ABSTRACT
Selective antagonism of N-methyl-d-aspartate (NMDA) 2B subunit containing receptors has been suggested to have potential therapeutic application for multiple CNS disorders. The amino terminal NR2B residues 1 to 282 were found to be both necessary and sufficient for the binding and function of highly NR2B subunit specific antagonists like ifenprodil and CP-101,606. Using a genetic approach in mice, we successfully replaced the murine NR2B gene function by "knocking-in" (KI) a chimeric human NR2A/B cDNA containing the minimal domain abolishing ifenprodil binding into the endogenous NR2B locus. Patch-clamp recording from hippocampal cultures of the NR2B KI mice demonstrated that their NMDA receptors have reduced sensitivity to both ifenprodil and CP-101,606, as predicted, but also have a lower affinity for glycine. The NR2B KI mice exhibited normal locomotor activity making this ifenprodil-insensitive mouse model a valuable tool to test the specificity of NR2B selective antagonists in vivo.
Subject(s)
Excitatory Amino Acid Antagonists/metabolism , Piperidines/metabolism , Protein Subunits/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Cells, Cultured , Dizocilpine Maleate/metabolism , Excitatory Amino Acid Agonists/metabolism , Female , Gene Targeting , Hippocampus/cytology , Humans , Male , Mice , Mice, Transgenic , Motor Activity/physiology , N-Methylaspartate/metabolism , Neurons/cytology , Neurons/metabolism , Patch-Clamp Techniques , Protein Subunits/genetics , Receptors, N-Methyl-D-Aspartate/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Xenopus laevisABSTRACT
We have previously identified the 7,8,9,10-tetrahydro-7,10-ethano-1,2,4-triazolo[3,4-a]phthalazine (1) as a potent partial agonist for the alpha(3) receptor subtype with 5-fold selectivity in binding affinity over alpha(1). This paper describes a detailed investigation of the substituents on this core structure at both the 3- and 6-positions. Despite evaluating a wide range of groups, the maximum selectivity that could be achieved in terms of affinity for the alpha(3) subtype over the alpha(1) subtype was 12-fold (for 57). Although most analogues showed no selectivity in terms of efficacy, some did show partial agonism at alpha(1) and antagonism at alpha(3) (e.g., 25 and 75). However, two analogues tested (93 and 96), both with triazole substituents in the 6-position, showed significantly higher efficacy for the alpha(3) subtype over the alpha(1) subtype. This was the first indication that selectivity in efficacy in the required direction could be achieved in this series.
Subject(s)
GABA-A Receptor Agonists , Phthalazines/chemical synthesis , Triazoles/chemical synthesis , Animals , Binding Sites , Cell Line , Female , Humans , Models, Molecular , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Phthalazines/chemistry , Phthalazines/pharmacology , Protein Subunits/agonists , Protein Subunits/physiology , Radioligand Assay , Receptors, GABA-A/physiology , Structure-Activity Relationship , Triazoles/chemistry , Triazoles/pharmacology , Xenopus laevisABSTRACT
A series of substituted 3,4-dihydronaphthalen-1(2H)-ones with high binding affinity for the benzodiazepine site of GABAA receptors containing the alpha5-subunit has been identified. These compounds have consistently higher binding affinity for the GABAA alpha5 receptor subtype over the other benzodiazepine-sensitive GABAA receptor subtypes (alpha1, alpha2 and alpha3). Compounds with a range of efficacies for the benzodiazepine site of alpha5-containing GABAA receptors were identified, including the alpha5 inverse agonist 3,3-dimethyl-8-methylthio-5-(pyridin-2-yl)-3,4-dihydronaphthalen-1(2H)-one 22 and the alpha5 agonist 8-ethylthio-3-methyl-5-(1-oxidopyridin-2-yl)-3,4-dihydronaphthalen-1(2H)-one 19.
Subject(s)
Naphthalenes/pharmacology , Receptors, GABA-A/drug effects , Allosteric Site , Benzodiazepines/antagonists & inhibitors , Binding Sites , GABA-A Receptor Agonists , GABA-A Receptor Antagonists , Humans , Ligands , Naphthalenes/chemical synthesis , Protein Binding , Protein Subunits , Structure-Activity RelationshipABSTRACT
Two classes of 5-substituted benzimidazoles were identified as potent antagonists of the NR2B subtype of the N-methyl-d-aspartate (NMDA) receptor. Selected compounds show very good selectivity versus the NR2A, NR2C, and NR2D subtypes of the NMDA receptor as well as versus hERG-channel activity and alpha(1)-adrenergic binding. Benzimidazole 37a shows excellent activity in the carrageenan-induced mechanical hyperalgesia assay in rats as well as good pharmacokinetic behavior in dogs.
