Development of a complex scintillation proximity assay for highthroughput screening of PPARγmodulators

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 (PPARγ) modulators. Methods: Fulllength PPARγand retinoid X receptor alpha (RXRα), 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 PPARγ, were identified initially by the ‘ABCDE‘ method (microbead); (3) Secondary screening using FlashPlate and cross-reactivity studies with RARα, β,γand RXRα,β,γconfirmed that 12 compounds possessed specific PPARγbinding properties including 2 with IC50 values less than 0.5μmol/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 PPARγmodulators in HTS setting. Its application may be expanded to other nuclear receptors that form heterodimers upon activation.

Development of a complex scintillation proximity assay for high-throughput screening of PPARgamma modulators

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.