Abstract
To develop a complex high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel peroxisome proliferator-activated receptor gamma (PPARgamma) modulators. Full-length PPARgamma and retinoid X receptor alpha (RXRalpha), biotinylated PPAR response element (PPRE), [3H]BRL49653 and streptavidin-coated FlashPlate or microbead were used to develop an HTS assay based on SPA technology. This 'ABCDE' method was validated against conventional hydroxyapatite (HA) assay and applied to large-scale screening of 16,000 synthetic compounds and natural product extracts. (1) IC50 values of positive control compounds (BRL49653 and troglitazone) obtained from the 'ABCDE' method and HA assay were comparable and consistent with those reported elsewhere; (2) Approximately 178 compounds, showing more than 70% competitive inhibition on BRL49653 binding to PPARgamma, were identified initially by the 'ABCDE' method (microbead); (3) Secondary screening using FlashPlate and cross-reactivity studies with RARalpha, beta, gamma and RXRalpha,beta, gamma confirmed that 12 compounds possessed specific PPARgamma binding properties including 2 with IC50 values less than 0.5 micromol/L and novel chemical structures. The 'ABCDE' method using either FlashPlate or microbead, is a highly efficient, automatable, and robust tool to screen potential PPARgamma modulators in HTS setting. Its application may be expanded to other nuclear receptors that form heterodimers upon activation.
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