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BioAssay: AID 2765

Luminescence Cell-Based Dose Retest to Identify Inhibitors of A1 Apoptosis

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(1065)
 
 
Active(150)
 
 
Inactive(905)
 
 
Inconclusive(10)
 
 
 Tested Substances
 Tested Substances
All(1065)
 
 
Active(150)
 
 
Inactive(905)
 
 
Inconclusive(10)
 
 
AID: 2765
Data Source: Broad Institute (2045-01_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-04-15
Modify Date: 2010-07-14

Data Table ( Complete ):           Active    All
Targets
BioActive Compounds: 150
Depositor Specified Assays
AIDNameTypeComment
2462Luminescence Cell-Based Primary HTS to Identify Inhibitors of A1 Apoptosis.screeningPrimary HTS
2526Summary of Broad Institute MLPCN A1 Apoptosis ProjectsummaryProject Summary
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.

Expected Outcome: Compounds that cause caspase activation will show an increase in luminescence signal as measured by the caspase glo reagent. Additional assays will determine whether this caspase activation is being caused by general toxicity or off target effects (as is the case for the nonspecific positive control, Clofoctol) or due to the on-target disruption of the A1-BIM interaction (as is the case for the specific but weaker positive control, ABT-263, which mimics the BH3 homology domain of BIM.)
Protocol
1.MEF cells expressing A1-2A-BIM 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. MEF cells grow 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 50 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 net for 1 minute. Incubate at room temperature for 1h.
9.Measure luminescence in Envision (Perkin Elmer).
NOTE: Follow-up studies on a subset of 255 compounds use the US Lum aperture to reduce cross-talk (annotated as "alternative aperture read").
Comment
HTS Data Analysis:
Active A1 inhibitors result in increased Caspaseglo signal.
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.

EC50 values were calculated using the Smart Fit strategy of Genedata
Screener Condoseo (7.0.3). EC50 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(EC50)

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 activtity >30% but <70% ('undefined' in Genedata)
or
The fit was not valid due to poor fit quality ('invalid' in Genedata).
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_Qualifier'>', '=', or '<'String
2EC50_uM*the concentration at which activity reaches 50% of the maximum, in micromolarFloatμM
3Log_EC50_Mthe log of the EC50 (molar)Float
4Log_EC50_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)
Classification
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