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BioAssay: AID 953

Confirmation Concentration-Response 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(151)
 
 
Inactive(145)
 
 
Inconclusive(6)
 
 
 Tested Substances
 Tested Substances
All(151)
 
 
Inactive(145)
 
 
Inconclusive(6)
 
 
AID: 953
Data Source: NCGC (TSHR742.2.c)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2007-12-22

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
AIDNameTypeProbeComment
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
926qHTS Assay for Agonists of the Thyroid Stimulating Hormone ReceptorConfirmatory same project related to Summary assay
933Secondary Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: HTRF Activity DetectionConfirmatory same project related to Summary assay
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
939Confirmation Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone ReceptorConfirmatory same project related to Summary assay
2104Secondary Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: ELISA Activity DetectionConfirmatory 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

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 "Confirmation Concentration-Response 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.
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 signficant, 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 0.0000030488 uM (3.04883e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000039214 uM (3.92136e-06μM**)% Activity at given concentration.Float%
16Activity at 0.0000060977 uM (6.09766e-06μM**)% Activity at given concentration.Float%
17Activity at 0.0000078427 uM (7.84272e-06μM**)% Activity at given concentration.Float%
18Activity at 0.0000121953 uM (1.21953e-05μM**)% Activity at given concentration.Float%
19Activity at 0.0000156854 uM (1.56854e-05μM**)% Activity at given concentration.Float%
20Activity at 0.0000243906 uM (2.43906e-05μM**)% Activity at given concentration.Float%
21Activity at 0.0000313709 uM (3.13709e-05μM**)% Activity at given concentration.Float%
22Activity at 0.0000487813 uM (4.87813e-05μM**)% Activity at given concentration.Float%
23Activity at 0.0000627417 uM (6.27417e-05μM**)% Activity at given concentration.Float%
24Activity at 0.0000975626 uM (9.75626e-05μM**)% Activity at given concentration.Float%
25Activity at 0.0001254835 uM (0.000125483μM**)% Activity at given concentration.Float%
26Activity at 0.0001951252 uM (0.000195125μM**)% Activity at given concentration.Float%
27Activity at 0.0002509669 uM (0.000250967μM**)% Activity at given concentration.Float%
28Activity at 0.0003902504 uM (0.00039025μM**)% Activity at given concentration.Float%
29Activity at 0.0005019339 uM (0.000501934μM**)% Activity at given concentration.Float%
30Activity at 0.0007805007 uM (0.000780501μM**)% Activity at given concentration.Float%
31Activity at 0.00100 uM (0.00100387μM**)% Activity at given concentration.Float%
32Activity at 0.00156 uM (0.001561μM**)% Activity at given concentration.Float%
33Activity at 0.00201 uM (0.00200774μM**)% Activity at given concentration.Float%
34Activity at 0.00312 uM (0.003122μM**)% Activity at given concentration.Float%
35Activity at 0.00402 uM (0.00401547μM**)% Activity at given concentration.Float%
36Activity at 0.00624 uM (0.00624401μM**)% Activity at given concentration.Float%
37Activity at 0.00803 uM (0.00803094μM**)% Activity at given concentration.Float%
38Activity at 0.012 uM (0.012488μM**)% Activity at given concentration.Float%
39Activity at 0.016 uM (0.0160619μM**)% Activity at given concentration.Float%
40Activity at 0.025 uM (0.024976μM**)% Activity at given concentration.Float%
41Activity at 0.032 uM (0.0321238μM**)% Activity at given concentration.Float%
42Activity at 0.050 uM (0.049952μM**)% Activity at given concentration.Float%
43Activity at 0.064 uM (0.0642475μM**)% Activity at given concentration.Float%
44Activity at 0.100 uM (0.0999041μM**)% Activity at given concentration.Float%
45Activity at 0.128 uM (0.128495μM**)% Activity at given concentration.Float%
46Activity at 0.200 uM (0.199808μM**)% Activity at given concentration.Float%
47Activity at 0.257 uM (0.25699μM**)% Activity at given concentration.Float%
48Activity at 0.400 uM (0.399616μM**)% Activity at given concentration.Float%
49Activity at 0.514 uM (0.51398μM**)% Activity at given concentration.Float%
50Activity at 0.799 uM (0.799233μM**)% Activity at given concentration.Float%
51Activity at 1.028 uM (1.02796μM**)% Activity at given concentration.Float%
52Activity at 1.598 uM (1.59847μM**)% Activity at given concentration.Float%
53Activity at 2.056 uM (2.05592μM**)% Activity at given concentration.Float%
54Activity at 3.197 uM (3.19693μM**)% Activity at given concentration.Float%
55Activity at 4.112 uM (4.11184μM**)% Activity at given concentration.Float%
56Activity at 6.394 uM (6.39386μM**)% Activity at given concentration.Float%
57Activity at 8.224 uM (8.22368μM**)% Activity at given concentration.Float%
58Activity at 12.79 uM (12.7877μM**)% Activity at given concentration.Float%
59Activity at 16.45 uM (16.4474μM**)% Activity at given concentration.Float%
60Activity at 25.58 uM (25.5754μM**)% Activity at given concentration.Float%
61Activity at 32.89 uM (32.8947μM**)% Activity at given concentration.Float%
62Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

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