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A phosphorimager-based filter binding thyroid hormone receptor competition assay for chemical screening
Joseph Chapo1, Yanyu Peng, Kelly R Pitts
1Molecular Pharmacology, Myogen, Inc., Westminster, CO 80021, USA.
Journal of Pharmacological and Toxicological Methods
|February 6, 2007
Summary
A new filter binding assay using phosphorimaging accurately verifies thyroid hormone receptor (THR) competitors. This efficient method significantly reduces time and radioactive waste compared to traditional assays.
Area of Science:
- Biochemistry
- Molecular Endocrinology
- Assay Development
Background:
- Thyroid hormone receptors (THR) play crucial roles in metabolism and development.
- Verifying small molecule modulators of THR is essential for drug discovery.
- Existing assays for THR competition can be time-consuming and generate significant waste.
Purpose of the Study:
- To develop and validate a novel phosphorimager-based filter binding assay for THR competition.
- To assess the efficiency and throughput of this new assay compared to conventional methods.
Main Methods:
- Utilized in vitro translated ligand binding domains (LBDs) of THRalpha and THRbeta.
- Employed en masse filtration using a 96-well vacuum manifold and nitrocellulose filters.
- Quantified receptor-bound radioactivity via phosphorimaging.
Main Results:
- The assay demonstrated a linear response for [I(125)]T3 over the dynamic range.
- IC(50) and K(i) values for T3 and known competitors (GC-1, DITPA) were comparable to published data.
- Filtration and phosphorimaging yielded similar binding values to Sephadex G-25 column separation.
Conclusions:
- En masse filtration and phosphorimaging is a valid and reliable method for THR competition assays.
- The new method offers a 3-fold time reduction and a 40-fold decrease in radioactive waste.
- This approach enhances assay throughput for screening large compound libraries.
