Aim: 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.
Methods: 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.
Results: (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.
Conclusions: 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.