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BioAssay: AID 488914

Secondary assay of A1 inhibitors in Trichostatin A primed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-07_Inhibitor_Dose_DryPowder_Activity

The fate of cell survival versus apoptosis is determined by the balance of anti and pro-apoptotic proteins. Expression of activator BH3-only proteins, such as BIM or tBID, leads to downstream caspase activation and apoptosis. A1 can functionally bind to and sequester BIM or tBID, which can be artificially overexpressed or primed through treatment with pro-apoptotic chemicals or cytokines. In this more ..
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 Tested Compounds
 Tested Compounds
All(26)
 
 
Active(14)
 
 
Inactive(10)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(26)
 
 
Active(14)
 
 
Inactive(10)
 
 
Inconclusive(2)
 
 
AID: 488914
Data Source: Broad Institute (2045-07_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-26
Hold-until Date: 2011-10-22
Modify Date: 2011-10-22

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 14
Depositor Specified Assays
AIDNameTypeComment
2526Summary of Broad Institute MLPCN A1 Apoptosis ProjectsummarySummary assay
Description:
Keywords: apoptosis, BH3 domain, Bcl2-A1, BIM, caspase, cancer

Primary Collaborator: Todd Golub, Broad Institute, golub@broadinstitute.org

Assay Overview:
The fate of cell survival versus apoptosis is determined by the balance of anti and pro-apoptotic proteins. Expression of activator BH3-only proteins, such as BIM or tBID, leads to downstream caspase activation and apoptosis. A1 can functionally bind to and sequester BIM or tBID, which can be artificially overexpressed or primed through treatment with pro-apoptotic chemicals or cytokines. In this assay, the parental control cells express A1 and have been primed through treatment with an HDAC inhibitor, Trichostatin A. This causes a stress on the cells that is balanced out by the overexpression of A1; inhibition of A1 will allow the cellular stress to signal for cell death through apoptotic pathways.

Expected Outcome: Compounds that cause caspase activation in this cell line may be acting through inhibition of A1 allowing for cell death signals caused by TSA priming to proceed and are of interest for further development.
Protocol
Protocol:
1.#MEWO cells expressing A1 are cultured in 150mm TC dishes with 30mls of growth media supplemented with 0.5-1 ug/ml blasticidin in a 37oC incubator (5% CO2). Use 30 ml media for a 150 mm dish. Do let cells go beyond 95% confluency (about 30X10^6 cells per 150mm dish). Split cells 1 to 6-10 (3-4X10^6 cells) for subsequent passage every other day.
2.#Day1 morning. MEWO cells grown on T200 mm cell culture flasks are washed once with 1XPBS (Gibco), and digested with 1ml (or 3ml) 1X trypsin (CellGro Mediatech) for 1-2 minutes.
3.#Add 10ml complete growth media (RPMI-1640 (Cellgrow Mediatech), 10% heat inactivated FBS (Thermo), 1X penn/strep/glutamine (Gibco)) to the plate, mix cells and break clumps, then transfer the cells to a 50ml centrifuge tube through a cell strainer (BD Falcon #352340) to get rid of any clumps. Count the cells, and centrifuge cells at 1000 rpm for 4 minutes.
4. Aspirate off the supernatant, and resuspend the cells in complete media at density of 1X10^5 cells/ml.
5. Plate cells in white 384 well plates (Corning 3570), 30ul/well (2500 cells/well), with Combi (Thermo) while gently stirring the media. Add 2 uL 16 uM Trichostatin A.
6. Day2. Pin transfer 100 nL of compound to the cells and incubate 37 degrees 5% CO2 95% humidity for 3 hours.
7. Remove the plate from the incubator and cool down to room temperature for 30 minutes.
8. Add 10ul of 1:1 diluted CaspaseGlo (Promega) (diluted with 50mM HEPES) to each well with Combi multidrop (Thermo.) Shake the plate on the combi nest for 1 minute. Incubate at room temperature for 1h.
9. Measure luminescence in Envision (Perkin Elmer), std lum, 0.1s/well
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.


EXPECTED OUTCOME: Active compounds result in increasing readout signal.


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


NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
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 (Clofoctol only) was set to a normalized activity value of 100.
Experimental wells values were scaled to this range. Due to the high activity of Clofoctol, small values of the resulting normalized compound wells can still indicate significant activity.

MASKING:
Due to the toxic nature of high levels of caspase activation, several compounds result in bell-shaped curves in which caspases rise at moderate concentration but fall at toxic higher concentrations. For curves of this shape, the decreasing portion of the curve was masked and sigmoidal curves were fit to the increasing data range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) 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.


PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -5% and < 5% at all tested concentrations, or
b) AC > AC_limit, or
c) 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

Activity_Outcome = 2 (active) when:
AC <= AC_limit

Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= 30% but <= 70%, or
b) The fit was not valid due to poor fit quality.


PUBCHEM_ACTIVITY_SCORE:

PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)

PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:

120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M

PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
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
3Log_AC50_MThe log of the molar AC50Float
4Log_AC50_M_Standard_ErrorThe standard error for the log of the molar AC50 valueFloat
5Hill_SlopeThe slope at AC50Float
6S0The fitted activity value at zero concentrationFloat%
7SinfThe fitted activity value at infinite concentrationFloat%
8Num_PointsThe number of data points used to generate the plotInteger
9Max_ActivityThe maximum activity value observed, based on mean of replicates per concentrationFloat%
10Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
11Activity_at_0.056uM (0.056μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.06uM (0.06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.08uM (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.09uM (0.09μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.12uM (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.135uM (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.195uM (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.21uM (0.21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.285uM (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.3uM (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.42uM (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.5uM (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.68uM (0.68μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.75uM (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_1uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_1.1uM (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_1.5uM (1.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_1.6uM (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_2.1uM (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_2.35uM (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_3uM (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_3.5uM (3.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_5uM (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_5.6uM (5.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_7.5uM (7.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_8uM (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37Activity_at_11uM (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
38Activity_at_12uM (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_16uM (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_18uM (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_28.5uM (28.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_42uM (42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_60uM (60μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_90uM (90μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_135uM (135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA028853-01

Data Table (Concise)
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