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A high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract
Siqi Lin1, Cindy L Bock, Debra B Gardner
1Leads Discovery, Dupont Pharmaceuticals Company, E357/1132, Experimental Station, Wilmington, Delaware 19880-0400,USA. siqi.lin@dupontpharma.com
Analytical Biochemistry
|December 18, 2001
Summary
A new high-throughput assay uses fluorescence polarization to measure nuclear receptor binding to compounds. This validated method, utilizing crude extracts, enables efficient screening for drug discovery.
Area of Science:
- Biochemistry
- Pharmacology
- Assay Development
Background:
- Nuclear receptors are crucial drug targets.
- High-throughput screening (HTS) is vital for drug discovery.
- Developing robust HTS assays for nuclear receptor-ligand interactions is essential.
Purpose of the Study:
- To develop a homogenous high-throughput assay for nuclear receptor binding.
- To utilize fluorescence polarization (FP) for detecting these interactions.
- To validate the assay using the human glucocorticoid receptor (GR) as a model.
Main Methods:
- A fluorescence polarization assay was developed.
- Crude nuclear receptor extracts were used, eliminating purification steps.
- Assay conditions (DMSO tolerance, temperature, stability) were optimized.
- The assay was validated in a 384-well format.
Main Results:
- Optimized conditions yielded a signal-to-background ratio of 2:1 and a Z'-factor of 0.7.
- The assay successfully evaluated known strong and weak GR ligands.
- Potential interferences from fluorescent compounds were addressed.
Conclusions:
- A validated, homogenous, high-throughput FP assay for nuclear receptor binding was established.
- This assay system is adaptable for screening various soluble nuclear receptors.
- The method facilitates efficient identification of potential drug candidates.