRF-R2) and activation of the phospholipase C (PLC)-protein kinase C (PKC) pathway. We also find that this potentiation requires CRF binding protein (CRF-BP). Accordingly, CRF-like peptides, which do not bind the CRF-BP with high affinity, do not potentiate NMDARs. These results provide evidence of the first specific roles for CRF-R2 and CRF-BP in the modulation of neuronal activity and suggest that NMDARs in the VTA may be a target for both drugs of abuse and stress.     

Differential effect of NR2A and NR2B subunit selective NMDA receptor antagonists on striato-pallidal neurons: relationship to motor response in the 6-hydroxydopamine model of parkinsonism

We previously demonstrated that NMDA receptors containing the NR2A or NR2B subunits differentially regulate striatal output pathways. We now investigate whether such a differential control is altered under parkinsonian conditions and whether subunit selective antagonists have different abilities to attenuate parkinsonian-like motor deficits. Three microdialysis probes were simultaneously implanted in the dopamine-depleted striatum, globus pallidus and substantia nigra reticulata of 6-hydroxydopamine hemilesioned rats. The NR2A antagonist NVP-AAM077 perfused in the striatum reduced pallidal GABA, but not glutamate, levels whereas the NR2B antagonist Ro 25-6981 was ineffective. Neither antagonist affected striatal or nigral amino acid levels. To investigate whether these neurochemical responses were predictive of different antiparkinsonian activities, antagonists were administered systemically and motor activity evaluated in different motor tasks. Neither antagonist attenuated akinesia/bradykinesia in the bar and drag test. However, NVP-AAM077 dually modulated rotarod performance (low doses being facilitatory and higher ones inhibitory) while Ro 25-6981 monotonically improved it. Microdialysis revealed that motor facilitating doses reduced pallidal GABA levels while motor inhibiting doses increased them. We conclude that, under parkinsonian conditions, the striato-pallidal pathway is driven by striatal NR2A subunits. Motor improvement induced by NVP-AAM077 and Ro 25-6981 is accomplished by blockade of striatal NR2A and extrastriatal NR2B subunits, respectively.     

Store Ca2+ depletion enhances NMDA responses in cultured human astrocytes.

NMDA produced whole-cell membrane currents in cultured human astrocytes. The currents were not inhibited by the selective NMDA receptor antagonist, APV, while they were partially inhibited by the broad G-protein inhibitor, GDPbetaS. NMDA-induced currents were enhanced by either the microsomal Ca2+/ATPase inhibitors, thapsigargin and cyclopiazonic acid, or the ATP-uncoupler, dinitrophenol (DNP). In the Ca2+ assay, NMDA increased intracellular calcium concentration. The increase was inhibited by 26% in Ca2+-free extracellular solution, and it was not inhibited by APV. The results of the present study suggest that NMDA responses in human astrocytes are regulated by store Ca2+ depletion-associated signal.     

Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture

The well-characterized human teratocarcinoma line Ntera2 (NT2) can be differentiated into mature neurons. We have significantly shortened the time-consuming process for generating postmitotic neurons to approximately 4 weeks by introducing a differentiation protocol for free-floating cell aggregates and a subsequent purification step. Here, we characterize the neurochemical phenotypes of the neurons derived from this cell aggregate method. During differentiation, the NT2 cells lose immunoreactivity for vimentin and nestin filaments, which are characteristic for the immature state of neuronal precursors. Instead, they acquire typical neuronal markers such as β-tubulin type III, microtubule-associated protein 2, and phosphorylated tau, but no astrocyte markers such as glial fibrillary acidic protein. They grow neural processes that express punctate immunoreactivity for synapsin and synaptotagmin suggesting the formation of presynaptic structures. Despite their common clonal origin, neurons cultured for 2–4 weeks in vitro comprise a heterogeneous population expressing several neurotransmitter phenotypes. Approximately 40% of the neurons display glutamatergic markers. A minority of neurons is immunoreactive for serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase. We have found no evidence for a dopaminergic phenotype. Subgroups of NT2 neurons respond to the application of nitric oxide donors with the synthesis of cGMP. A major subset shows immunoreactivity to the cholinergic markers choline acetyl-transferase, vesicular acetylcholine transporter, and the non-phosphorylated form of neurofilament H, all indicative of motor neurons. The NT2 system may thus be well suited for research related to motor neuron diseases. G. Podrygajlo is a Marie Curie Actions Fellow and M.A. Tegenge received a Georg Christoph Lichtenberg Fellowship from the Federal State of Lower Saxony. M. Stern and G. Bicker were supported by the Federal Ministry for Education and Research (BMBF). F. Paquet-Durand was supported by grants from the Kerstan Foundation, Tübingen, Germany and the German Research Council (DFG; PA 1751/1-1).     

