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1.
Neuropharmacology ; 45(7): 895-906, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14573382

ABSTRACT

Group-III metabotropic glutamate receptors (mGluR4, -6, -7, and -8) modulate neurotoxicity of excitatory amino acids and beta-amyloid-peptide (betaAP), as well as epileptic convulsions, most likely via presynaptic inhibition of glutamatergic neurotransmission. Due to the lack of subtype-selective ligands for group-III receptors, we previously utilized knock-out mice to identify mGluR4 as the primary receptor mediating neuroprotection of unselective group-III agonists such as L-AP(4) or (+)-PPG, whereas mGluR7 is critical for anticonvulsive effects. In a recent effort to find group-III subtype-selective drugs we identified (+/-)-PHCCC as a positive allosteric modulator for mGluR4. This compound increases agonist potency and markedly enhances maximum efficacy and, at higher concentrations, directly activates mGluR4 with low efficacy. All the activity of (+/-)-PHCCC resides in the (-)-enantiomer, which is inactive at mGluR2, -3, -5a, -6, -7b and -8a, but shows partial antagonist activity at mGluR1b (30% maximum antagonist efficacy). Chimeric receptor studies showed that the binding site of (-)-PHCCC is localized in the transmembrane region.Finally, (-)-PHCCC showed neuroprotection against betaAP- and NMDA-toxicity in mixed cultures of mouse cortical neurons. This neuroprotection was additive to that induced by the highly efficacious mGluR1 antagonist CPCCOEt and was blocked by MSOP, a group-III mGluR antagonist. Our data provide evidence for a novel pharmacological site on mGluR4, which may be used as a target-site for therapeutics.


Subject(s)
Benzopyrans/pharmacology , Neuroprotective Agents/pharmacology , Receptors, Metabotropic Glutamate/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/toxicity , Calcium/metabolism , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cloning, Molecular , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Excitatory Amino Acid Agonists/toxicity , Excitatory Amino Acids/toxicity , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , N-Methylaspartate/antagonists & inhibitors , N-Methylaspartate/toxicity , Neuroglia/drug effects , Neuroglia/metabolism , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Receptors, Metabotropic Glutamate/drug effects
2.
Farmaco ; 56(1-2): 95-9, 2001.
Article in English | MEDLINE | ID: mdl-11347975

ABSTRACT

We have investigated the mechanism of inhibition of the new group I mGluR antagonists CPCCOEt and MPEP and determined that both compounds have a non-competitive mode of inhibition. Furthermore using chimeric/mutated receptors constructs we have found that these antagonists act at a novel pharmacological site located in the trans-membrane (TM). Specific non-conserved amino acid residues in the TM domain have been identified which are necessary for the inhibition by CPCCOEt and MPEP of the mGlul and mGlu5 receptors, respectively. Using molecular modeling a model of the TM domain was built for both mGlu1 and mGlu5 receptor subtypes. Docking of CPCCOEt and MPEP into their respective model allowed the modelisation of the novel binding site.


Subject(s)
Receptors, Metabotropic Glutamate/antagonists & inhibitors , Binding Sites , Models, Molecular , Receptors, Metabotropic Glutamate/chemistry
3.
J Med Chem ; 43(23): 4428-36, 2000 Nov 16.
Article in English | MEDLINE | ID: mdl-11087567

