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BioAssay: AID 504389

qHTS Assay for Antagonists of the Thyroid Stimulating Hormone Receptor: SAR for Probe

TSH is an alpha/beta heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family. The actions of TSH are mediated by a seven-transmembrane receptor, which upon TSH binding couples preferentially to the G-alpha (s) protein (Gs), resulting in activation of adenylate cyclase and increase in cyclic adenosine 3', 5' monophosphate more ..
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 Tested Compounds
 Tested Compounds
All(118)
 
 
Active(4)
 
 
Inactive(43)
 
 
Inconclusive(71)
 
 
 Tested Substances
 Tested Substances
All(119)
 
 
Active(4)
 
 
Inactive(43)
 
 
Inconclusive(72)
 
 
AID: 504389
Data Source: NCGC (TSHR508)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-01
Hold-until Date: 2011-08-25
Modify Date: 2011-08-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 4
Related Experiments
AIDNameTypeComment
488980qHTS Assay for Antagonists of the Thyroid Stimulating Hormone ReceptorConfirmatorydepositor-specified cross reference: Primary screen.
488990qHTS Assay for Inverse Agonists of the Thyroid Stimulating Hormone Receptor: SummarySummarydepositor-specified cross reference: Summary assay for project.
504379Concentration-response assay for inverse agonists of the thyroid stimulating hormone receptor: ELISA activity detectionConfirmatorysame project related to Summary assay
504380RT-PCR Analysis of TSH-dependent transcripts in primary cultures of human thyrocytesOthersame project related to Summary assay
504384Counterscreen for antagonists of the thyroid stimulating hormone receptor: ELISA activity in a Follicle Stimulating Hormone receptor cell lineConfirmatorysame project related to Summary assay
504385Counterscreen for antagonists of the thyroid stimulating hormone receptor: ELISA activity in a Luteinizing Hormone Receptor cell lineConfirmatorysame project related to Summary assay
504387Concentration-response assay for antagonists of the thyroid stimulating hormone receptor: ELISA activity detectionConfirmatorysame project related to Summary assay
504812Inverse Agonists of the Thyroid Stimulating Hormone Receptor: HTS campaignScreeningsame project related to Summary assay
Description:
TSH is an alpha/beta heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family. The actions of TSH are mediated by a seven-transmembrane receptor, which upon TSH binding couples preferentially to the G-alpha (s) protein (Gs), resulting in activation of adenylate cyclase and increase in cyclic adenosine 3', 5' monophosphate (cAMP). The TSH receptor (THSR) is mainly expressed in thyroid follicular cells and regulates their growth and function. This cell based assay was conducted on a TSHR transfected HEK 293 cell line, measuring cAMP stimulation using antibody-based Homogeneous Time Resolved Fluorescence (HTRF).
Our goal is to identify small molecules that will inhibit (antagonize) the thyroid-stimulating hormone receptor (TSHR), a member of the 7 transmembrane-spanning receptor (7TMR) family of cell surface receptors. TSHR-mediated hyperthyroidism is important in thyroid pathology. The development of small molecule antagonists of TSHR may lead to therapeutic approaches for TSHR-mediated hyperthyroidism caused by constitutively activating mutations or stimulating auto-antibodies associated with Graves' disease. Graves' disease is the leading cause of hyperthyroidism and is caused by autoantibodies that stimulate TSHR (TsAbs) causing thyroid growth and unregulated overproduction of thyroid hormones and existing treatments have liabilities (agranulocytosis). In addition, TSHRs are known to be expressed in multiple extrathyroidal tissues including bone, brain, kidney, testis, fat and cells of the immune system but the role of the TSHR in these tissues is not clear. Therefore, TSHR antagonists/inverse agonists could be used as probes of extrathyroidal TSHR function. Finally, TSHR inverse agonists, which are a subclass of antagonists that inhibit agonist-independent (or basal or constitutive) TSHR signaling, could be used to inhibit TSH-independent signaling in patients with recurrent or metastatic thyroid cancer who are receiving thyroid hormone to suppress TSH.
The present assay presents confirmation data for analogues of series of interest in the primary screening assay.
Protocol
An anti cAMP antibody contains a fluorescent cryptate tag (excitation 337 nm; emission 620 nm) and the acceptor is cAMP-d2 (excitation 620 nm; emission 665 nm). When antibody and cAMP-d2 interact and are excited by a 337 nm light, a FRET occurs resulting in emission at 665 nm. The assay is competition between cAMP-d2 and cAMP (from the cell lysate). The homogeneous protocol allows for one step dispense after stimulation that is automation friendly.

Step, Parameter, Value, Description
1, Cells, 3 uL, 2000 TSHR freshly harvested cells/well 1536 TC treated White solid bottom plate (Cells resuspended in 10 mM HEPES buffered HBSS and 70 uM RO 20-1724)
2, Compound, 23 nL, Libraries and controls
3, Compound, 1 uL, EC80 TSH (4 nM final)
4, Time, 2 hours, 37C
5, Reagent, 2 uL, cAMP antibody diluted 1:20 in lysis buffer
6, Time, 30 minutes, Room temperature
7, Detector, HTRF settings, Viewlux
Comment
Compound Ranking:

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, apparent inhibitors are ranked higher than compounds that showed apparent activation.
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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0003244270 uM (0.000324427μM**)% Activity at given concentration.Float%
15Activity at 0.0009732809 uM (0.000973281μM**)% Activity at given concentration.Float%
16Activity at 0.00292 uM (0.00291984μM**)% Activity at given concentration.Float%
17Activity at 0.00876 uM (0.00875953μM**)% Activity at given concentration.Float%
18Activity at 0.026 uM (0.0262786μM**)% Activity at given concentration.Float%
19Activity at 0.079 uM (0.0788358μM**)% Activity at given concentration.Float%
20Activity at 0.237 uM (0.236507μM**)% Activity at given concentration.Float%
21Activity at 0.710 uM (0.709522μM**)% Activity at given concentration.Float%
22Activity at 2.129 uM (2.12857μM**)% Activity at given concentration.Float%
23Activity at 6.386 uM (6.3857μM**)% Activity at given concentration.Float%
24Activity at 19.16 uM (19.1571μM**)% Activity at given concentration.Float%
25Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
26Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH090855-01

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