AID	Name	Type	Probe	Comment
1469	qHTS for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2	confirmatory
1479	Total Fluorescence Counterscreen for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2	confirmatory
1570	Concentration Response Confirmation Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2, Fluorescein Fluoroprobe	confirmatory
1571	Total Fluorescence Confirmation Counterscreen for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2, Fluorescein Fluoroprobe	confirmatory
1572	Total Fluorescence Confirmation Counterscreen for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2	confirmatory
1573	Concentration Response Confirmation Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2	confirmatory
1485	Quantitative High-Throughput Screen for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2: Summary	summary
2512	qHTS Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2: Probe Summary	summary	1

NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLPCN Grant: DK058080
Assay Provider: R. Kip Guy, St. Jude Children's Research Hospital

NCGC Assay Overview:

Thyroid receptor (TR) regulates many homeostatic processes including basal metabolism, cardiovascular function, body weight, and lipid trafficking. TR modulators are potential therapeutics for obesity and hyperlipidemias but current thyroid analogs have undesirable side effects, particularly cardiac stimulation. To identify inhibitors that specifically prevent the interaction of TR with the steroid receptor coregulator 2 (SRC2), a fluorescence polarization assay was screened (Arnold et al., 2006). This assay detects interaction of the ligand-binding domain of human TRb with a fluorescein labeled SRC2 peptide, corresponding to a 20 amino acid region of the nuclear receptor interaction domain. Small molecule inhibitors that block the interaction of TR and SRC2 are detected by a decrease in fluorescence polarization.

NCGC Assay Protocol Summary:

Twenty uL/well 0.6 uM TRb and 20 nM fluorescein labeled SRC2-2 in protein buffer (20mM Tris hydrochloride, pH 7.4, 100mM NaCl, 10% glycerol, 1mM EDTA, 0.01% NP-40, 1mM DTT, 1uM T3 and 4% DMSO) was dispensed into black solid 384-well plates (Costar 3710) using a Biomek FX (Beckman Coulter) liquid handling system. Following transfer of 260 nL compound (ranging from 7 nM to 130 uM), the plates were incubated 3 hr at ambient temperature. The plates were read by an Envision (Perkin Elmer) to detect fluorescence polarization of SRC2 Fluorescein (480 nm excitation and 535 nm emission).

Arnold, L. A.; Estebanez-Perpina, E.; Togashi, M.; Shelat, A.; Ocasio, C. A.; McReynolds, A. C.; Nguyen, P.; Baxter, J. D.; Fletterick, R. J.; Webb, P.; Guy, R. K. A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding. Sci STKE 2006, p 13.

Compound Ranking:

Active compounds were assigned a score between 50 and 100 based on compound potency. Inactive compounds were assigned a score of 0 and inconclusive compounds were assigned a score of 25.

Grant Number: DK058080