Total Fluorescence Counterscreen for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2
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 more ..
BioActive Compounds: 806
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. This assay detects interaction of the ligand-binding domain of human TRb with a Texas Red labeled SRC2 peptide, corresponding to a 20 amino acid region of the nuclear receptor interaction domain (Arnold et al., 2006). Total fluorescence (555 nm excitation and 632 nm emission) was measured to identify potential fluorescent and light absorbing compounds. Such compounds may be artifacts that interfere with the fluorescence polarization assay.
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.
NCGC Assay Protocol Summary:
For screening, 5 uL/well 0.6 uM TRb and 20 nM SRC2 Texas Red in protein buffer (20mM Tris hydrochloride, 100mM NaCl, 10% glycerol, 1mM EDTA, 0.01% NP-40, 1mM DTT, 1uM T3 and 5% DMSO) was dispensed into black solid 1536-well plates (Grenier) using a solenoid-based dispenser. Following transfer of 23 nL compound or DMSO vehicle by a pin tool, the plates were centrifuged 15 s at 1000 RPM and incubated 5 hr at ambient temperature. The plates were read by an Envision (Perkin Elmer) to detect fluorescence polarization of SRC2 Texas Red (555 nm excitation and 632 nm emission). Data were normalized to unbound (all components except TRb) and bound SRC2 Texas Red controls. Total fluorescence (TF) was calculated as follows: TF = S + 2P where S and P are fluorescence readings in the parallel and perpendicular channels, respectively.
Keywords: NIH Roadmap, MLPCN, MLI, MLSMR, qHTS, NCGC, qHTS, thyroid hormone receptor, steroid receptor coactivator, fluorescence polarization
In this assay we provide the total fluorescence data, which include % activity (the fluorescence intensity has been normalized to positive and negative controls) for each concentration as well as potencies for the active compounds.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, compounds that showed apparent activation are likely fluorescent and apparent inhibitors are likely absorbent compounds.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)