ABSTRACT

Racemic CPCCOEt ((1aRS,7aRS)-2-hydroxyimino-1a, 2-dihydro-1H-7-oxacyclopropa[b]naphthalene-7a-carboxylic acid ethyl ester, (+/-)-1) derivatives have been shown to be subtype-selective metabotropic glutamate (mGlu) 1 receptor antagonists (Annoura et al. Bioorg. Med. Chem. Lett. 1996, 6, 763-766). The optical isomers of (+/-)-1 have been separated by chromatography on a chiral stationary phase. The absolute configuration at the C-1a and C-7a positions was determined using X-ray crystallography of an amide derivative with the methyl ester of L-phenylalanine (L-PheOMe) ((+)-6). In a phosphoinositol (PI) turnover assay at the cloned human mGlu1b receptor, (-)-1 and the new amide derivatives (-)-5 and (-)-6, all of which have (1aS,7aS)-stereochemistry on the chromane ring system, showed IC(50) values of 1.5, 0.43, and 0.93 microM, respectively. In contrast, (+)-1 and the new amide derivatives (+)-5 and (+)-6were found to be inactive up to a concentration of 30 microM indicating a selectivity for the (-)-enantiomers of at least 70-fold. In a previous study (Litschig et al. Mol. Pharmacol. 1999, 55, 453-461) we demonstrated using site-directed mutagenesis that the interaction site of (+/-)-1 is located in the transmembrane (TM) domain of hmGlu1b. To suggest a plausible binding mode of (-)-1, we have built a molecular mechanics model of the putative seven TM domain of hmGlu1 based on the alpha-carbon template of the TM helices of rhodopsin. A receptor docking hypothesis suggests that the OH of T815 (TMVII) comes in close contact with the oxime OH of (-)-1 and (-)-5, whereas no such close interactions could be demonstrated by docking of (+)-1.


Subject(s)
Chromones/chemical synthesis , Excitatory Amino Acid Antagonists/chemical synthesis , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Chromones/chemistry , Chromones/pharmacology , Cricetinae , Crystallography, X-Ray , Excitatory Amino Acid Antagonists/chemistry , Excitatory Amino Acid Antagonists/pharmacology , Hydrolysis , Inositol Phosphates/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Receptors, Metabotropic Glutamate/chemistry , Stereoisomerism , Structure-Activity Relationship
4.
J Neurosci ; 20(17): 6413-20, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10964947

ABSTRACT

Activation of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, and mGluR8) has been established to be neuroprotective in vitro and in vivo. To disclose the identity of the receptor subtype(s) that exert(s) the protective effect, we have used group III agonists in combination with mGluR4 subtype-deficient mice (-/-). In cortical cultures prepared from wild-type (+/+) mice and exposed to a toxic pulse of NMDA, the selective group III agonist (+)-4-phosphonophenylglycine [(+)-PPG] reversed excitotoxicity with an EC(50) value of 4.9 microm, whereas its enantiomer (-)-PPG was inactive. This correlated closely with the potency of (+)-PPG in activating recombinant mGluR4a. In cortical neurons from -/- mice, (+)-PPG showed no protection against the NMDA insult up to 300 microm, whereas group I/II mGluR ligands still retained their protective activity. Classical group III agonists (l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate) were also substantially neuroprotective against NMDA toxicity in +/+ and heterozygous (+/-) cultures but were inactive in -/- cultures. Interestingly, -/- cultures were more vulnerable to low concentrations of NMDA and showed higher extracellular glutamate levels compared with +/+ cultures. We have also examined neurodegeneration induced by intrastriatal infusion of NMDA in wild-type or mGluR4-deficient mice. Low doses of (R,S)-PPG (10 nmol/0.5 microl) substantially reduced NMDA toxicity in +/+ mice but were ineffective in -/- mice. Higher doses of (R,S)-PPG were neuroprotective in both strains of animals. Finally, microdialysis studies showed that intrastriatal infusion of NMDA increased extracellular glutamate levels to a greater extent in -/- than in +/+ mice, supporting the hypothesis that the mGluR4 subtype is necessary for the maintenance of the homeostasis of extracellular glutamate levels.


