Original Articles

Development of a complex scintillation proximity assay for highthroughput screening of PPARγ modulators

Authors: Bin WU, Jie GAO, Ming-wei WANG


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
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. 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.

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