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BioAssay: AID 588513

qHTS assay for small molecule antagonists of estrogen receptor alpha signaling

Estrogen receptor alpha (ER-alpha) is an important member of nuclear hormone receptors. ER-alpha plays important role in breast cancer as well as in other women's health issues. The majority of breast cancers is estrogen receptor (ER) positive and depends on estrogen for growth. There is also growing concern for some of environmental chemicals which may contribute the increasing the incidence of breast cancer. Therefore, it is important to understand the effect of environmental chemicals in ER-alpha signaling pathway. ..more
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 Tested Compounds
 Tested Compounds
All(2325)
 
 
Active(70)
 
 
Inactive(1708)
 
 
Inconclusive(727)
 
 
 Tested Substances
 Tested Substances
All(2858)
 
 
Active(75)
 
 
Inactive(2002)
 
 
Inconclusive(781)
 
 
 Related BioAssays
 Related BioAssays
AID: 588513
Data Source: NCGC (ERN101)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: Other
Deposit Date: 2011-10-14

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 70
Description:
NIH Chemical Genomics Center [NCGC]
U.S. Environmental Protection Agency [EPA]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]

NCGC Assay Overview

Estrogen receptor alpha (ER-alpha) is an important member of nuclear hormone receptors. ER-alpha plays important role in breast cancer as well as in other women's health issues. The majority of breast cancers is estrogen receptor (ER) positive and depends on estrogen for growth. There is also growing concern for some of environmental chemicals which may contribute the increasing the incidence of breast cancer. Therefore, it is important to understand the effect of environmental chemicals in ER-alpha signaling pathway.
Protocol
NCGC Assay Protocol Summary
Using a beta-lactamase reporter-gene under control of the ER response element, a cell-based assay [ER-alpha-UAS-bla GripTiteTM cell-Based Assay from Invitrogen] was used to measure ER-alpha signaling pathway in antagonist mode. ER-alpha-UAS-bla-GripTite HEK 293 cells were used with assay medium containing 2% charcoal/dextran treated FBS, 0.1 mM NEAA and 1 mM sodium pyruvate. Cells were cultured in this assay medium overnight in the flasks before the assay. The assay was performed in clear bottom black Greiner 1536-well plates. 17-beta-estradiol was used as a positive control in the screen. Library compounds were measured for their ability to inhibit the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an EnVision plate reader. Data was normalized relative to 17-beta-estradiol control (0.5nM, 100%), and DMSO only wells (basal, 0%). Concentration-response titration points for each compound were fitted to the Hill equation yielding concentrations of half-maximal inhibition (IC50) and maximal response (efficacy) values.
qHTS protocol for ER-alpha beta-lactamase assay in antagonist mode
[Step] [Parameter] [Value] [Description]
1. Reagent; 5 uL; 5000 cells/well
2. Incubation; 37 C for 4 hrs
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls (4-hydroxytamoxifen); 23 nL
- Dose response, Start 38uM, 1:2 dilutions with duplicates
- Two columns with 38 and 19uM (final) concentration.
5. Agonist (Stimulator); 1uL; 17-beta-estradiol at 0.5nM (final) concentration
6. Incubation; 37 C for 18 hrs
7. Detection reagent; 1 uL of CCF4-dye
8. Incubation; RT for 2 hrs
9. Detection; Fluorescence; Envision plate reader or Acumen plate reader
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, active compounds are expected to have the inhibitor phenotype and 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, 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 ChEMBL:
Assay Type: Functional
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
5W460-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 significant, but below 80% of control.String
6W460-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7W460-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8W460-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9W460-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10W460-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11W460-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12W460-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
13W460-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14W460-Activity at 0.0004329502 uM (0.00043295μM**)% Activity at given concentration.Float%
15W460-Activity at 0.00129 uM (0.00129224μM**)% Activity at given concentration.Float%
16W460-Activity at 0.00387 uM (0.00386725μM**)% Activity at given concentration.Float%
17W460-Activity at 0.00491 uM (0.00490613μM**)% Activity at given concentration.Float%
18W460-Activity at 0.012 uM (0.0116724μM**)% Activity at given concentration.Float%
19W460-Activity at 0.035 uM (0.0345767μM**)% Activity at given concentration.Float%
20W460-Activity at 0.055 uM (0.0548492μM**)% Activity at given concentration.Float%
21W460-Activity at 0.106 uM (0.105565μM**)% Activity at given concentration.Float%
22W460-Activity at 0.314 uM (0.313757μM**)% Activity at given concentration.Float%
23W460-Activity at 0.638 uM (0.638334μM**)% Activity at given concentration.Float%
24W460-Activity at 0.967 uM (0.967051μM**)% Activity at given concentration.Float%
25W460-Activity at 2.847 uM (2.84688μM**)% Activity at given concentration.Float%
26W460-Activity at 5.457 uM (5.45724μM**)% Activity at given concentration.Float%
27W460-Activity at 8.491 uM (8.49104μM**)% Activity at given concentration.Float%
28W460-Activity at 24.93 uM (24.9298μM**)% Activity at given concentration.Float%
29W460-Activity at 35.68 uM (35.6785μM**)% Activity at given concentration.Float%
30W460-Activity at 76.79 uM (76.7909μM**)% Activity at given concentration.Float%
31W460-Activity at 182.0 uM (182.01μM**)% Activity at given concentration.Float%
32W460-Activity at 407.0 uM (406.974μM**)% Activity at given concentration.Float%
