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BioAssay: AID 504380

RT-PCR Analysis of TSH-dependent transcripts in primary cultures of human thyrocytes

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(1)
 
 
Active(1)
 
 
 Tested Substances
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Active(1)
 
 
AID: 504380
Data Source: NCGC (TSHR507)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-02-28
Hold-until Date: 2011-08-25
Modify Date: 2011-08-25

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
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
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
504389qHTS Assay for Antagonists of the Thyroid Stimulating Hormone Receptor: SAR for ProbeConfirmatorysame 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.

This assays looks for effects on TSH-dependent functions in primary human thyrocytes. Total RNA is purified using RNeasy Micro kits. RT-PCR is performed in 25-ul reactions using cDNA prepared from 100 ng or less of total RNA and Universal PCR Master Mix (Applied Biosystems). mRNA levels are measured using primers and probes from Applied Biosystems. Quantitative RT-PCR results are normalized to GAPDH to correct for differences in RNA input.
Protocol
Thyroid tissue samples were collected from normal thyroid tissue from patients undergoing total thyroidectomy for thyroid cancer at the National Institutes of Health Clinical Center. Surgical specimens were maintained in Hanks' balanced salt solution (HBSS) on ice, minced into small pieces, and digested with 3 mg/ml collagenase type IV (Life Technologies). Monodispersed cells were plated in 10 ml DMEM with 10% FBS in 10-cm tissue culture dishes and incubated at 37C in a humidified5%CO2 incubator, and after 24 h, the primary cultures of adherent thyroid cells were obtained. For determination of thyroglobulin (TG), thyroid peroxidase (TPO), TSHR, sodium iodide symporter (NIS), or deiodinase type 2 (DIO2) mRNAexpression, thyrocytes were seeded into 12-well plates at a density of 1 x 105 cells per well. The cells were incubated in DMEM with 2% FBS and 1 mM 3-isobutyl-1-methylxanthine (IBMX) for 48 h in the presence or absence of compound.
Comment
Active compounds which modulated TSHR pathway transcripts were given a score of 100, inactive compounds were given a score of 0.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1TSHR Probe ActivityPercent change in probe abundance following compound incubation.Float%
2TPO Probe ActivityPercent change in probe abundance following compound incubation.Float%
3TG Probe ActivityPercent change in probe abundance following compound incubation.Float%
4NIS Probe ActivityPercent change in probe abundance following compound incubation.Float%
5DIO2 Probe ActivityPercent change in probe abundance following compound incubation.Float%
Additional Information
Grant Number: MH090855-01

Data Table (Concise)
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