Pari Malherbe

Fenobam: A Clinically Validated Nonbenzodiazepine Anxiolytic Is a Potent, Selective, and Noncompetitive mGlu5 Receptor Antagonist with Inverse Agonist Activity

Fenobam [N-(3-chlorophenyl)-N′-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] is an atypical anxiolytic agent with unknown molecular target that has previously been demonstrated both in rodents and human to exert anxiolytic activity. Here, we report that fenobam is a selective and potent metabotropic glutamate (mGlu)5 receptor antagonist acting at an allosteric modulatory site shared with 2-methyl-6-phenylethynyl-pyridine (MPEP), the protypical selective mGlu5 receptor antagonist. Fenobam inhibited quisqualate-evoked intracellular calcium response mediated by human mGlu5 receptor with IC50 = 58 ± 2 nM. It acted in a noncompetitive manner, similar to MPEP and demonstrated inverse agonist properties, blocking 66% of the mGlu5 receptor basal activity (in an over expressed cell line) with an IC50 = 84 ± 13 nM. [3H]Fenobam bound to rat and human recombinant receptors with Kd values of 54 ± 6 and 31 ± 4 nM, respectively. MPEP inhibited [3H]fenobam binding to human mGlu5 receptors with a Ki value of 6.7 ± 0.7 nM, indicating a common binding site shared by both allosteric antagonists. Fenobam exhibits anxiolytic activity in the stress-induced hyperthermia model, Vogel conflict test, Geller-Seifter conflict test, and conditioned emotional response with a minimum effective dose of 10 to 30 mg/kg p.o. Furthermore, fenobam is devoid of GABAergic activity, confirming previous reports that fenobam acts by a mechanism distinct from benzodiazepines. The non-GABAergic activity of fenobam, coupled with its robust anxiolytic activity and reported efficacy in human in a double blind placebo-controlled trial, supports the potential of developing mGlu5 receptor antagonists with an improved therapeutic window over benzodiazepines as novel anxiolytic agents.

Positive allosteric modulators of metabotropic glutamate 1 receptor: characterization, mechanism of action, and binding site.

We have identified two chemical series of compounds acting as selective positive allosteric modulators (enhancers) of native and recombinant metabotropic glutamate 1 (mGlu1) receptors. These compounds did not directly activate mGlu1 receptors but markedly potentiated agonist-stimulated responses, increasing potency and maximum efficacy. Binding of these compounds increased the affinity of a radiolabeled glutamate-site agonist at its extracellular N-terminal binding site. Chimeric and mutated receptors were used to localize amino acids in the receptor transmembrane region critical for these enhancing properties. Finally, the compounds potentiated synaptically evoked mGlu1 receptor responses in rat brain slices. The discovery of selective positive allosteric modulators of mGlu1 receptors opens up the possibility to develop a similar class of compounds for other family 3 G protein-coupled receptors.

Molecular cloning and functional characterization of human vesicular glutamate transporter 3

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. It is loaded into synaptic vesicles by a proton gradient‐dependent uptake system and is released by exocytosis upon stimulation. Recently, two mammalian isoforms of a vesicular glutamate transporter, VGLUT1 and VGLUT2, have been identified, the expression of which enables quantal release of glutamate from glutamatergic neurons. Here, we report a novel isoform of a human vesicular glutamate transporter (hVGLUT3). The predicted amino acid sequence of hVGLUT3 shows 72% identity to both hVGLUT1 and hVGLUT2. hVGLUT3 functions as a vesicular glutamate transporter with similar properties to the other isoforms when it is heterologously expressed in a neuroendocrine cell line. Although mammalian VGLUT1 and VGLUT2 exhibit a complementary expression pattern covering all glutamatergic pathways in the CNS, expression of hVGLUT3 overlaps with them in some brain areas, suggesting molecular diversity that may account for physiological heterogeneity in glutamatergic synapses.

Functional characteristics and sites of gene expression of the alpha 1, beta 1, gamma 2-isoform of the rat GABAA receptor

GABAA receptors, the major synaptic targets for the neurotransmitter GABA, constitute gated chloride channels. By their allosteric, drug- induced modulation, they serve as control elements for the regulation of anxiety, vigilance, and epileptiform activity. The structural requirements of fully functional GABAA receptors in the mammalian brain have remained elusive so far. We report here on the cloning of the gamma 2-subunit cDNA of rat brain and its functional analysis by coexpression with the alpha 1- and beta 1-subunits in Xenopus oocytes, and on the sites of gene expression of the 3 subunits in the rat brain. The recombinant receptor displayed GABA-inducible currents (Imax = 6 microA; Ka = 75 microM) which were allosterically modulated by benzodiazepine receptor ligands (enhancement and inhibition by diazepam and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, respectively). In the absence of GABA, pentobarbital elicited a maximal current amplitude similar to that of GABA. A minor population of channels is expressed which is open in the absence of GABA or pentobarbital. Mapping subunit gene expression by in situ hybridization histochemistry suggests that the alpha 1-, beta 1-, and gamma 2- subunits are likely receptor constituents in some neuronal populations, e.g., mitral cells of the olfactory bulb, pyramidal cells of the hippocampus, and granule cells of the dentate gyrus and cerebellum.

Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification.

