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BioAssay: AID 602293

qHTS for antagonists of the Thyroid Stimulation Hormone Receptor: Hit Validation in HTRF Activity in a Luteinizing Hormone Receptor Cell Line

The thyroid-stimulating hormone receptor (TSHR) is a member of the 7 trans-membrane-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 more ..
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 Tested Compounds
 Tested Compounds
All(1282)
 
 
Active(29)
 
 
Inactive(1128)
 
 
Inconclusive(125)
 
 
 Tested Substances
 Tested Substances
All(1284)
 
 
Active(29)
 
 
Inactive(1130)
 
 
Inconclusive(125)
 
 
AID: 602293
Data Source: NCGC (TSH721)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-05

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 29
Related Experiments
AIDNameTypeComment
504813Antagonists of the Thyroid Stimulating Hormone Receptor: SummarySummarydepositor-specified cross reference: Summary AID
504810Antagonists of the Thyroid Stimulating Hormone Receptor: HTS campaignScreeningsame project related to Summary assay
504821Antagonists of the Thyroid Stimulating Hormone Receptor: ValidationConfirmatorysame project related to Summary assay
602292qHTS for antagonists of the Thyroid Stimulation Hormone Receptor: Hit Validation in Primary ScreenConfirmatorysame project related to Summary assay
602441qHTS for antagonists of the Thyroid Stimulation Hormone Receptor: Hit Validation in Parental Cell LineConfirmatorysame project related to Summary assay
602442qHTS for antagonists of the Thyroid Stimulation Hormone Receptor: Hit Validation for Inverse AgonismOthersame project related to Summary assay
602447qHTS for antagonists of the Thyroid Stimulation Hormone Receptor: Hit Validation with Beta-2 Adrenergic ReceptorOthersame project related to Summary assay
602448qHTS for antagonists of the Thyroid Stimulation Hormone Receptor: Hit Validation with Follickle Stimulating Hormone Receptor (FSHR)Othersame project related to Summary assay
Description:
The thyroid-stimulating hormone receptor (TSHR) is a member of the 7 trans-membrane-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.

In collaboration between scientist from the NCGC and the NIDDK, a miniaturized HTRF high-throughput screen was developed. It's a cell based assay, where 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. In order to confirm specificity of the compounds to the TSHR, a confirmatory assay was conducted on a similar family member, the Luteinizing Hormone (LH) receptor, transfected into a HEK 293 cell line, using an orthogonal detection technology to measure cAMP stimulation: antibody-based Homogeneous Time Resolved Fluorescence (HTRF). This line did not contain the CNG and was therefore incapable of being used in the primary assay format.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

Grant: MH090855
Assay Provider: Marvin Gershengorn, NIDDK
Protocol
Homogeneous Time Resolved Fluorescence (HTRF) cAMP Assay. HEK 293 cells stably transfected with a vector containing the LH receptor and maintained under hygromycin selection were obtained from the laboratory of Dr. Marvin Gershengorn. Compounds were assayed using a HTRF cAMP detection kit (Cisbio) on the LH receptor transfected cell line. Briefly, 750 cells were plated in 2.5 ul/well of complete media (DMEM containing 10 % FCS, and 100 uM RO 20-1724) in 1536 well solid bottom white plates and 23 nl/well compound in DMSO solution or controls was added. Following 30 minute incubation at room temperature, 2.5 ul/well of labeled d2 cAMP and 2.5 ul/well of anti-cAMP antibody (both diluted 1:20 in lysis buffer) were added to each well using a flying reagent dispenser (Aurora Discovery, San Diego) . Plates were measured using the Envision plate reader (PerkinElmer, Boston, MA) with excitation at 330 nm and emissions of 615 nm and 660 nm.
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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
From ChEMBL:
Assay Type: Functional
Result Definitions
Show more
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 0.00368 uM (0.00368μM**)% Activity at given concentration.Float%
15Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
16Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
17Activity at 0.460 uM (0.46μM**)% Activity at given concentration.Float%
18Activity at 2.300 uM (2.3μM**)% Activity at given concentration.Float%
19Activity at 11.55 uM (11.5499μM**)% Activity at given concentration.Float%
20Activity at 56.90 uM (56.8968μM**)% Activity at given concentration.Float%
21Activity at 58.00 uM (58μM**)% Activity at given concentration.Float%
22Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

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