ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Subject(s)
Humans , Base Sequence , Cell Line , DNA Primers , DNA, Complementary , DNA-Binding Proteins , Chemistry , Genetics , Hypolipidemic Agents , Plasmids , Receptors, Cytoplasmic and Nuclear , Chemistry , Genetics , Reproducibility of Results , Transcription Factors , Chemistry , Genetics , TransfectionABSTRACT
To establish a new high-throughput screening model for the agonist of PPARdelta, PPARdelta gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and subcloned to pGEM-T Vector for sequencing, then the PPARdelta fragment was excised by restriction enzymes, and inserted into pTARGET Vector to construct expression vector pTARGET-ppARdelta. Insert three copies of PPRE into pGl3-promoter vector to construct expression vector pGl3-PPRE x 3-luc. The vector pTARGET-ppARdelta was transiently cotransfected with pGl3-PPRE x 3-luc into different cell lines to assay the expression levels of luciferase. The PPARdelta agonist screening model was established and optimized. Bezafibrate and linoleic acid can induce the expression of luciferase significantly and in a dose-dependent manner. This method can be used for high throughput screening for the agonist of PPARdelta, which might become lead compounds for new anti-atheroscleriosis or anti-adiposity drugs.
Subject(s)
Animals , Humans , Mice , 3T3 Cells , Bezafibrate , Pharmacology , Cell Line , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Methods , Genetic Vectors , Chemistry , Genetics , HeLa Cells , Linoleic Acid , Pharmacology , Lipids , Chemistry , Luciferases , Genetics , Metabolism , PPAR delta , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , Transfection , MethodsABSTRACT
Human leukocyte elastase is an important selection target of inflammation and cancer. In this paper, a high throughput screening model was established for screening human leukocyte elastase inhibitors from thousands of strains of actinomycetes. As a result, a strain, N01WA-735 with potent suppression activity was isolated. Firstly, the strain N01WA-735 was identified as Streptomyces according to morphology and biochemical analysis. The Streptomyces N01WA-735 was processed by solvent extraction, silica column chromatography, Sephadex LH-20 column chromatography and crystallization to get a pure active compound named N01WA-735E. Its chemical structure was elucidated as the same as that of the compound named BE-52440A by physicochemical properties and spectral data of UV, MS, 1H-NMR and 13C-NMR respectively. The compound showed a strong inhibitory activity against human leukocyte elastase with IC50 of 3.02 micromol/L. The compound is reported as a human leukocyte elastase inhibitor for the first time.