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BioAssay: AID 488885

Inhibitors of A1 in HMC1-8 Mcl1 Dependent Cells Measured in Cell-Based System Using Plate Reader - 2045-03_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. In this assay, the parental control cells do not depend on A1 for survival, but are naturally dependent on a different anti-apoptotic protein (Mcl1) to sequester these anti-apoptotic factors. ..more
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 Tested Compounds
 Tested Compounds
All(26)
 
 
Active(2)
 
 
Inactive(9)
 
 
Inconclusive(15)
 
 
 Tested Substances
 Tested Substances
All(26)
 
 
Active(2)
 
 
Inactive(9)
 
 
Inconclusive(15)
 
 
AID: 488885
Data Source: Broad Institute (2045-03_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-25
Hold-until Date: 2011-10-18
Modify Date: 2011-10-18

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
2526Summary of Broad Institute MLPCN A1 Apoptosis ProjectSummary1 depositor-specified cross reference: Summary assay
2462Luminescence Cell-Based Primary HTS to Identify Inhibitors of A1 Apoptosis.Screening same project related to Summary assay
2465Luminescence Cell-Based Primary HTS to Identify Compounds which Inhibit A1 ApoptosisScreening same project related to Summary assay
2765Luminescence Cell-Based Dose Retest to Identify Inhibitors of A1 ApoptosisConfirmatory same project related to Summary assay
449754Luminescence Cell-Based Counterscreen to Identify Inhibitors of A1 Apoptosis (Bax-Bak knockout)Confirmatory same project related to Summary assay
449755Luminescence Cell-Based Counterscreen to Identify Inhibitors of A1 Apoptosis (HMC 1-8 low)Confirmatory same project related to Summary assay
449757Luminescence Cell-Based Secondary Screen to Identify Inhibitors of A1(alternate construct)Confirmatory same project related to Summary assay
449761Luminescence Cell-Based Counterscreen to Identify Inhibitors of A1 Apoptosis (non-primed)Confirmatory same project related to Summary assay
488858Inhibitors of A1-dependent Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
488891Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate A1 construct Measured in Cell-Based System Using Plate Reader - 2045-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488897Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate pro-apoptotic inducer Measured in Cell-Based System Using Plate Reader - 2045-08_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488898Inhibitors of Bcl2-A1 in Bax/Bak -/- Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488902Counterscreen of A1 inhibitors in wild-type Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488914Secondary assay of A1 inhibitors in Trichostatin A primed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-07_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488934Secondary assay of A1 inhibitors in A1 dependent Mel501 cells Measured in Cell-Based System Using Plate Reader - 2045-09_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488948Counterscreen of A1 inhibitors in unprimed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
504342Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate A1 construct Measured in Cell-Based System Using Plate Reader - 2045-05_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504343Counterscreen of A1 inhibitors in HMC1-8 Mcl1 dependent cells Measured in Cell-Based System Using Plate Reader - 2045-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504344Counterscreen of A1 inhibitors in Bax/Bak -/- Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504345Inhibitors of A1-dependent Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504346Counterscreen of A1 inhibitors in unprimed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-06_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504347Inhibitors of A1-dependent Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
504348Counterscreen of A1 inhibitors in HMC1-8 Mcl1 dependent cells Measured in Cell-Based System Using Plate Reader - 2045-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504353Counterscreen of A1 inhibitors in wild-type Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-04_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
504354Counterscreen of A1 inhibitors in unprimed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504356Secondary 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_Set2Confirmatory same project related to Summary assay
504359Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate pro-apoptotic inducer Measured in Cell-Based System Using Plate Reader - 2045-08_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504360Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate pro-apoptotic inducer Measured in Cell-Based System Using Plate Reader - 2045-08_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504365Secondary assay of A1 inhibitors in A1 dependent Mel501 cells Measured in Cell-Based System Using Plate Reader - 2045-09_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504392Counterscreen of A1 inhibitors in wild-type Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504403Counterscreen of A1 inhibitors in Bax/Bak -/- Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504405Counterscreen of A1 inhibitors in wild-type Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-04_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504407Counterscreen of A1 inhibitors measured by cytochrome c ELISA in CHL-1 wild type cells Measured in Biochemical System Using Plate Reader - 2045-18_Inhibitor_SinglePoint_DryPowder_ActivityScreening same project related to Summary assay
504409Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate A1 construct Measured in Cell-Based System Using Plate Reader - 2045-05_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504412Secondary screen of A1 inhibitors measured by cytochrome c ELISA in CHL-1 cells expressing A1-2A-BIM Measured in Biochemical System Using Plate Reader - 2045-19_Inhibitor_SinglePoint_DryPowder_ActivityOther same project related to Summary assay
504413Inhibitors of A1-dependent Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-01_Inhibitor_Dose_DryPowder_Activity_3Confirmatory same project related to Summary assay
504415Counterscreen of A1 inhibitors in HMC1-8 Mcl1 dependent cells, viability readout Measured in Cell-Based System Using Plate Reader - 2045-12_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary 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. In this assay, the parental control cells do not depend on A1 for survival, but are naturally dependent on a different anti-apoptotic protein (Mcl1) to sequester these anti-apoptotic factors.
Cells expressing low A1 or with Bax-Bak knocked out should not show caspase activation if the compound is A1-specific. This cell line, HMC 1-8, is a human cancer cell line with low levels of A1 expression and is immortalized due to factors other than the anti-apoptotic A1.

