Luminescence Cell-Based Secondary Screen to Identify Inhibitors of A1(alternate construct)
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
BioActive Compounds: 147
Keywords: apoptosis, BH3 domain, Bcl2-A1, BIM, caspase, cancer
Primary Collaborator: Todd Golub, Broad Institute, firstname.lastname@example.org
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.
Cells expressing low A1 or with Bax-Bak knocked out should not show caspase activation if the compound is A1-specific. An A1 inhibitor causes the release of A1-bound BIM, which activates BAX/BAK, leading to downstream caspase activation. Similar to the primary screening cell line, the cells in this experiment have been primed with A1-BIM in the form of a different construct to validate specificity for A1 rather than an artifact of the specific construct used.
Expected Outcome: Compounds that cause caspase activation in this cell line may be due to direct inhibition of A1 and are of interest. In conjunction with counterscreens and the primary screen, the mode of action can be more specifically determined.
1. MEF cells expressing Flag-A1-IRES-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 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
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.
Inactive compounds = 0
Active compounds = -10*Log(AC50)
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)
The fit was not valid due to poor fit quality ('invalid' in Genedata).
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)