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BioAssay: AID 687027

HTS for PAX8 inhibitors using PAX8 luciferase reporter gene assay in RMG-I cells Measured in Cell-Based System Using Plate Reader - 7054-01_Inhibitor_Dose_CherryPick_Activity

A cellular assay that measures Pax8 activity using a Luciferase-based reporter of a well characterized PAX8 transcriptional target, thyroperoxidase (TPO), was used in the primary assay. A 420 bp fragment of the rat TPO promoter that was sensitive to Pax8 levels in RMG-I ovarian cancer cells was stably introduced to create a reporter cell line (RMG-1:TPO-Luc)with a biologically-relevant context. Cells were treated with compound and controls for 24 hours. Following compound treatment, TPO promoter activity (via luciferase light production) was measured using Promega SteadyGlo. ..more
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 Tested Compounds
 Tested Compounds
All(1807)
 
 
Active(1635)
 
 
Inactive(173)
 
 
 Tested Substances
 Tested Substances
All(1810)
 
 
Active(1637)
 
 
Inactive(173)
 
 
AID: 687027
Data Source: Broad Institute (7054-01_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-06-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1635
Related Experiments
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AIDNameTypeComment
652157Broad Institute Discovering modulators of PAX8 for targeting ovarian cancer Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
652154HTS for PAX8 inhibitors using PAX8 luciferase reporter gene assay in RMG-I cells Measured in Cell-Based System Using Plate Reader - 7054-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
687029PAX8: PAX8-dependent cytoxicity Measured in Cell-Based System Using Plate Reader - 7054-06_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
687032SA5-Pax8: Cytotoxicity IOSE-T80 Measured in Cell-Based System Using Plate Reader - 7054-07_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
743020Counterscreen:CMV reporter Measured in Cell-Based System Using Plate Reader - 7054-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743021PAX8: non-specific cytotoxicity Measured in Cell-Based System Using Plate Reader - 7054-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743043SA10 PAX8: cytotoxicity IGROV1 Measured in Cell-Based System Using Plate Reader - 7054-12_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743044SA9-PAX8: cytotoxicity NIHOVCAR3 Measured in Cell-Based System Using Plate Reader - 7054-11_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743045SA5-Pax8: Cytotoxicity IOSE-T80 Measured in Cell-Based System Using Plate Reader - 7054-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743046SA6-Pax8: cytotoxicity A2780 Measured in Cell-Based System Using Plate Reader - 7054-08_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743047SA2 PAX8:selectivity PAX5 Measured in Cell-Based System Using Plate Reader - 7054-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743048Pax8 Selectivity: PAx2 Measured in Cell-Based System Using Plate Reader - 7054-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743049PAX8: PAX8-dependent cytoxicity Measured in Cell-Based System Using Plate Reader - 7054-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743144SA12 PAX8: cytotoxicity COV362 Measured in Cell-Based System Using Plate Reader - 7054-16_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743145SA7-Pax8: cytotoxicity COV434 Measured in Cell-Based System Using Plate Reader - 7054-09_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743149SA8-Pax8: cytotoxicity OV-90 Measured in Cell-Based System Using Plate Reader - 7054-10_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743150SA11 PAX8: cytotoxicity OVCAR4 Measured in Cell-Based System Using Plate Reader - 7054-13_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743197SA13 PAX8: cytotoxicity OVCAR8 Measured in Cell-Based System Using Plate Reader - 7054-15_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords: ovarian cancer, Pax8, luciferase, SteadyGlo, RMG-1 cells, thyroperoxidase


Assay Overview:
A cellular assay that measures Pax8 activity using a Luciferase-based reporter of a well characterized PAX8 transcriptional target, thyroperoxidase (TPO), was used in the primary assay. A 420 bp fragment of the rat TPO promoter that was sensitive to Pax8 levels in RMG-I ovarian cancer cells was stably introduced to create a reporter cell line (RMG-1:TPO-Luc)with a biologically-relevant context. Cells were treated with compound and controls for 24 hours. Following compound treatment, TPO promoter activity (via luciferase light production) was measured using Promega SteadyGlo.

