代谢型谷氨酸受体4亚型(mGluR4)高通量药物筛选模型的建立

Development of a functional cell-based HTS assay for the identification mGluR4 modulators

摘要:

为发现代谢型谷氨酸受体4亚型(mGluR4)的调节剂, 通过荧光检测胞内钙浓度的方法, 建立一个基于细胞功能性检测的高通量筛选(HTS)系统。将人mGluR4基因转染稳定表达Ga15蛋白的人胚肾细胞(HEK-293), 用Zeocin筛选获得稳定表达mGluR4的细胞株, 并通过钙流检测试验证实该细胞系的生物学功能。优化了实验系统中荧光染料的孵育时间, 溶剂二甲基亚砜(DMSO)耐受性, 以及溶剂氢氧化钠(NaOH)耐受性, 建立了可靠稳定的筛选系统。钙流检测试验数据表明, mGluR4细胞系对其激动剂的活性程度排序是: L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4)> L-Serine-O-phosphate (L-SOP)>L-Glutamic acid (L-Glu); 拮抗剂是: (RS)-a-Methylserine-O-phosphate (MSOP)> (RS)-a-Methyl-4-phosphonophenylglycine (MPPG)。在96和384细胞微孔培养板中, 得到该筛选系统Z’因子分别是0.80和0.65。结果表明, 该稳定细胞系拥有一个稳定的检测系统, 适合于mGluR4激动剂/拮抗剂的筛选。

Abstract:

To identify metabotropic glutamate receptor 4 (mGluR4) modulators by Ca2+ influx assay, we developed the functional cell-based high throughput-screening (HTS) assay. The human mGluR4 cDNA was transfected into HEK-293 stably expressing promiscuous G-protein (Ga15) cells. Recombinant stable mGluR4 cell line was selected under Zeocin and validated by Ca2+ influx assay. The assay was optimized on loading time of Fluo Calcium Indicator, Dimethyl sulfoxide (DMSO) tolerance and sodium hydroxide (NaOH) tolerance using agonist (L-Glutamic acid (L-Glu)) of mGluR4. The rank order of the agonist potency for the stable human mGluR4 cell line was L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4)>L-Serine-O-phosphate (L-SOP)>L-Glu, and of the antagonist potency was (RS)-a-Methylserine-O-phosphate (MSOP)>(RS)-a-Methyl-4-phosphonophenylglycine (MPPG). Z’ factor value of the cell line in 96- and 384-well plate format was 0.80 and 0.65. Our data indicate a successful development of functional human mGluR4 recombinant stable cell line that was suitable for high throughput screening to identify mGluR4 agonist/antagonist.