Subject(s)
Aminobutyrates/pharmacology , Cerebral Cortex/cytology , Glycine/analogs & derivatives , N-Methylaspartate/toxicity , Neurons/physiology , Neurotoxins/pharmacology , Receptors, Metabotropic Glutamate/physiology , Animals , Cells, Cultured , Cerebral Cortex/physiology , Excitatory Amino Acid Agonists/pharmacology , Glutamic Acid/metabolism , Glycine/pharmacology , Heterozygote , Mice , Mice, Knockout , Nerve Degeneration/chemically induced , Nerve Degeneration/physiopathology , Neurons/cytology , Neurons/drug effects , Neuroprotective Agents/pharmacology , Receptors, Metabotropic Glutamate/deficiency , Receptors, Metabotropic Glutamate/genetics , Stereoisomerism
5.
Bioorg Med Chem Lett ; 10(11): 1241-4, 2000 Jun 05.
Article in English | MEDLINE | ID: mdl-10866390

ABSTRACT

A new synthesis of (R,S)-PPG (4-phosphonophenylglycine) and the separation of the protected enantiomers leading after deprotection to (+)- and (-)-PPG are described. Pharmacological characterization at the group III metabotropic glutamate receptors hmGluR4a and hmGluR7b revealed (+)-PPG as the active enantiomer.


Subject(s)
Excitatory Amino Acid Agonists/pharmacology , Glycine/analogs & derivatives , Receptors, Metabotropic Glutamate/agonists , Glycine/chemical synthesis , Glycine/chemistry , Glycine/pharmacology
6.
J Pharmacol Exp Ther ; 289(3): 1678-87, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10336568

ABSTRACT

Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of excitatory amino acids, via mechanisms of presynaptic inhibition, such as regulation of neurotransmitter release. Here, we describe (R,S)-4-phosphonophenylglycine (PPG) as a novel, potent, and selective agonist for group III mGluRs. In recombinant cell lines expressing the human receptors hmGluR4a, hmGluR6, hmGluR7b, or hmGluR8a, EC50 values for (R,S)-PPG of 5.2 +/- 0.7 microM, 4.7 +/- 0.9 microM, 185 +/- 42 microM, and 0.2 +/- 0.1 microM, respectively, were measured. The compound showed EC50 and IC50 values of >/=200 microM at group I and II hmGluRs and was inactive at cloned human N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, and kainate receptors (>300 microM). On the other hand, it showed micromolar affinity for a Ca2+/Cl--dependent L-glutamate binding site in rat brain, similar to other phosphono-substituted amino acids like L-2-amino-4-phosphonobutyrate. In cultured cortical neurons, (R, S)-PPG provided protection against a toxic pulse of N-methyl-D-aspartate (EC50 = 12 microM), which was reversed by the group III mGluR antagonist (R,S)-alpha-methylserine-O-phosphate but not by the group II antagonist (2S)-alpha-ethylglutamate. Moreover, (R,S)-PPG protected against N-methyl-D-aspartate- and quinolinic acid-induced striatal lesions in rats and was anticonvulsive in the maximal electroshock model in mice. In contrast to the group III mGluR agonists L-2-amino-4-phosphonobutyrate and L-serine-O-phosphate, (R,S)-PPG showed no proconvulsive effects (2200 nmol i.c.v.). These data provide novel in vivo evidence for group III mGluRs as attractive targets for neuroprotective and anticonvulsive therapy. Also, (R,S)-PPG represents an attractive tool to analyze the roles of group III mGluRs in nervous system physiology and pathology.


Subject(s)
Anticonvulsants/pharmacology , Brain/metabolism , Glycine/analogs & derivatives , Neuroprotective Agents/pharmacology , Receptors, Metabotropic Glutamate/agonists , Animals , Cell Line , Cell Membrane/metabolism , Colforsin/pharmacology , Corpus Striatum/drug effects , Corpus Striatum/pathology , Corpus Striatum/physiology , Cyclic AMP/metabolism , Electroshock , Glutamic Acid/metabolism , Glycine/chemistry , Glycine/pharmacology , Humans , Kinetics , Male , Mice , Mice, Inbred Strains , N-Methylaspartate/toxicity , Phosphatidylinositols/metabolism , Protein Isoforms/agonists , Protein Isoforms/genetics , Quinolinic Acid/toxicity , Rats , Rats, Sprague-Dawley , Receptors, Metabotropic Glutamate/genetics , Recombinant Proteins/agonists , Second Messenger Systems/drug effects , Seizures/physiopathology , Seizures/prevention & control , Structure-Activity Relationship
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