title: Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors.
abstract: NR1/NR2A is a subtype of N-methyl-d-aspartate receptors (NMDARs), which are glutamate and glycine-gated Ca(2+)-permeable channels highly expressed in the central nervous system. A high-throughput screening (HTS) campaign using human osteosarcoma (U-2 OS) cells transiently transduced with NR1/NR2A NMDAR subunits, tested in a specifically designed fluorometric imaging plate reader (FLIPR)/Ca(2+) assay, identified sulfonamide derivative series, exemplified by 3-chloro-4-fluoro-N-[(4-{[2-(phenylcarbonyl)hydrazino]carbonyl}phenyl)methyl]benzenesulfonamide (compound 1) and thiodiazole derivative N-(cyclohexylmethyl)-2-({5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio)acetamide (compound 13) as novel NR1/NR2A receptor antagonists. Compounds 1 and 13 displayed submicromolar and micromolar potency at NR1/NR2A receptor, respectively, although they did not show activity at NR2B-containing receptor up to 50 μM concentration. Addition of 1 mM glycine, but not 1 mM l-glutamate, was able to surmount compound 1 and 13 inhibitory effects in FLIPR NR1/NR2A assay. However, compounds 1 and 13 displaced a glutamate site antagonist [(3)H]d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid ([(3)H]CGP 39653) to a greater extent than the glycine site antagonist [(3)H]3-[(E)-2-carboxy-2-phenylethenyl]-4,6-dichloro-1H-indole-2-carboxylic acid ([(3)H]MDL 105,519), in rat brain cortex binding assay. Results of FLIPR cell-based, electrophysiological, and biochemical binding assays suggest that compounds 1 and 13 are the prototypes of novel classes of NMDAR ligands, which to the best of our knowledge are the first selective antagonists at NR1/NR2A over NR1/NR2B receptor, and might constitute useful tools able to elucidate the relative role of the NR2A subunit in physiological and pathological conditions.
authors.affiliation: Screening and Compound Profiling, GlaxoSmithKline Medicines Research Centre, Verona, Italy. ezio.bettini@aptuit.com
authors.forename: Ezio
authors.initials: E
authors.lastname: Bettini
authors.forename: Anna
authors.initials: A
authors.lastname: Sava
authors.forename: Cristiana
authors.initials: C
authors.lastname: Griffante
authors.forename: Corrado
authors.initials: C
authors.lastname: Carignani
authors.forename: Alberto
authors.initials: A
authors.lastname: Buson
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Capelli
authors.forename: Michele
authors.initials: M
authors.lastname: Negri
authors.forename: Filippo
authors.initials: F
authors.lastname: Andreetta
authors.forename: Sergio A
authors.initials: SA
authors.lastname: Senar-Sancho
authors.forename: Lorena
authors.initials: L
authors.lastname: Guiral
authors.forename: Francesca
authors.initials: F
authors.lastname: Cardullo
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D020013:Calcium Signaling / Q000187:drug effects; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D005470:Fluorometry / Q000379:methods; D018698:Glutamic Acid / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D015394:Molecular Structure; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D018691:Excitatory Amino Acid Antagonists; C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D016202:N-Methylaspartate; D005998:Glycine