33W460-Activity at 910.0 uM (909.994μM**)% Activity at given concentration.Float%
34W460-Activity at 2034.7 uM (2034.75μM**)% Activity at given concentration.Float%
35W460-Activity at 4832.4 uM (4832.4μM**)% Activity at given concentration.Float%
36W460-Activity at 10805.3 uM (10805.3μM**)% Activity at given concentration.Float%
37W460-Activity at 24160.5 uM (24160.5μM**)% Activity at given concentration.Float%
38W460-Activity at 54023.0 uM (54023μM**)% Activity at given concentration.Float%
39Ratio-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 significant, but below 80% of control.String
40Ratio-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
41Ratio-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
42Ratio-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
43Ratio-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
44Ratio-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
45Ratio-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
46Ratio-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
47Ratio-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
48Ratio-Activity at 0.0004339386 uM (0.000433939μM**)% Activity at given concentration.Float%
49Ratio-Activity at 0.00129 uM (0.00128589μM**)% Activity at given concentration.Float%
50Ratio-Activity at 0.00380 uM (0.00379846μM**)% Activity at given concentration.Float%
51Ratio-Activity at 0.00490 uM (0.00490421μM**)% Activity at given concentration.Float%
52Ratio-Activity at 0.012 uM (0.0116697μM**)% Activity at given concentration.Float%
53Ratio-Activity at 0.034 uM (0.0340419μM**)% Activity at given concentration.Float%
54Ratio-Activity at 0.056 uM (0.0560707μM**)% Activity at given concentration.Float%
55Ratio-Activity at 0.107 uM (0.106572μM**)% Activity at given concentration.Float%
56Ratio-Activity at 0.312 uM (0.3122μM**)% Activity at given concentration.Float%
57Ratio-Activity at 0.645 uM (0.644698μM**)% Activity at given concentration.Float%
58Ratio-Activity at 0.993 uM (0.993188μM**)% Activity at given concentration.Float%
59Ratio-Activity at 2.854 uM (2.85412μM**)% Activity at given concentration.Float%
60Ratio-Activity at 5.105 uM (5.10538μM**)% Activity at given concentration.Float%
61Ratio-Activity at 8.462 uM (8.4618μM**)% Activity at given concentration.Float%
62Ratio-Activity at 24.24 uM (24.2388μM**)% Activity at given concentration.Float%
63Ratio-Activity at 36.04 uM (36.0389μM**)% Activity at given concentration.Float%
64Ratio-Activity at 76.93 uM (76.9316μM**)% Activity at given concentration.Float%
65Ratio-Activity at 182.0 uM (181.982μM**)% Activity at given concentration.Float%
66Ratio-Activity at 406.9 uM (406.925μM**)% Activity at given concentration.Float%
67Ratio-Activity at 909.9 uM (909.911μM**)% Activity at given concentration.Float%
68Ratio-Activity at 2034.6 uM (2034.62μM**)% Activity at given concentration.Float%
69Ratio-Activity at 4832.0 uM (4831.96μM**)% Activity at given concentration.Float%
70Ratio-Activity at 10804.6 uM (10804.6μM**)% Activity at given concentration.Float%
71Ratio-Activity at 24159.8 uM (24159.8μM**)% Activity at given concentration.Float%
72Ratio-Activity at 54023.0 uM (54023μM**)% Activity at given concentration.Float%
73W530-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 significant, but below 80% of control.String
74W530-Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
75W530-Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
76W530-Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
77W530-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
78W530-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
79W530-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
80W530-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
81W530-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
82W530-Activity at 0.0004329502 uM (0.00043295μM**)% Activity at given concentration.Float%
83W530-Activity at 0.00129 uM (0.00129224μM**)% Activity at given concentration.Float%
84W530-Activity at 0.00387 uM (0.00386721μM**)% Activity at given concentration.Float%
85W530-Activity at 0.00490 uM (0.00490421μM**)% Activity at given concentration.Float%
86W530-Activity at 0.012 uM (0.0116723μM**)% Activity at given concentration.Float%
87W530-Activity at 0.035 uM (0.0345763μM**)% Activity at given concentration.Float%
88W530-Activity at 0.055 uM (0.0548307μM**)% Activity at given concentration.Float%
89W530-Activity at 0.106 uM (0.105564μM**)% Activity at given concentration.Float%
90W530-Activity at 0.314 uM (0.313754μM**)% Activity at given concentration.Float%
91W530-Activity at 0.638 uM (0.63814μM**)% Activity at given concentration.Float%
92W530-Activity at 0.967 uM (0.967037μM**)% Activity at given concentration.Float%
93W530-Activity at 2.847 uM (2.84686μM**)% Activity at given concentration.Float%
94W530-Activity at 5.456 uM (5.45602μM**)% Activity at given concentration.Float%
95W530-Activity at 8.491 uM (8.49099μM**)% Activity at given concentration.Float%
96W530-Activity at 24.93 uM (24.9296μM**)% Activity at given concentration.Float%
97W530-Activity at 35.67 uM (35.6731μM**)% Activity at given concentration.Float%
98W530-Activity at 76.79 uM (76.7904μM**)% Activity at given concentration.Float%
99W530-Activity at 182.0 uM (181.982μM**)% Activity at given concentration.Float%
100W530-Activity at 406.9 uM (406.925μM**)% Activity at given concentration.Float%
101W530-Activity at 909.9 uM (909.911μM**)% Activity at given concentration.Float%
102W530-Activity at 2034.6 uM (2034.62μM**)% Activity at given concentration.Float%
103W530-Activity at 4832.0 uM (4831.96μM**)% Activity at given concentration.Float%
104W530-Activity at 10804.6 uM (10804.6μM**)% Activity at given concentration.Float%
105W530-Activity at 24159.8 uM (24159.8μM**)% Activity at given concentration.Float%
106W530-Activity at 54023.0 uM (54023μM**)% Activity at given concentration.Float%
107Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

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