ABSTRACT
The glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor, given its potential as pharmacological target, has been thoroughly studied by structure-activity relationships, which has made possible its description in terms of spatial limits and interactions of various types. A structural model, based on mutational analysis and sequence alignements, has been proposed. Yet, the amino acid residues responsible for the interactions with the ligand have not been unambiguously characterized. To evidence nucleophilic pocket-lining residues, we have designed and synthesized reactive glycine-site ligands derived from 3-substituted 4-hydroxy-quinolin-2(1H)-ones by introducing various electrophilic groups at different positions of the molecule. These ligands were found to have high affinity at the glycine site and to be functional antagonists by inhibiting glycine/glutamate-induced currents in transfected oocytes. The correlation between their potency and their substitution pattern was strictly consistent with previously established structure-activity relationships. Most ligands displayed intrinsic reactivity toward cysteine, but none inactivated wild-type receptors. This is consistent with the model since it indicates the absence of exposed cysteine in the glycine-binding site. A strategy of cysteine incorporation by point mutations at selected polypeptide positions will create unambiguously localized targets for our reactive probes.
Subject(s)
Quinolines/chemical synthesis , Receptors, Glycine/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Binding, Competitive , Brain/metabolism , Electrophysiology , Excitatory Amino Acid Antagonists/chemical synthesis , Excitatory Amino Acid Antagonists/chemistry , Excitatory Amino Acid Antagonists/metabolism , Excitatory Amino Acid Antagonists/pharmacology , Glycine/pharmacology , In Vitro Techniques , Ligands , Oocytes , Quinolines/chemistry , Quinolines/metabolism , Quinolines/pharmacology , Radioligand Assay , Rats , Receptors, Glycine/biosynthesis , Receptors, N-Methyl-D-Aspartate/biosynthesis , Structure-Activity Relationship , Xenopus laevisABSTRACT
Novel analogs of the allosteric AMPA receptor modulator SYM 2206 have been prepared. Structure/activity correlations of these novel analogs and other dihydrophthalazines (DHPs) reveal the important contribution of the heteroatom-based aryl substituents in this class of noncompetitive inhibitors. One of the analogs (6, SYM 2189) is equipotent with the early series, but with reduced sedation.
Subject(s)
Phthalazines/pharmacology , Receptors, AMPA/drug effects , Allosteric Regulation , Animals , Cells, Cultured , Mice , Neurons/drug effects , Phthalazines/chemistry , Rats , Structure-Activity RelationshipABSTRACT
The glycine co-agonist binding site of the NMDA receptor is a target for the prevention and treatment of neurotoxic and neurodegenerative conditions. Until now, the interactions taking place at this site, and its structure, have been investigated by ligand structure-activity relationships and by site-directed mutagenesis. On the basis of a structural model which is currently proposed for this site, we have designed and synthesized six affinity markers by substituting electrophilic reactive groups in the 4, the 7 and the 3' positions of L 701,324, a high-affinity glycine site antagonist. These compounds compete with 3H-DCKA binding to rat brain membranes at equilibrium with nanomolar to low-micromolar affinities, and antagonize glycine-evoked currents in oocytes transfected with wild-type NR1-NR2B. However, they do not induce a time-shift in binding equilibria, and do not inactivate irreversibly the glycine evoked currents. Since they react only with cysteine at physiological pH, we conclude that there is no such residue in the site, in agreement with the model. Our affinity markers therefore represent potential topological probes for NMDA receptors with sequence positions related to the glycine-binding site mutated into cysteine.
