Abstract
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 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.
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Project supported by grants from the Ministry of Science and Technology of China (2002AA-2Z343A), Chinese Academy of Sciences (KSCX1-SW-11-2) and Shanghai Municipality Science and Technology Development Fund (03dz19224).
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Wu, B., Gao, J. & Wang, Mw. Development of a complex scintillation proximity assay for high throughput screening of PPARγ modulators. Acta Pharmacol Sin 26, 339–344 (2005). https://doi.org/10.1111/j.1745-7254.2005.00040.x
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DOI: https://doi.org/10.1111/j.1745-7254.2005.00040.x
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