Expected Outcome: Compounds that cause caspase activation in this HMC 1-8 low A1 line are most likely not working through A1 inhibition. Compounds causing a robust caspase response in this line are not of further interest.
Protocol
Protocol:

1. Human cancer cell line HMC 1-8 are cultured in 150 mm TC dishes with 30 mls of growth media supplemented with 0.5-1 ug/ml blasticidin in a 37 deg C incubator (5% CO2). Use 30 ml media for a 150 mm dish. Do not let cells go beyond 95% confluency (about 30X10^6 cells per 150 mm dish). Split cells 1 to 6-10 (3-4X10^6 cells) for subsequent passage every other day.

DAY 1 MORNING

2. HMC 1-8 cells grown on T200 mm cell culture flasks are washed once with 1X PBS (Gibco), and digested with 1 ml (or 3 ml) 1X trypsin (CellGro Mediatech) for 1-2 minutes.

3. Add 10 ml 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 50 ml 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), 30 ul/well (2500 cells/well), with Combi (Thermo) while gently stirring the media.

DAY 2

6. Pin transfer 100 nL of compound to the cells and incubate 37 deg C, 5% CO2 and 95% humidity for 4 hours.

7. Remove the plate from the incubator and cool down to room temperature for 30 minutes.

8. Add 10 ul of 1:1 diluted CaspaseGlo (Promega) (diluted with 50 mM HEPES) to each well with Combi multidrop (Thermo.) Shake the plate on the combi nest for 1 minute. Incubate at room temperature for 1 h.

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 (10)(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.

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 Format: Cell-based
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_0.06uM (0.06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.068uM (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.09uM (0.09μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.1uM (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.135uM (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.15uM (0.15μM**)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_0.235uM (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.3uM (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.35uM (0.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.46uM (0.46μM**)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_1.2uM (1.2μM**)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_1.8uM (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_2.35uM (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_2.6uM (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_3.5uM (3.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_3.8uM (3.8μM**)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_6uM (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_8uM (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_9uM (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37Activity_at_12uM (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
38Activity_at_13.5uM (13.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_18uM (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_19.5uM (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_26uM (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_30uM (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_38uM (38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_46uM (46μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_60uM (60μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46Activity_at_68uM (68μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
47Activity_at_90uM (90μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
48Activity_at_100uM (100μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
49Activity_at_135uM (135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
50Activity_at_150uM (150μM**)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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