Evaluation and Biological Properties of Reactive Ligands for the Mapping of the Glycine Site on the<i>N</i>-Methyl-<scp>d</scp>-aspartate (NMDA) Receptor

Kreimeyer, Annett; Laube, Bodo; Sturgess, Mike; Goeldner, Maurice; Foucaud, Bernard

doi:10.1021/jm9910730

Cited by 35 publications

(19 citation statements)

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“…27). Indeed, the agonist responses of Xenopus oocytes injected with wild-type NR1 and NR2B cRNAs (15)(16) were blocked by Ͼ95% upon incubation with saturating concentrations (50 M) of these reactive antagonists but fully recovered after a 3-5 min washout as reported (27). In contrast, when cysteine mutant receptor-expressing Xenopus oocytes were incubated with these ligands under identical conditions, inhibition of agonist-induced currents often could not be reversed upon prolonged washing for up to 20 min with some of the mutants.…”

Section: Resultssupporting

confidence: 68%

“…1), were selected out of 12 compounds described previously, on the basis of their high reactivity toward cysteine, their stability in FR solution, and their affinity for wild-type NMDA receptors (27): 7-CAA, 7-NCS, and m 1 -NCS bind reversibly to rat brain membranes with K i s of ϳ280, 64, and 48 nM, respectively (27), as compared with 14 nM for L-701,324. 7-CAA, 7-NCS, and m 1 -NCS all have been shown to reversibly and dose-dependently inhibit the glycine binding site of the wild-type NMDA receptor (IC 50 s in the nano-to micromolar range, Ref.…”

Section: Resultsmentioning

confidence: 99%

“…The reactive ligands, derived from L-701,324 (Fig. 1), have been described previously (27). NR1 subunit positions substituted into cysteines were selected using an improved three-dimensional model of the glycine binding site generated by homology modeling to the crystal structure of the GluR2 "S1-S2" fusion protein (28).…”

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confidence: 99%

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Structural Model of the N-Methyl-D-aspartate Receptor Glycine Site Probed by Site-directed Chemical Coupling

Foucaud

Laube

Schemm

et al. 2003

Journal of Biological Chemistry

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The N-methyl-D-aspartate (NMDA) receptor is a ligand-gated ion channel that requires both glutamate and glycine for efficient activation. Here, a strategy combining cysteine scanning mutagenesis and affinity labeling was used to investigate the glycine binding site located on the NR1 subunit. Based on homology modeling to the crystal structure of the glutamate binding site of the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid receptor GluR2, cysteines were introduced into the NR1 subunit as chemical sensors for three thiolreactive derivatives of the competitive antagonist L-701,324. After coexpressing the mutant NR1 with wildtype NR2B subunits in Xenopus oocytes, agonist-induced currents were recorded to monitor irreversible receptor inactivation by the reactive antagonists. For each derivative, glycine site-specific inactivations were observed with a distinct subset of cysteine-substituted receptors. Together these inactivating substitutions identified seven NR1 residues (Ile-385, Gln-387, Glu-388, Thr-500, Asn-502, Ala-696, and Val-717) that undergo proximity-induced covalent coupling with specific regions of the bound antagonist and disclose its mode of docking in the glycine binding pocket of the NMDA receptor. Our approach may help to unravel the structural basis of distinct NMDA receptor subtype pharmacologies.The N-methyl-D-aspartate (NMDA) 1 subtype of glutamate receptors is a ligand-gated cation channel characterized by a high Ca 2ϩ /Na ϩ permeability ratio (1, 2). It requires both glutamate and the co-agonist glycine for efficient channel gating (3, 4) and is composed of two NR1 and two NR2 subunits, each (5). The NR1 subunit binds glycine with submicromolar affinity, whereas the NR2 subunit harbors the glutamate binding site (6, 7). Various splice variants of the NR1 subunit and four isoforms of the NR2 subunit (A-D) generate different subtypes of tetrameric NMDA receptors, which differ in their pharmacologies (8) and expression patterns (9, 10).NMDA receptors are crucially implicated in synaptic plasticity and memory formation as well as in many neurological and psychiatric disorders (11). Classic NMDA receptor antagonists have therefore been examined for their therapeutic potential; in most cases, however, severe side-effects (reviewed by Kornhuber and Weller, Ref. 12), have prevented their clinical use. An alternative strategy focuses on glycine site antagonists that display considerable subtype selectivity (13). Structureactivity studies have defined a pharmacophore that delineates different ligand interactions within the glycine binding site of NMDA receptors (reviewed in Ref. 14). However, the structure of this site and the amino acid residues responsible for these interactions have only been deduced by indirect approaches.Mutational analysis has revealed residues whose substitution reduces the efficacies of glycine site agonists and antagonists (15)(16)(17)(18)(19). Notably, these residues are located within ϳ200 positions N-terminal to the first putative transmembrane domain (segme...

