A yeast two-hybrid technology-based system for the discovery of PPARγ 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 MELI 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 EC50 values of the PPARgamma agonists rosiglitazone, troglitazone, pioglitazone, indomethacin, 15-deoxy-Delta12,14-prostaglan din J(2) (15d-PGJ(2)) and GI262570 were investigated, and the quantitatively antagonistic effect by IC50 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 EC50 and IC50 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 microliter 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. (C) 2004 Elsevier Inc. All rights reserved.