Subject(s)
Affinity Labels/chemical synthesis , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/metabolism , Affinity Labels/chemistry , Affinity Labels/metabolism , Animals , Binding Sites , Brain/metabolism , Excitatory Amino Acid Antagonists/chemical synthesis , Excitatory Amino Acid Antagonists/chemistry , Excitatory Amino Acid Antagonists/metabolism , Glycine/chemistry , In Vitro Techniques , Kinetics , Ligands , Molecular Structure , Protein Conformation , Quinolones/chemistry , Quinolones/metabolism , Quinolones/pharmacology , Rats , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitorsABSTRACT
NMDA receptors require both L-glutamate and the coagonist glycine for efficient channel activation. The glycine binding site of these heteromeric receptor proteins is formed by regions of the NMDAR1 (NR1) subunit that display sequence similarity to bacterial amino acid binding proteins. Here, we demonstrate that the glutamate binding site is located on the homologous regions of the NR2B subunit. Mutation of residues within the N-terminal domain and the loop region between membrane segments M3 and M4 significantly reduced the efficacy of glutamate, but not glycine, in channel gating. Some of the mutations also decreased inhibition by the glutamate antagonists, D-AP5 and R-CPP. Homology-based molecular modeling of the glutamate and glycine binding domains indicates that the NR2 and NR1 subunits use similar residues to ligate the agonists' alpha-aminocarboxylic acid groups, whereas differences in side chain interactions and size of aromatic residues determine ligand selectivity.
Subject(s)
Excitatory Amino Acid Agonists/pharmacology , Glutamic Acid/metabolism , Ion Channel Gating/drug effects , Receptors, N-Methyl-D-Aspartate/agonists , 2-Amino-5-phosphonovalerate/pharmacology , Amino Acid Sequence , Binding Sites , Glutamic Acid/pharmacology , Glycine/pharmacology , Models, Molecular , Molecular Sequence Data , Piperazines/pharmacology , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
An immunochemiluminometric assay has been developed for the measurement of free T4 concentrations in serum. The assay uses chemiluminescent acridinium ester labelled monoclonal antibodies which react with free T4 in the sample. A T4-rabbit immunoglobulin G conjugate competes for antibody binding sites, immune-complexes containing this being isolated using an anti-immunoglobulin G antibody coupled to paramagnetic particles. Associated chemiluminescence intensity is thus dependent on the free T4 concentration. The assay distinguishes patients with primary thyroid disease from euthyroid subjects and is unaffected by abnormal binding proteins which compromise the diagnostic accuracy of radiolabelled analogue immunoassays. the test yields results which accurately reflect the clinical thyroid status of euthyroid patients with a variety of acute and chronic non-thyroid illnesses. This is again in marked contrast to the aberrant results seen using certain radiolabelled analogue procedures.
Subject(s)
Immunologic Techniques , Thyroxine/blood , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal , Euthyroid Sick Syndromes/blood , Female , Humans , Luminescent Measurements , Male , Middle Aged , Thyroid Diseases/bloodABSTRACT
A chemiluminescent labeled-antibody immunoassay has been developed for measurement of total thyroxin (T4) in serum. Monoclonal antibodies to T4 labeled with a chemiluminescent acridinium ester are used. Serum samples are incubated with the labeled antibodies and a thyroxin-rabbit immunoglobulin conjugate, then reacted with magnetizable particles containing sheep anti-rabbit immunoglobulin. The total reaction time is 40 min. The chemiluminescence intensity of the solid-phase immune complexes is inversely proportional to the concentration of T4 in the sample. The sensitivity of the assay is 1 nmol/L, and the working range of 20-190 nmol/L is characterized by CVs less than or equal to 10%.
Subject(s)
Thyroxine/blood , Animals , Antibodies, Monoclonal , Humans , Hyperthyroidism/blood , Hypothyroidism/blood , Immunochemistry , Luminescent Measurements , Rabbits , SheepABSTRACT
A two-site immunochemiluminometric assay (ICMA) has been developed for the measurement of human thyrotrophin (TSH). The procedure involves reaction of serum samples with monoclonal antibodies to TSH which have been labelled with a chemiluminescent acridinium ester followed by reaction with solid-phase polyclonal antibodies to TSH. The total reaction time is 2 h and bound labelled antibody is quantified luminometrically. The assay has an absolute sensitivity of 0.004 mU/l and a linear dose-response range up to 60 mU/l. Circulating TSH concentrations in 84 normal subjects were in the range 0.4 to 4.0 mU/l and in 16 hyperthyroid patients were less than 0.03 mU/l. This assay should thus prove useful as a first line test of thyroid function.
