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 confirm that inhibitors specifically prevent the interaction of TR with the steroid receptor coregulator 2 (SRC2), an orthogonal TR-SRC2 interaction assay using Alpha Screen (PerkinElmer) was employed. This assay detects the interaction of the ligand-binding domain of human TR# with a 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 chemiluminescence.

NCGC Assay Protocol Summary:

Fifteen uL/well of 100 nM TRb and 100 nM SRC2-2-peg2-biotin in buffer (25 mM HEPES, 100 mM NaCl, 1 mM DTT, 0.1% BSA, 0.01% NP-40) was dispensed into white 384 well Optiplates (PerkinElmer). Following transfer of 135 nL compound (ranging from 90 nM to 200 uM), plates were incubated 1 hr, and 5 uL acceptor beads (6.3 ug/ml TRb antibody [Santa Cruz #sc-32754] and 40 ug/ml protein A beads in buffer) were added. After 30 min incubation, 5 uL of streptavidin donor beads were added and after 90 min incubation, the plates were read by an Envision (Perkin Elmer) to detect luminescence of the donor and acceptor bead interaction (680 nm excitation and 520-620 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