The effects of NMDA receptor antagonists on acute morphine antinociception in mice

Summary.  Antagonists of the N-methyl-d-aspartate (NMDA) receptor complex inhibit the development of tolerance to antinociceptive effects of morphine and upon acute administration, influence morphine antinociceptive activity. The analysis of numerous studies investigating acute interaction between NMDA receptor antagonists and morphine in mice indicate a variety of procedural differences and reveal that these compounds may potentiate, attenuate and produce no effect on morphine antinociception. The conditions responsible for such conflicting experimental outcome of acute interaction remain unclear. It appears that the effects of NMDA receptor antagonists on morphine tolerance are not causally related to their acute effects on morphine antinociception. Received July 6, 2001 Accepted August 6, 2001 Published online August 9, 2002     

Development of 3-substituted-1H-indole derivatives as NR2B/NMDA receptor antagonists

A combined ligand-based and structure-based approach has previously allowed us to identify NR2B/NMDA receptor antagonists containing indole scaffold. In order to further explore the main structure activity relationships of this class of derivatives we herein report the design, synthesis and biological evaluation of new analogues. Some derivatives demonstrated to produce significant anticonvulsant properties and NMDA antagonism. The most active of them (3d) showed NR2B binding affinity equipotent to that of ifenprodil. These results were also corroborated by computational studies.     

Neuroligins/LRRTMs prevent activity- and Ca2+/calmodulin-dependent synapse elimination in cultured neurons

Neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs) are postsynaptic cell adhesion molecules that bind to presynaptic neurexins. In this paper, we show that short hairpin ribonucleic acid-mediated knockdowns (KDs) of LRRTM1, LRRTM2, and/or NL-3, alone or together as double or triple KDs (TKDs) in cultured hippocampal neurons, did not decrease synapse numbers. In neurons cultured from NL-1 knockout mice, however, TKD of LRRTMs and NL-3 induced an ～40% loss of excitatory but not inhibitory synapses. Strikingly, synapse loss triggered by the LRRTM/NL deficiency was abrogated by chronic blockade of synaptic activity as well as by chronic inhibition of Ca(2+) influx or Ca(2+)/calmodulin (CaM) kinases. Furthermore, postsynaptic KD of CaM prevented synapse loss in a cell-autonomous manner, an effect that was reversed by CaM rescue. Our results suggest that two neurexin ligands, LRRTMs and NLs, act redundantly to maintain excitatory synapses and that synapse elimination caused by the absence of NLs and LRRTMs is promoted by synaptic activity and mediated by a postsynaptic Ca(2+)/CaM-dependent signaling pathway.     

Molecular electrostatic potentials in aromatic substituted 4-hydroxyquino-2-lones: Glycine/NMDA receptor antagonists

Hydroxyquinolone derivatives have proven to be useful for inhibition at the glycine binding site of N-methyl-D-aspartate (NMDA) receptor. In this work the electronic structure, molecular electrostatic potential (MESP) and vibrational characteristics of a set of C₃ substituted 4-hydroxyquino-2-lone (HQ) derivatives, which act as Glycine/NMDA receptor antagonists, have been investigated using the density functional calculations. In the optimized structures a substituent at the C₃ site of HQ tends to adopt a helical structure. MESP investigations reveal that the ligands showing better inhibition activity should possess electron-rich regions extending over the substituent and carbonyl group of HQ. A correlation of inhibitory activity to the molecular electrostatic potential topography at the carbonyl oxygen as well as to the molecular electron density topography turns out to be a significant output of the investigation. Figure Quantam chemical approach has been employed to understand the reactivity of a set of hydroxyquinolone derivatives known for their inhibition activity towards Glycine/NMDA receptor. Molecular electrostatic potential topography has been used as a tool to understand the reactivity pattern