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BioAssay: AID 926

qHTS Assay for Agonists of the Thyroid Stimulating Hormone Receptor

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(72024)
 
 
Active(352)
 
 
Inactive(56359)
 
 
Inconclusive(12276)
 
 
Unspecified(3423)
 
 
 Tested Substances
 Tested Substances
All(73162)
 
 
Active(352)
 
 
Inactive(57004)
 
 
Inconclusive(12374)
 
 
Unspecified(3432)
 
 
AID: 926
Data Source: NCGC (TSHR742)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-20
Modify Date: 2010-07-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 352
Depositor Specified Assays
AIDNameTypeComment
1402Counterscreen for Agonists of Thyroid Stimulating Hormone Receptor: HTRF Activity in a Luteinizing Hormone Receptor Cell Lineconfirmatory
1403Counterscreen for Agonists of Thyroid Stimulating Hormone Receptor: HTRF Activity in a Follicle Stimulating Hormone Receptor Cell Lineconfirmatory
1401Quantitative High-Throughput Screen for Agonists of Thyroid Stimulating Hormone Receptor: Summarysummary
2104Secondary Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: ELISA Activity Detectionconfirmatory
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:

Thyroid Stimulating Hormone Receptor

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.
This cell based assay utilized a cyclic nucleotide gated ion channel (CNG) as a biosensor for cAMP induction by potential TSHR agonists.


TSHR cell line
HEK 293 cells stably expressing the TSHR as well as a modified CNG were purchased from BD biosciences. Stimulation of cAMP production causes the CNG to open and subsequent membrane depolarization to occur. A fluorescent membrane potential dye (BD biosciences) was utilized to detect cAMP stimulation in TSHR cells.
The cells were maintained in DMEM medium (Invitrogen) containing 10 % FBS (Hyclone), 100 units/ml Penicillin, 100 ug/ml Streptomycin (Invitrogen), and 250 ug/ml Geneticin (Invitrogen) and 1 ug/ml Puromycin (Invitrogen) at 37C in 5% CO2. HEK 293 cells expressing CNG but without TSHR (parental cells) were used in concert to control for compounds signaling through endogenous receptors and targets of HEK 293 cells.
Protocol
NCGC Assay Protocol Summary:

High Throughput Screening. Freshly passaged cells (TSHR or parental) were dispensed into 1536-well assay plates at 4 ul/well in DMEM containing phenol red (Invitrogen), 10% FCS (Hyclone), and 100 units/ml Penicillin, 100 ug/ml Streptomycin (Invitrogen) and incubated at 37 C with 5% CO2 for 20 to 36 hrs before the robotic screening. The screen was initiated by addition of 4 ul/well membrane potential dye containing 100 uM RO 20-1724 (Sigma Aldrich), followed by 60 min incubation at room temperature to allow for dye equilibration across the cell membrane. 23 nl/well compound or control was then added to the assay plates, which were incubated for additional 30 min before reading a Envision fluorescence plate reader (Perkin Elmer) in bottom-read mode for fluorescence intensity (emission at 590 nm).

Keywords: Thyroid-stimulating hormone TSH, TSHR, TSHR agonist, quantitative high throughput screening, qHTS, HTS, probe identification, CNG, cAMP, cyclic nucleotide gated ion channel PubChem
Comment
Compound Ranking:

1. Compounds are first classified as activators, inconclusive, and inactive based on activity in the TSHR and parental cell lines. TSHR agonists (activators) are expected to increase signal in TSHR cells, but not to potentiate signal in parental cells. Signal activators potentiate signal in both cell lines (inconclusive).

2. Compounds are then 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 Classification". 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 (activators), 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
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
5Parental-Curve_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 signficant, but below 80% of control.String
6Parental-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
7Parental-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Parental-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Parental-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Parental-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
11Parental-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Parental-Excluded_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
13Parental-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Parental-Activity at 0.0007000000 uM (0.0007μM**)% Activity at given concentration.Float%
15Parental-Activity at 0.00160 uM (0.0016μM**)% Activity at given concentration.Float%
16Parental-Activity at 0.00370 uM (0.0037μM**)% Activity at given concentration.Float%
17Parental-Activity at 0.00820 uM (0.0082μM**)% Activity at given concentration.Float%
18Parental-Activity at 0.018 uM (0.0183μM**)% Activity at given concentration.Float%
19Parental-Activity at 0.041 uM (0.0409μM**)% Activity at given concentration.Float%
20Parental-Activity at 0.092 uM (0.0915μM**)% Activity at given concentration.Float%
21Parental-Activity at 0.205 uM (0.2045μM**)% Activity at given concentration.Float%
22Parental-Activity at 0.457 uM (0.4572μM**)% Activity at given concentration.Float%
23Parental-Activity at 1.022 uM (1.0224μM**)% Activity at given concentration.Float%
24Parental-Activity at 2.286 uM (2.286μM**)% Activity at given concentration.Float%
25Parental-Activity at 5.112 uM (5.1115μM**)% Activity at given concentration.Float%
26Parental-Activity at 11.43 uM (11.4293μM**)% Activity at given concentration.Float%
27Parental-Activity at 25.56 uM (25.5558μM**)% Activity at given concentration.Float%
28Parental-Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
29TSHR-Curve_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 signficant, but below 80% of control.String
30TSHR-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
31TSHR-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
32TSHR-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
33TSHR-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
34TSHR-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
35TSHR-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
36TSHR-Excluded_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
37TSHR-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
38TSHR-Activity at 0.0007000000 uM (0.0007μM**)% Activity at given concentration.Float%
39TSHR-Activity at 0.00160 uM (0.0016μM**)% Activity at given concentration.Float%
40TSHR-Activity at 0.00370 uM (0.0037μM**)% Activity at given concentration.Float%
41TSHR-Activity at 0.00820 uM (0.0082μM**)% Activity at given concentration.Float%
42TSHR-Activity at 0.018 uM (0.0183μM**)% Activity at given concentration.Float%
43TSHR-Activity at 0.041 uM (0.0409μM**)% Activity at given concentration.Float%
44TSHR-Activity at 0.092 uM (0.0915μM**)% Activity at given concentration.Float%
45TSHR-Activity at 0.205 uM (0.2045μM**)% Activity at given concentration.Float%
46TSHR-Activity at 0.457 uM (0.4572μM**)% Activity at given concentration.Float%
47TSHR-Activity at 1.022 uM (1.0224μM**)% Activity at given concentration.Float%
48TSHR-Activity at 2.286 uM (2.286μM**)% Activity at given concentration.Float%
49TSHR-Activity at 5.112 uM (5.1115μM**)% Activity at given concentration.Float%
50TSHR-Activity at 11.43 uM (11.4293μM**)% Activity at given concentration.Float%
51TSHR-Activity at 25.56 uM (25.5558μM**)% Activity at given concentration.Float%
52TSHR-Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
53Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Classification
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