A yeast two-hybrid technology-based system for the discovery of PPARgamma agonist and antagonist.
Peroxisome proliferator-activated receptor gamma (PPARgamma) is an important therapeutic drug target against several diseases such as diabetes, inflammation, dyslipidemia, hypertension, and cancer. Ligand binding to PPARgamma is responsible for controlling the biological functions, and developing new technology to measure ligand-PPARgamma binding is significant for both the function study of the receptor and ligand discovery. In this study, we exploited an efficient approach for the discovery of PPARgamma agonist and antagonist via a yeast two-hybrid system based on the fact that PPARgamma interacts with the coactivator CBP (CREP-binding protein) ligand-dependently. We employed the MEL1 reporter gene instead of the traditionally used LacZ gene to evaluate the protein-protein interactions by conducting a convenient alpha-galactosidase assay in the yeast strain AH109 with genes of PPARgamma-LBD (ligand-binding domain) and CBP N terminus introduced. With this built screening platform, the EC(50) values of the PPARgamma agonists rosiglitazone, troglitazone, pioglitazone, indomethacin, 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)), and GI262570 were investigated, and the quantitatively antagonistic effect by IC(50) of the PPARgamma typical antagonist GW9662 on the rosiglitazone agonistic activity was fully examined. The reliability of this presented system evaluated by the comparable agreement of EC(50) and IC(50) values for the test compounds with the reported ones indicated that this yeast two-hybrid-based approach is powerful for PPARgamma agonist and antagonist screening. In addition, because this screening system is designed for use in a microtiter plate format where numerous chemicals could be readily screened, it is hoped that this yeast two-hybrid screening approach may be adaptable for high-throughput settings.
Chen
Qing
Q
Drug Discovery Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Chen
Jing
J
Sun
Tao
T
Shen
Jianhua
J
Shen
Xu
X
Jiang
Hualiang
H
D000813:Anilides / Q000494:pharmacology; D050882:CREB-Binding Protein; D002478:Cells, Cultured; D002839:Chromans / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D017930:Genes, Reporter; D007213:Indomethacin / Q000494:pharmacology; D008024:Ligands; D009687:Nuclear Proteins / Q000378:metabolism; D010080:Oxazoles / Q000494:pharmacology; D047495:PPAR gamma / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000077205:Pioglitazone; D015230:Prostaglandin D2 / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding; D000077154:Rosiglitazone; D045162:Thiazolidinediones / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism; D000077288:Troglitazone; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology; D015003:Yeasts; D000519:alpha-Galactosidase / Q000378:metabolism
C477819:15-deoxyprostaglandin J2; C457499:2-chloro-5-nitrobenzanilide; D000813:Anilides; D002839:Chromans; D008024:Ligands; D009687:Nuclear Proteins; D010080:Oxazoles; D047495:PPAR gamma; D045162:Thiazolidinediones; D015534:Trans-Activators; D000077154:Rosiglitazone; C422415:farglitazar; D014443:Tyrosine; D050882:CREB-Binding Protein; D000519:alpha-Galactosidase; D000077288:Troglitazone; D015230:Prostaglandin D2; D000077205:Pioglitazone; D007213:Indomethacin