Subject(s)
Analgesics/chemical synthesis , Benzimidazoles/chemical synthesis , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Analgesics/pharmacokinetics , Analgesics/pharmacology , Animals , Benzimidazoles/pharmacokinetics , Benzimidazoles/pharmacology , Brain/metabolism , Calcium/metabolism , Carrageenan , Cell Line , Dogs , Female , Humans , Hyperalgesia/blood , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , In Vitro Techniques , Male , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/physiology , Structure-Activity RelationshipABSTRACT
The specific mechanisms underlying general anesthesia are primarily unknown. The intravenous general anesthetic etomidate acts by potentiating GABA(A) receptors, with selectivity for beta2 and beta3 subunit-containing receptors determined by a single asparagine residue. We generated a genetically modified mouse containing an etomidate-insensitive beta2 subunit (beta2 N265S) to determine the role of beta2 and beta3 subunits in etomidate-induced anesthesia. Loss of pedal withdrawal reflex and burst suppression in the electroencephalogram were still observed in the mutant mouse, indicating that loss of consciousness can be mediated purely through beta3-containing receptors. The sedation produced by subanesthetic doses of etomidate and during recovery from anesthesia was present only in wild-type mice, indicating that the beta2 subunit mediates the sedative properties of anesthetics. These findings show that anesthesia and sedation are mediated by distinct GABA(A) receptor subtypes.
Subject(s)
Anesthetics/pharmacology , Etomidate/pharmacology , Hypnotics and Sedatives/pharmacology , Receptors, GABA-A/metabolism , Animals , Anticonvulsants/pharmacology , Behavior, Animal/drug effects , Binding, Competitive/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Cell Separation , Consciousness/drug effects , Dose-Response Relationship, Drug , Electroencephalography/drug effects , Gene Targeting , In Vitro Techniques , Male , Mice , Mice, Mutant Strains , Motor Activity/drug effects , Patch-Clamp Techniques , Protein Isoforms/genetics , Protein Isoforms/metabolism , Purkinje Cells/cytology , Purkinje Cells/drug effects , Purkinje Cells/physiology , Receptors, GABA-A/drug effects , Receptors, GABA-A/genetics , Recovery of Function/drug effects , Recovery of Function/genetics , Triazoles/pharmacologyABSTRACT
Novel (E)-N(1)-(benzyl)cinnamamidines were prepared and evaluated as NR2B subtype NMDA receptor ligands. Excellent affinity was achieved by appropriate substitution of either phenyl ring. The 2-methoxybenzyl compound 1h had approximately 1,000-fold lower IC(50) in NR2B than NR2A-containing cells. Replacement of the styryl unit by 2-naphthyl was well tolerated.
Subject(s)
Amidines/chemical synthesis , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Amidines/metabolism , Benzamidines/chemical synthesis , Benzamidines/metabolism , Drug Evaluation, Preclinical , Humans , Naphthalenes/chemical synthesis , Naphthalenes/metabolism , Protein Binding , Radioligand Assay , Receptors, N-Methyl-D-Aspartate/metabolism , Structure-Activity RelationshipABSTRACT
A novel series of benzamidines was synthesized and shown to exhibit NR2B-subtype selective NMDA antagonist activity. Compound 31 is orally active in a carrageenan-induced rat hyperalgesia model of pain and shows no motor coordination side effects.
Subject(s)
Benzamidines/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Administration, Oral , Animals , Benzamidines/chemical synthesis , Carrageenan , Drug Evaluation, Preclinical , Hyperalgesia/drug therapy , Pain/drug therapy , Psychomotor Performance/drug effects , Rats , Structure-Activity RelationshipABSTRACT
Homoquinolinate, a derivative of the endogenous NMDA agonist, quinolinate, has been shown to display higher affinity for Xenopus oocytes expressing NR2A- and NR2B-containing receptors, compared to NR2C- and NR2D-containing receptors, whilst autoradiographical experiments subsequently showed that [3H]homoquinolinate labelled a subpopulation of NMDA receptors in rat brain sections, with a similar distribution to NR2B-containing receptors. In this study, we have shown that NMDA-specific [3H]homoquinolinate binding to rat brain membranes comprised 44% of total binding with a Bmax value of 5.73 pmol/mg protein, which was inhibited by NMDA with Ki=0.867 micro m. However, NMDA-specific [3H]homoquinolinate binding was not observed for a number of human recombinant NMDA receptors investigated, suggesting that there are subtle differences between the binding sites of recombinant and native receptors. Electrophysiological experiments revealed that homoquinolinate activated human recombinant NR1a/NR2A, NR1a/NR2B and NR1a/NR2A/NR2B receptors with EC50 values of 25.2, 13.8 and 9.04 micro m, respectively, with intrinsic activities of 148, 93.3 and 125%, respectively, compared to glutamate (=100%). In contrast to an autoradiographical study, these radioligand binding and electrophysiological experiments suggest that homoquinolinate is not highly selective for NR2B-containing receptors.
