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BioAssay: AID 2104

Secondary Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: ELISA Activity Detection

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(32)
 
 
Active(12)
 
 
Inactive(9)
 
 
Inconclusive(11)
 
 
 Tested Substances
 Tested Substances
All(33)
 
 
Active(13)
 
 
Inactive(9)
 
 
Inconclusive(11)
 
 
AID: 2104
Data Source: NCGC (TSHR746)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2009-11-05
Modify Date: 2010-12-20

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: thyroid stimulating hormone receptor [Homo sapiens]
Description ..   
Protein Family: G protein-coupled glucose receptor regulating Gpa2

Gene:TSHR     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 12
Related Experiments
AIDNameTypeProbeComment
926qHTS Assay for Agonists of the Thyroid Stimulating Hormone ReceptorConfirmatory depositor-specified cross reference
933Secondary Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: HTRF Activity DetectionConfirmatory depositor-specified cross reference
939Confirmation Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone ReceptorConfirmatory depositor-specified cross reference
1401Quantitative High-Throughput Screen for Agonists of Thyroid Stimulating Hormone Receptor: SummarySummary2 depositor-specified cross reference
1402Counterscreen for Agonists of Thyroid Stimulating Hormone Receptor: HTRF Activity in a Luteinizing Hormone Receptor Cell LineConfirmatory depositor-specified cross reference
1403Counterscreen for Agonists of Thyroid Stimulating Hormone Receptor: HTRF Activity in a Follicle Stimulating Hormone Receptor Cell LineConfirmatory depositor-specified cross reference
938qHTS Assay for Agonists of the Thyroid Stimulating Hormone Receptor: Activators of Intracellular cAMP Concentrations in Parental HEK 293Confirmatory same project related to Summary assay
953Confirmation Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: Activators of Intracellular cAMP Concentrations in Parental HEK 293Confirmatory same project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
MLSCN Grant: 1 X01 MH080680-01
PI Name: Dr. Marvin Gershengorn, NIH

NCGC Assay Overview:

Confirmation of Thyroid Stimulating Hormone Receptor Agonists

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. Recombinant TSH is used to activate TSHR in patients with thyroid cancer receiving thyroid hormone suppression therapy and to screen for residual tumor after surgery, but it is expensive and must be administered intramuscularly. An orally active small molecule TSHR agonist would serve as an invaluable research tool for studying TSHR pharmacology and physiology, and would have multiple advantages in therapeutic settings. However, no selective small molecule agonist of the TSH receptor exists, and no small molecule screen for TSHR agonists has ever been reported.
A confirmatory assay of putative TSHR agonists was conducted on a HEK 293 cell line stably expressing the TSHR, using an orthogonal detection technology to measure cAMP stimulation: an ELISA based cAMP competition assay using cAMP-Screen Direct System (Applied Biosystems, Foster City , CA).
Protocol
NCGC Assay Protocol Summary:
HEK 293 cells expressing TSHR were cultured for 24 h before incubation for 1 h in serum-free DMEM containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) (SIGMA) and small molecule ligands in a humidified 5% CO2 incubator at 37C. Following aspiration of the medium, cells were lysed using lysis buffer of the cAMP-Screen DirectTM System (Applied Biosystems). The cAMP content of the cell lysate was determined using the method described in the manufacturer#s protocol. An enzyme substrate with a luminescent enhancer generates glow light emission kinetics. Light signal intensity, which is inversely proportional to the cAMP level in the sample, is measured in a luminometer (Perkin Elmer, 1420 Multilabel, Victor 3) for 3 seconds/well 30 min after substrate addition. Potencies (i.e., EC50) of the ligands were obtained from dose-response curves by data analysis with GraphPad Prism 4 software for Windows.
Comment
Compound Ranking:
Compounds tested with an AC50 below one micomolar were given a score of 100. Less active compounds were given a score of 10 and inactive compounds were given a score of 0.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
From ChEMBL:
Assay Type: Functional
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Potency*Concentration at which compound exhibits half-maximal efficacy, AC50.FloatμM

* Activity Concentration.
Additional Information
Grant Number: MH080680-01

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