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Section: Resultssupporting

confidence: 68%

Section: Resultsmentioning

confidence: 99%

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Structural Model of the N-Methyl-D-aspartate Receptor Glycine Site Probed by Site-directed Chemical Coupling

Foucaud

Laube

Schemm

et al. 2003

Journal of Biological Chemistry

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“…52) introduced site specific mutation to cysteine in combination with nonspecific cysteine-reactive agents for the study of proteins. Recently, a novel technique to elucidate relative position of a ligand in its binding pocket has been successfully applied in several cases (53)(54)(55)(56)(57)(58)(59). The technique has been described in detail (60).…”

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confidence: 99%

On the Benzodiazepine Binding Pocket in GABAA Receptors

Berezhnoy

Nyfeler

Gonthier

et al. 2004

Journal of Biological Chemistry

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Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the ␥-aminobutyric acid, type A (GABA A ) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA A receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of ␣ 1 H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated ␣ 1 H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that ␥ 2 Ala-79 is probably located in the access pathway of the ligand to its binding pocket.

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“…[1][2][3][4][5][6] The biological importance of quinolinones stimulated an intensive research work for the synthesis of many members of this class of compounds. [7][8][9][10] In continuation of our previous reports on the synthesis of novel 4-hydroxyquinolin-2(1H)-ones starting from 3-(1-ethy1-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)-3-oxopropanoic acid (1), 11 the present work aims to synthesize the 3-(nitroacetyl)-1-ethyl-4-hydroxyquinolin-2(1H)-one (4) and to study its reactivity towards a variety of chemical reagents.…”

Section: Introductionmentioning

confidence: 99%

Studies on the chemical behavior of 3-(nitroacetyl)-1-ethyl-4-hydroxyquinolin-2(1H)-one towards some electrophilic and nucleophilic reagents

Ibrahim¹,

Hassanin²,

Gabr³

et al. 2012

J. Braz. Chem. Soc.

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Uma variedade de sistemas heterocíclicos contendo o sistema 1-etilquinolin-2(1H)-ona, foi preparada a partir da reação de 3-(nitroacetil)-1-etil-4-hidroxiquinolin-2(1H)-ona com alguns reagentes eletrofílicos e nucleofílicos. Além da sua ciclização para 5-etil-2-(hidroxiimino)-2,3,4,5-tetrahydrofuro[3,2-c]quinolina-3,4-diona, o composto 3-(nitroacetil)-1-etil-4-hidroxiquinolina-2(1H)-ona foi bromado, clorado, formilado, acetilado, e condensado com cromona-3-carbonitrila e 2-amino-3-formilcromona. Alguns novos pirazolo [4,3-c]

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Evaluation and Biological Properties of Reactive Ligands for the Mapping of the Glycine Site on theN-Methyl-d-aspartate (NMDA) Receptor

Cited by 35 publications

References 50 publications

Structural Model of the N-Methyl-D-aspartate Receptor Glycine Site Probed by Site-directed Chemical Coupling

Structural Model of the N-Methyl-D-aspartate Receptor Glycine Site Probed by Site-directed Chemical Coupling

On the Benzodiazepine Binding Pocket in GABAA Receptors

Studies on the chemical behavior of 3-(nitroacetyl)-1-ethyl-4-hydroxyquinolin-2(1H)-one towards some electrophilic and nucleophilic reagents

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