Development of a complex scintillation proximity assay for high throughput screening of PPARγ modulators
AbstractAim:To develop a complex high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel peroxisome proliferators-activated receptor gamma (PPARγ) modulators.Methods:Full length PPARγ and retinoid X receptor alpha (RXRα), biotinylated PPAR response element (PPER), [3H]BRL49653 and streptavidin-coated Flash Plate 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 then 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, γ and RXRα, β, γ confirmed that 12 compounds possessed specific PPARγ binding properties including 2 with IC50 values less then 0.5 μmol/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 PPARγ modulators in HTS setting. Its application May be expanded to other nuclear receptors that form heterodimers upon activation.