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. This secondary assay characterized compounds identified in the qHTS for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2 (PubChem AID 1485) for selectivity against the vitamin D receptor (VDR). This assay detects interaction of the ligand-binding domain of human VDR with an Alexa Fluor 647 labeled SRC2-3 peptide, corresponding to a 20 amino acid region of the nuclear receptor interaction domain (Teichert et al., 2009). Small molecule inhibitors that block the interaction of VDR and SRC2-3 are detected by a decrease in fluorescence polarization.

Teichert A, Arnold LA, Otieno S, Oda Y, Augustinaite I, Geistlinger TR, Kriwacki RW, Guy RK, Bikle DD. Quantification of the vitamin D receptor-coregulator interaction. Biochemistry. 2009 48(7):1454-61.

NCGC Assay Protocol Summary:

Twenty uL/well 1 uM VDR and 5 nM Alexa Fluor 647 SRC2-3 peptide in buffer (25 mM PIPES, pH 6.75, 50 mM NaCl, 0.01% NP-40, 6 uM LG190178, 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 Alexa Fluor 647 (620 nm excitation and 688 nm emission).

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