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首页> 外文期刊>Journal of biomolecular screening: The official journal of the Society for Biomolecular Screening >Validation of a fluorescent imaging plate reader membrane potential assay for high-throughput screening of glycine transporter modulators
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Validation of a fluorescent imaging plate reader membrane potential assay for high-throughput screening of glycine transporter modulators

机译：用于高通量筛选甘氨酸转运蛋白调节剂的荧光成像板读取器膜电位测定法的验证

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摘要

A fluorescent imaging plate reader (FLIPR) membrane potential (V-m) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT(2)) in a stable rGlyT(2)-HEK cell line. Data show that glycine activation of rGlyT(2) consistently results in a concentration-dependent V-m response on the FLIPR that is blocked by the potent and selective GlyT(2) antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [H-3]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT(2) physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT(2) inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based V-2 assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT(2).
机译：评价了荧光成像板读数器（FLIPR）膜电位（Vm）分析在稳定的rGlyT（2）-HEK细胞系中对大鼠2型甘氨酸转运蛋白（rGlyT（2））的药理特性和高通量筛选（HTS） 。数据显示，rGlyT（2）的甘氨酸激活始终导致FLIPR上的浓度依赖性Vm反应，该反应被有效和选择性的GlyT（2）拮抗剂4-苄氧基-3,5-二甲氧基-N- [1-二甲基氨基-环戊基）甲基]-苯甲酰胺（Org-25543）。激动剂和拮抗剂药理学与使用常规[H-3]甘氨酸摄取测定和电生理学报道的那些相匹配。甘氨酸反应取决于与GlyT（2）生理学一致的缓冲离子组成。分析的信噪比和变异系数符合进行HTS的足够统计标准。筛选化学药品清单的结果表明，该测定法能够成功识别和确认GlyT（2）抑制剂。该测定法的优点是其均一性，与96孔和384孔形式的相容性以及缺乏放射活性。因此，作者得出的结论是，在FLIPR上进行基于荧光的V-2检测是一种可行的方法，该方法可用于鉴定和鉴定电转运蛋白rGlyT（2）的小分子调节剂。

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