Expected Outcome: If a compound inhibits Pax8 function, there will be less TPO expression in RMG-1 cells and this will lead to a decrease in luciferase signal.
Protocol
PAX8 Primary Assay Protocol
Cell maintenance and passaging:
RMG-I-Pax8Luc cells: Stably express a Pax8-Luciferase reporter that contains a portion of the thyroperoxidase (TPO) gene promoter. Cells are grown in phenol red free RPMI-1640 media supplemented with 10% FBS (Sigma, F4135), Hygromycin (75 ug/mL) and 1X Pen/Strep (Gibco 15140-122). Cells are grown in a humidified incubator, 37 degrees C, 5% CO2.
Upon thawing, cells are allowed to recover for at least 1 week before any experimental procedure. When flasks reach ~80% confluent, cells are passaged, at a 1:4 ratio:
1.Remove media.
2.Briefly wash with PBS.
3.Add Trypsin (0.25%,Gibco 25200-056)
4.Place flask in incubator for 2-3 minutes.
5.Stop trypsin by adding serum containg medium. Transfer cells to a 50 or 250 mL conical tube. Centrifuge 5 minutes at 1200 rpm.
6.Re-plate 25% of the cells into a new plate containing fresh medium.
Note: The cells are very sensitive to long incubation with trypsin or to insufficient dilution of trypsin in FBS-containing medium.
Important: Medium needs to be replaced every 2-3 days between passages (Avoid yellow color of medium, highly acidic).

Day 1: Preparing cells for experiment:
Cells are trypsinized as stated above, centrifuged, aspirate trypsin/media, resuspended in fresh media and counted. Cells are diluted in fresh medium to a final concentration of 167,000 cells/ml. Cells are dispensed into barcoded 384 well plates (Corning 8867 white, opaque), 30 ul/well (5000 cells/well) using a Thermo-Fisher Multidrop Combi and a standard Combi cassette.

Day 2: Treatment with compounds:
100 nL of compounds or controls are added to the plates by pinning method. The positive control used is 2 uM mitoxantrone (BRD-K21680192-001-01-5), a known cytotoxin that will reduce signal of luciferase in the reporter assay due to loss of cells.

Day3: SteadyGlo Luciferase assay:
Measure luciferase activity 24h after compound pinning. Steady-Glo solution (Promega E2550 for assay development and med. chem experiments; X1006 pre-made solution for the HTS) is added using a Thermo-Fisher Multidrop Combi, 10 ul/well (standard cassette at medium speed). Plates are shaken briefly and allowed to incubate for 10 minutes at room temperature. Luminescence levels are read on a Perkin-Elmer EnVision plate reader, using the USLum (ultra-sensitive luminescence) protocol. (Time= 0.5s/well)
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: RMG-I
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
5ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
6ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.String
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_0.26uM_(%)_TEST1 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.5uM_(%)_TEST1 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_1uM_(%)_TEST1 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_2.1uM_(%)_TEST1 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_4.2uM_(%)_TEST1 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_8uM_(%)_TEST1 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_16uM_(%)_TEST1 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_35uM_(%)_TEST1 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
26ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
27ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
28AC50_Qualifier_TEST2'>','=', or '<'String
29AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
30pAC50_M_TEST2Equal to -1*log10(AC50)String
31Hill_Slope_TEST2The slope at AC50Float
32S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
33Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
34Num_Points_TEST2The number of data points used to generate the plotInteger
35Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
36Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
37Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
38Activity_at_0.235uM_(%)_TEST2 (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_0.26uM_(%)_TEST2 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_0.285uM_(%)_TEST2 (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_0.46uM_(%)_TEST2 (0.46μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_0.5uM_(%)_TEST2 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_0.56uM_(%)_TEST2 (0.56μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_0.9uM_(%)_TEST2 (0.9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_1uM_(%)_TEST2 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46Activity_at_1.1uM_(%)_TEST2 (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
47Activity_at_1.8uM_(%)_TEST2 (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
48Activity_at_1.95uM_(%)_TEST2 (1.95μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
49Activity_at_2.1uM_(%)_TEST2 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
50Activity_at_2.35uM_(%)_TEST2 (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
51Activity_at_3.8uM_(%)_TEST2 (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
52Activity_at_4.2uM_(%)_TEST2 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
53Activity_at_4.6uM_(%)_TEST2 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
54Activity_at_7.5uM_(%)_TEST2 (7.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
55Activity_at_8uM_(%)_TEST2 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
56Activity_at_9uM_(%)_TEST2 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
57Activity_at_15uM_(%)_TEST2 (15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
58Activity_at_16uM_(%)_TEST2 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
59Activity_at_18uM_(%)_TEST2 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
60Activity_at_30uM_(%)_TEST2 (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
61Activity_at_35uM_(%)_TEST2 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH097485-01A1

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