[Development of a functional cell-based HTS assay for the identification mGluR4 modulators].
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 (Ga alpha15) 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)-alpha-Methylserine-O-phosphate (MSOP) > (RS)-alpha-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.
Zhang
Yaling
Y
Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101300, China. zhangyl@big.ac.cn
Bai
Yanqiu
Y
D000613:Aminobutyrates / Q000494:pharmacology*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D010768:Phosphoserine / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D018094:Receptors, Metabotropic Glutamate / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000235:genetics; D014162:Transfection
D000613:Aminobutyrates; D018076:DNA, Complementary; D018094:Receptors, Metabotropic Glutamate; C120877:methylserine phosphate; D010768:Phosphoserine; C012729:2-amino-4-phosphonobutyric acid; C108823:metabotropic glutamate receptor 4