Ligand-gated ion channels generally display a heterooligomeric subunit structure. The present report describes an electrophysiological method that provides criteria indicating the subunit stoichiometry of a recombinant GABAA receptor composed of alpha 3, beta 2 and gamma 2 subunits. Our results exclude the stoichiometries 3 alpha 1 beta 1 gamma, 1 alpha 3 beta 1 gamma, 1 alpha 1 beta 3 gamma and suggest that the possible subunit stoichiometries for this receptor are 2 alpha 1 beta 2 gamma, 2 alpha 2 beta 1 gamma or 1 alpha 2 beta 2 gamma, of which the alpha subunit composition 2 alpha 1 beta 2 gamma may be favoured. The method is based on the quantification of the outward rectification of the GABA-evoked current induced by point mutation of charged amino acids located near the ion channel pore.

cDNA cloning of a novel secreted isoform of the human receptor for advanced glycation end products and characterization of cells co-expressing cell-surface scavenger receptors and Swedish mutant amyloid precursor protein.

The receptor for advanced glycation end products (RAGE) has been proposed as a cell surface receptor that binds amyloid-beta protein (Abeta), thereby triggering its cytotoxic effects [S.D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, A.M. Schmidt, RAGE and amyloid-beta peptide neurotoxicity in Alzheimers disease, Nature 382 (1996) 685-691.]. A cDNA library of human lung was screened for RAGE with an appropriate hybridization probe. In addition to cell surface RAGE, one clone was found which encodes a new version of RAGE, termed hRAGEsec, which lacks the 19 amino acids of the membrane-spanning region and is therefore secreted. Comparison with the genomic sequence revealed that the synthesis of the secreted isoform requires alternative splicing. The deduced protein sequence of the mature hRAGEsec consists of 321 amino acids with a predicted molecular mass of 35.66 kDa. The pattern of expression of hRAGEsec in human brain was analyzed by in situ hybridization histochemistry. The most intense expression of the gene in contrast to cell surface RAGE was detected in hippocampal CA3 pyramidal cells, dentate gyrus granule cells, cortical neurons as well as glial cells in white matter. To investigate the interaction between Abeta and RAGE and another scavenger receptor, SRA, under physiological conditions, they were co-expressed with human betaAPP(695)-SFAD in a human cell and the level of Abeta in the condition medium was assessed by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) analysis. A nearly 100% reduction of Abeta from the conditioned medium of hRAGE cells and approximately 40% reduction from the SRA-cells implied that hRAGE could be a prominent cell surface receptor interacting with Abeta.

Recombinant GABAA receptor function and ethanol.

Different combinations of cloned subunits of the rat brain GABAA receptor were expressed in Xenopus oocytes. Possible effects of ethanol on the expressed GABA‐induced chloride current were determined. The consequence of replacing the γ2s subunit by the alternatively spliced variant γ2L was specifically tested on the responsiveness to ethanol. A significant stimulation of the GABA response was only observed at very high concentrations (> 60 mM) of ethanol. No differential response was observed between subunit combinations containing different γ2 subunit splice variants.

Activation of protein kinase C results in down-modulation of different recombinant GABAA-channels.

Different combinations of cloned rat brain subunits of the GABAA receptor were expressed in Xenopus oocytes. The effect of the phorbol ester PMA, an activator of protein kinase C, on the expressed GABA‐gated ion current was determined. Ion currents were diminished by β‐PMA, but not by the control substance α‐PMA, irrespective of the subunit combination studied. The mechanism of current decrease was investigated in more detail for the subunit combination α5β2γ2. The reversal potential of the current remained unaffected, while the maximal current amplitude was decreased and the apparent K a for GABA‐dependent channel gating was shifted to higher concentrations.

GABAA-receptor expressed from rat brain α- and β-subunit cDNAs displays potentiation by benzodiazepine receptor ligands

In mammalian brain, the activation of GABAA-receptors is associated with the opening of chloride channels, whose function can be allosterically modulated by drugs, in particular by ligands of the benzodiazepine receptor. Agonistic ligands potentiate while inverse agonists reduce the efficiency of GABA. We have cloned cDNAs encoding α1- and β1-subunits of the GABAA-receptor from rat brain. When the corresponding RNAs were co-expressed in Xenopus oocytes, GABA-induced currents were recorded which were inhibited by bicuculline and potentiated by pentobarbital. GABA activated the channel in a weakly cooperative manner. Furthermore, the GABA-response was modulated by benzodiazepine receptor ligands. However, not only various agonists but also the antagonist flumazenil and the inverse agonist DMCM potentiated the GABA-response. Thus, α1- and β1-subunits are sufficient to form GABAA-receptors which contain benzodiazepine binding sites, although in a functionally restricted form.

Characterization of [3H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using Semliki Forest virus vectors

The binding properties of [(3)H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave K(D) values of 20+/-5 and 53+/-8 nM and B(max) values of 474+/-161 and 667+/-89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl(2), DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [(3)H]-LY354740 binding with similar potencies on both receptors. L-CCG I, L-AP4, L-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [(3)H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and L-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. ZnCl(2), at 10 mM, inhibited more than 70% of [(3)H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [(3)H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPgammaS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [(3)H]-LY354740 binding on mGlu3 receptors.