Implementation of a Fluorescence-Based Screening Assay Identifies Histamine H3 Receptor Antagonists Clobenpropit and Iodophenpropit as Subunit-Selective N-Methyl-D-Aspartate Receptor Antagonists
Implementation of a Fluorescence-Based Screening Assay Identifies Histamine H3 Receptor Antagonists Clobenpropit and Iodophenpropit as Subunit-Selective N-Methyl-D-Aspartate Receptor Antagonists
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DOI：
10.1124/jpet.110.166256
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发表时间：
2010-06-01
影响因子：
3.5
通讯作者：
Traynelis, Stephen F.
中科院分区：
文献类型：
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作者：
Hansen, Kasper B.;Mullasseril, Praseeda;Traynelis, Stephen F.
N-甲基-D-天冬氨酸 (NMDA) 受体是配体门控离子通道，介导中枢神经系统兴奋性突触传递的缓慢、Ca2+渗透性成分，在突触可塑性、神经元发育和多种神经系统疾病中发挥关键作用。我们描述了一种基于荧光的测定法，用于测量 BHK-21 细胞系中 NMDA 受体介导的细胞内钙变化，该细胞系在四环素诱导型启动子 (Tet-On) 的控制下稳定表达 NMDA 受体 NR2D 和 NR1。该测定通过使用超最大浓度的谷氨酸和甘氨酸来选择性地鉴定变构调节剂，以尽量减少竞争性拮抗剂的检测。通过在来自两个小型重点库（包括市售的药理学活性化合物库）的 1800 种筛选化合物中成功识别已知的非竞争性但非竞争性 NMDA 受体拮抗剂，对该测定进行了验证。来自初级筛选的命中通过二级筛选进行验证，二级筛选使用双电极电压钳记录非洲爪蟾卵母细胞中表达的重组 NMDA 受体。该策略鉴定了几种新型 NMDA 受体功能调节剂，包括组胺 H3 受体拮抗剂 clobenpropit 和 iodophenpropit，以及香草酸受体瞬时受体电位阳离子通道、亚家族 V、成员 1 (TRPV1) 拮抗剂 capsazepine。这些化合物是非竞争性拮抗剂，组胺 H3 受体配体对 NR1/NR2B NMDA 受体显示出亚微摩尔效力，这提高了开发同时对谷氨酸和组胺受体系统发挥高效作用的化合物的可能性。此外，体内组胺 H3 受体抑制的一些作用也可能涉及 NMDA 受体拮抗作用。
N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supra-maximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.