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BioAssay: AID 2512

qHTS Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2: Probe Summary

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 ..
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Probe(1)
 
 
Active(5)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Probe(1)
 
 
Active(5)
 
 
Inactive(1)
 
 
AID: 2512
Data Source: NCGC (TR326)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-11
Modify Date: 2011-03-03

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 1    Active: 5
Related Experiments
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AIDNameTypeComment
1469qHTS for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Confirmatorydepositor-specified cross reference: Primary screen tested at 6 concentrations from 92 uM to 74 nM
1479Total Fluorescence Counterscreen for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Confirmatorydepositor-specified cross reference: Primary counterscreen tested at 6 concentrations from 92 uM to 74 nM, total fluorescence readout
1567Cell-based Gene Reporter Secondary Assay to Characterize qHTS Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Confirmatorydepositor-specified cross reference: Primary screen confirmation
1568Cell Viability Secondary Assay to Characterize qHTS Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Confirmatorydepositor-specified cross reference: Primary screen confirmation cytotoxicity readout
1570Concentration Response Confirmation Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2, Fluorescein FluoroprobeConfirmatorydepositor-specified cross reference: Primary screen confirmation: TR-beta inhibition measured by the fluorescein fluoroprobe
1571Total Fluorescence Confirmation Counterscreen for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2, Fluorescein FluoroprobeConfirmatorydepositor-specified cross reference: Primary screen confirmation: TR-beta inhibition measured by the fluorescein fluoroprobe, total fluor
1573Concentration Response Confirmation Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Confirmatorydepositor-specified cross reference: Primary screen confirmation: TR-beta inhibition measured by the texas red fluoroprobe
2444Protein Interaction Secondary Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2, Alpha ScreenConfirmatorydepositor-specified cross reference: Secondary assay 3: Alpha screen for protein interaction inhibition
2447Cell Viability Secondary Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Confirmatorydepositor-specified cross reference: Secondary assay 4: Cytotoxicity measured by HepG2 cell viability
2448Androgen Receptor Selectivity Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Confirmatorydepositor-specified cross reference: Selectivity secondary assay 3: AR inhibition
2449PPAR gamma Selectivity Assay For Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Confirmatorydepositor-specified cross reference: Selectivity secondary assay: PPAR-gamma inhibition
2455Vitamin D Receptor Selectivity Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Confirmatorydepositor-specified cross reference: Selectivity secondary assay 2: VDR inhibition
2479Gene Reporter Secondary Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2 in a Thyroid Receptor AssayConfirmatorydepositor-specified cross reference: Secondary assay 2: Gene reporter assay for transcription inhibition
2487Secondary Confirmation Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2, Fluorescein FluoroprobeConfirmatorydepositor-specified cross reference: Secondary assay: TR-beta inhibition measured by the fluorescein fluoroprobe
2490Secondary Confirmation Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2, Texas Red FluoroprobeConfirmatorydepositor-specified cross reference: Confirmation assay: retest of selected compounds
2552Irreversible Binding Assay for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2Otherdepositor-specified cross reference: Primary screen confirmation: Irreversible binding assay
Description:
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). Small molecule inhibitors that block the interaction of TR and SRC2 are detected by a decrease in fluorescence polarization. Total fluorescence (555 nm excitation and 632 nm emission) was also measured to identify potential fluorescent and light absorbing compounds. Such compounds may be artifacts that interfere with the fluorescence polarization assay.

The assay was screened against the MLSMR in a quantitative high-throughput screen (qHTS) of six library concentrations where FP (AID 1469) and FP total fluorescence (AID 1479) readouts were collected. The total fluorescence measurement for the FP assay served as a counterscreen to identify fluorescent compounds. The titration-response data were curve-fit and classified to identify actives and nascent SAR analysis was performed. Compounds with quality curve-fit in the FP measurement and little or no activity in the total fluorescence readout were selected for follow up. The follow up compounds were tested in the original screening assay (AID 2490) as well as an orthogonal assay where FP was measured by a fluorescein fluoroprobe (AID 2487).

A nitrosulfonyl benzoate series that showed good performance in the follow-up assays and suitable chemical tractability was chosen for probe optimization. Analogs from this series were tested in several secondary assays to further confirm their activity and determine the selectivity of TR-beta inhibition, including an Alpha Screen (AID 2444), a cell based gene reporter assay for transcription inhibition (AID 2479), a cell viability assay that measures cytotoxicity (AID 2447), and other nuclear receptor assays: PPAR-gamma (AID 2449), VDR (AID 2455) and AR (AID 2448) for selectivity. These assays indicated the nitrosulfonyl benzoate series were selective inhibitors of TR-beta and SRC-2 interaction in vitro. The most active compound (ML151) from this series was nominated as the probe for TR inhibition.

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.
Protocol
Please refer to other AIDs (1469, 1479, 2490, 2487, 2479, 2444, 2447, 2449, 2455, 2448) for detailed assay protocols.
Comment
This summary is written for the purposes of summarizing the probe activities from the project. MLSCN probes are given a score of 100. Molecules in the prior art are given a score of 80. Other, less active molecules in the same chemical series as the probe molecules are given a score of 50. Molecules pending validation are given a score of 10. Inactive analogues from these series are given a score of 0.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML151
PubChem Substance ID (SID) for probe 1: 87550851
PubChem Compound ID (CID) for probe 1: 5184800
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 1400
Target for probe 1: TR-SRC2 (gi: 189491771)
Disease relevance for probe 1: Obesity and hyperlipidemias
Anti-target for probe 1: AR-SRC2; VDR-SRC2; PPARg- DRIP
Fold selectivity for probe 1: > 92; 38; > 92
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK56238/ (ID: 2510184)
Grant number for probe 1: DK058080
NCBI Book chapter title for probe 1: qHTS for Inhibitors of the Interaction of Thyroid Hormone Receptor and Steroid Receptor Coregulator 2
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity SummaryIndicates type of activity observed.String
2Fl-SRC2-2 Potency (uM)Potency observed in the TR inhibition fluorescence polarization assay (fluorescein probe).FloatμM
3Tx-SRC2-2 Potency (uM)*Potency observed in the TR inhibition fluorescence polarization assay (Texas red probe).FloatμM
4Transcription Inhibition Potency (uM)Potency observed in the TR transcription assay.FloatμM
5Transcription Inhibition Efficacy (%)Efficacy (% inhibition) observed in the TR transcription assay.Float%
6Alpha Screen Potency (uM)Potency observed in the TR inhibition assay (alpha screen).FloatμM
7PPAR-gamma ActivityActivity observed in the PPAR-gamma inhibition assay.String
8VDR Potency (uM)Potency observed in the VDR inhibition assay.FloatμM
9AR ActivityActivity observed in the AR inhibition assay.String
10HepG2 Cell Viability Potency (uM)Potency observed in the HepG2 cell viability assay.FloatμM
11Compound QCCompound QC information.String

* Activity Concentration.
Additional Information
Grant Number: DK058080

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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