A cell-based high-throughput screening assay for Farnesoid X receptor agonists.
OBJECTIVE
To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library.


METHODS
cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid.


RESULTS
After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z' factor value of 0.65.


CONCLUSIONS
A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.
Zheng
Zhi-Hui
ZH
Insitute of Medicinal Biotechnology, Peking Union Medical College / Chinese Academy of Medical Sciences, Beijing 100050, China. z_zhihui2003@yahoo.com.cn
Lv
Guo-Ping
GP
Si
Shu-Yi
SY
Dong
Yue-Sheng
YS
Zhao
Bao-Hua
BH
Zhang
Hua
H
He
Jian-Gong
JG
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000819:agonists* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D000960:Hypolipidemic Agents / Q000032:analysis*; D010957:Plasmids; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists* / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D014157:Transcription Factors / Q000819:agonists* / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D000099150:Receptor, Farnesoid X-Activated
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000960:Hypolipidemic Agents; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D000099150:Receptor, Farnesoid X-Activated