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BioAssay: AID 938

qHTS Assay for Agonists of the Thyroid Stimulating Hormone Receptor: Activators of Intracellular cAMP Concentrations in Parental HEK 293

This cell based assay utilized a cyclic nucleotide gated ion channel (CNG) as a biosensor for cAMP induction. HEK 293 cells stably expressing the modified CNG were purchased from BD biosciences (http://www.atto.com/products/actone/features_benefits.shtml) . 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 cells. ..more
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 Tested Compounds
 Tested Compounds
All(72024)
 
 
Active(1794)
 
 
Inactive(60806)
 
 
Inconclusive(9670)
 
 
Unspecified(6)
 
 
 Tested Substances
 Tested Substances
All(73162)
 
 
Active(1807)
 
 
Inactive(61633)
 
 
Inconclusive(9716)
 
 
Unspecified(6)
 
 
AID: 938
Data Source: NCGC (TSHR742.2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-21
Modify Date: 2010-07-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1794
Depositor Specified Assays
AIDNameTypeComment
1401Quantitative High-Throughput Screen for Agonists of Thyroid Stimulating Hormone Receptor: Summarysummary
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
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
MLSCN Grant: 1 X01 MH080680-01
PI Name: Dr. Marvin Gershengorn

NCGC Assay Overview:

This cell based assay utilized a cyclic nucleotide gated ion channel (CNG) as a biosensor for cAMP induction. HEK 293 cells stably expressing the modified CNG were purchased from BD biosciences (http://www.atto.com/products/actone/features_benefits.shtml) . 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 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. This assay was used as a counterscreen to "qHTS Assay for Agonists of the Thyroid Stimulating Hormone Receptor", to control for compounds signaling through endogenous receptors and other targets of HEK 293 cells.
Protocol
NCGC Assay Protocol Summary:

High Throughput Screening. Freshly passaged cells 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: cAMP, CNG Ion Channel, 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 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 activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds increasing signal (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
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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 signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation (calculated based on molar units).Float
7Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio 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 0.0007000000 uM (0.0007μM**)% Activity at given concentration.Float%
15Activity at 0.00160 uM (0.0016μM**)% Activity at given concentration.Float%
16Activity at 0.00370 uM (0.0037μM**)% Activity at given concentration.Float%
17Activity at 0.00820 uM (0.0082μM**)% Activity at given concentration.Float%
18Activity at 0.018 uM (0.0183μM**)% Activity at given concentration.Float%
19Activity at 0.041 uM (0.0409μM**)% Activity at given concentration.Float%
20Activity at 0.092 uM (0.0915μM**)% Activity at given concentration.Float%
21Activity at 0.205 uM (0.2045μM**)% Activity at given concentration.Float%
22Activity at 0.457 uM (0.4572μM**)% Activity at given concentration.Float%
23Activity at 1.022 uM (1.0224μM**)% Activity at given concentration.Float%
24Activity at 2.286 uM (2.286μM**)% Activity at given concentration.Float%
25Activity at 5.112 uM (5.1115μM**)% Activity at given concentration.Float%
26Activity at 11.43 uM (11.4293μM**)% Activity at given concentration.Float%
27Activity at 25.56 uM (25.5558μM**)% Activity at given concentration.Float%
28Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
29Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Classification
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