Development of a complex scintillation proximity assay for high throughput screening of PPAR_γ modulators¹

Aim: 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 16000 synthetic compounds and natural product extracts.

Results: (1) IC₅₀ values of positive control compounds (BRL4963 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 ‘ABBCDE’ method (microbead); (3) Secondary screening using FlashPlate and cross-reactivity studies with RARα, β, γ and RXRαβ, 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 Flash Plate 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.