Acta Pharmacologica Sinica 2005 March; 26 (3): 339-344; doi: 10.1111/j.1745-7254.2005.00040.x

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:  Full-length PPARγ and 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, γ and RXRα, β, γ confirmed that 12 compounds possessed specific PPARγ binding properties including 2 with IC₅₀ values less than 0.5 µmol/L and novel chemical structures.

Conclusion: 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.

Keywords: peroxisome proliferator-activated receptor gamma; retinoid X receptor alpha; scintillation proximity assay; high-throughput screening