Identification and Characterization of Novel NMDA Receptor Antagonists Selective for NR2A-over NR2B-Containing Receptors
Identification and Characterization of Novel NMDA Receptor Antagonists Selective for NR2A-over NR2B-Containing Receptors
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DOI：
10.1124/jpet.110.172544
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发表时间：
2010-12-01
影响因子：
3.5
通讯作者：
Cardullo, Francesca
中科院分区：
文献类型：
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作者：
Bettini, Ezio;Sava, Anna;Cardullo, Francesca
NR 1/NR 2A是N-甲基-D-天冬氨酸受体（NMDARs）的一个亚型，其是在中枢神经系统中高度表达的谷氨酸和甘氨酸门控的Ca（2+）渗透性通道。使用瞬时转导有NR 1/NR 2A NMDAR亚基的人骨肉瘤（U-2 OS）细胞的高通量筛选（HTS）活动，在专门设计的荧光成像板读数器（FLIPR）/Ca（2+）测定中进行测试，鉴定了磺酰胺衍生物系列，例如3-氯-4-氟-N-[（4-{[2-（苯羰基）肼基]羰基}苯基）甲基]苯磺酰胺（化合物1）和噻二唑衍生物N-（环己基甲基）-2-（{5[（苯甲基）氨基]-1，3，4-噻二唑-2-基}硫基）乙酰胺（化合物13）作为新的NR 1/NR 2A受体拮抗剂。化合物1和13分别对NR 1/NR 2A受体表现出亚微摩尔和微摩尔的效力，尽管它们在高达50 μ M浓度的含NR 2B受体下没有表现出活性。在FLIPR NR 1/NR 2A测定中，添加ImM甘氨酸而不是ImM L-谷氨酸能够克服化合物1和13的抑制作用。然而，化合物1和13取代谷氨酸位点拮抗剂[（3）H]D，L-（E）-2-氨基-4-丙基-5-膦酰基-3-戊烯酸（[（3）H]CGP 39653）的程度大于甘氨酸位点拮抗剂[（3）H]3-[（E）-2-羧基-2-苯基乙烯基]-4，6-二氯-1H-吲哚-2-羧酸（[（3）H]MDL 105，519），在大鼠脑皮质结合试验中。基于FLIPR细胞的电生理和生物化学结合测定的结果表明，化合物1和13是新型NMDAR配体的原型，据我们所知，它们是NR 1/NR 2A上的第一选择性拮抗剂，而不是NR 1/NR 2B受体，并且可能构成能够阐明NR 2A亚基在生理和病理条件下的相对作用的有用工具。
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 mu M 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]-4,6dichloro-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.