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

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Gα15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-phosphonobutyricacid(L-AP4)>L-Serine-O-phosphate(L-SOP)>L-Glutamicacid(L-Glu);拮抗剂是:(RS)-α-Methylserine-O-phosphate(MSOP)>(RS)-α-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。

To identify metabotropic glutamate receptor 4 （mGluR4） modulators by Ca^2＋ 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 （Gα15） cells. Recombinant stable mGluR4 cell line was selected under Zeocin and validated by Ca^2＋ 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）-α-Methylserine-O-phosphate （MSOP）〉（RS）-α-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.