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BioAssay: AID 449755

Luminescence Cell-Based Counterscreen to Identify Inhibitors of A1 Apoptosis (HMC 1-8 low)

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. However, they can be primed to depend on A1 by co-expressing A1 and BIM. The primed cells still maintain a balance between anti and pro-apoptotic proteins, but rely on A1 to sequester BIM. ..more
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 Tested Compounds
 Tested Compounds
All(255)
 
 
Active(82)
 
 
Inactive(102)
 
 
Inconclusive(71)
 
 
 Tested Substances
 Tested Substances
All(255)
 
 
Active(82)
 
 
Inactive(102)
 
 
Inconclusive(71)
 
 
AID: 449755
Data Source: Broad Institute (2045-03_INHIBITORS_DOSE-TITRATION_MLPCN-CHEERYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-07-19
Hold-until Date: 2010-08-20
Modify Date: 2010-08-20

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 82
Related Experiments
Show more
AIDNameTypeProbeComment
2462Luminescence Cell-Based Primary HTS to Identify Inhibitors of A1 Apoptosis.Screening depositor-specified cross reference: Primary HTS
2526Summary of Broad Institute MLPCN A1 Apoptosis ProjectSummary1 depositor-specified cross reference: Project Summary
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
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
488885Inhibitors of A1 in HMC1-8 Mcl1 Dependent Cells Measured in Cell-Based System Using Plate Reader - 2045-03_Inhibitor_Dose_DryPowder_ActivityConfirmatory 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. However, they can be primed to depend on A1 by co-expressing A1 and BIM. The primed cells still maintain a balance between anti and pro-apoptotic proteins, but rely on A1 to sequester BIM.
An A1 inhibitor causes the release of A1-bound BIM, which activates BAX/BAK, and leads to caspase activation that can be quantitatively measured using a luciferin-linked caspase substrate (peptide sequence DEVD) available commercially as Promega's Caspase Glo 3/7 reagent. 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 response in this line are not of further interest.
Protocol
1. Human cancer cell line HMC 1-8 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 30X106 cells per 150mm dish). Split cells 1 to 6-10 (3-4X106 cells) for subsequent passage every other day.
2. Day1 morning. HMC 1-8 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 1X105 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.
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 luminescence, 0.1s/well
Comment
Secondary Screen Data Analysis

32 negative control wells (DMSO) were included on every plate. 24 nonspecific positive control wells (clofoctol) were included on every plate. 12 pathway-specific positive control wells (ABT-263) were included on every plate. Compound activity was scaled to the negative and nonspecific positive control, with DMSO activity measuring 0% and clofoctol measuring 100% activity.

The activity score at each concentration was derived using the follow procedure:
1. A background-subtracted value was calculated for each well by subtracting the median value of the negative control wells on each plate from the value of each well on that plate.

2. An activity score was derived for each well by dividing the background-subtracted value for each well by the median of the background-subtracted value of the clofoctol positive control wells in the same run and multiplying the resulting fraction by 100.

3. No plate pattern correction was applied.

AC50 values were calculated using the Smart Fit strategy of Genedata
Screener Condoseo (7.0.3). AC50 values were extrapolated up to 1 log
over the highest tested concentration.

Due to the nature of the screen, it is likely that certain compounds will display the desired phenotype (caspase activation) at moderate concentrations but show no caspase activation at higher concentrations due to toxicity. Therefore curves which shows an increase-decrease shape were masked at the highest concentrations and curves were fit to the increasing portion of the curve only.

PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(AC50)

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70% ('undefined' in Genedata)
or
The fit was not valid due to poor fit quality ('invalid' in Genedata).
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 maximum, in micromolarFloatμM
3Log_AC50_Mthe log of the EC50 (molar)Float
4Log_AC50_M_Standard_Errorthe standard error for the log of the EC50 valueFloat
5Hill_Slopethe slope at EC50Float
6S0the fitted activity level at zero concentrationFloat%
7Sinfthe fitted activity level at infinite concentrationFloat%
8Num_Pointsthe number of data points included in the plotInteger
9Max_Activitythe maximum activity value observedFloat
10Activity_at_0.26uM (0.026μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.5uM (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_1uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_2.1uM (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_4.2uM (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_8uM (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_16uM (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_35uM (35μM**)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)
Data Table ( Complete ):     View Active Data    View All Data
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