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BioAssay: AID 924

qHTS Screen for Compounds that Selectively Target Cancer Cells with p53 Mutations: Cytotoxicity of p53ts Cells at the Permissive Temperature

Tumor suppressor gene p53 serves as a major cellular barrier against tumorigenesis. It has been reported that p53 mutations occurs in half of all tumors, and the other half possess alterations in the p53 pathway that impair p53 function. The goal of this screening is to identify small molecule probes that selectively kill cancer cells with mutations in p53. This screening was carried out using more ..
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 Tested Compounds
 Tested Compounds
All(124021)
 
 
Active(1156)
 
 
Inactive(118845)
 
 
Inconclusive(4094)
 
 
 Tested Substances
 Tested Substances
All(125218)
 
 
Active(1168)
 
 
Inactive(119940)
 
 
Inconclusive(4110)
 
 
 Related BioAssays
 Related BioAssays
AID: 924
Data Source: NCGC (P53T796.1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-20
Modify Date: 2010-07-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1156
Depositor Specified Assays
AIDNameTypeComment
902qHTS Screen for Compounds that Selectively Target Cancer Cells with p53 Mutations: Cytotoxicity of p53ts Cells at the Nonpermissive Temperatureconfirmatory
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]

MLSCN Grant: 1 X01 MH079844-01
Assay Submitter (PI): Dr. SUN, YI, University of Michigan

NCGC Assay Overview:

Synthetic Lethal Screen for Compounds to Kill Cancer Cells with p53 Mutation.

Tumor suppressor gene p53 serves as a major cellular barrier against tumorigenesis. It has been reported that p53 mutations occurs in half of all tumors, and the other half possess alterations in the p53 pathway that impair p53 function. The goal of this screening is to identify small molecule probes that selectively kill cancer cells with mutations in p53. This screening was carried out using two lines of H1299 lung cancer cells. The H1299-p53 A138V line express a temperature sensitive p53 mutant (p53ts) in which the mutant p53 protein (A138V) is in the mutant confirmation when grown at 39C (the nonpermissive temperature) and in the wild type conformation when grown at 32C (the permissive temperature). A H1299-neo line (p53 null) grown at both 39C and 32C were used as counter-screens to filter out false positive associated with temperature shifting. The small molecular compounds that selectively target mutant p53-containing cancer cells were selected as candidate probes for further study.

A cytotoxicity assay based on the measurement of ATP contents in cells was used in this screen. Concentration-responses of all the compounds in the library collection were determined in qHTS using the cell lines and temperature conditions as above.
Protocol
NCGC Assay Protocol Summary:

Both the H1299-p53A138V and H1299-neo cell lines were provided by Dr.Yi Sun, a PI at the University Michigan. The cells were maintained and cultured at 37C. The resuspended frozen cells were used to plate the cells in the 1536-well assay plates at a density of 600 cells per well in 5 ul medium. The assay plates with the cells were incubated at 37C for 16 to 30 hours before the experiments. The library compounds in DMSO solution were added to the cells in assay plates and incubated 24 hours at 39C (for mutant p53 conformation) or 32C (for normal p53 conformation as control). An ATP_lite assay kit (PerkinElmer) was used to determine the cell viability. Tomaxifen (100uM) was used as the positive control for cell killing. Data were normalized to the controls for basal activity (DMSO only) and 100% killing (100uM tamoxifen). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

P53 assay protocol:
1. Reagent; 5 uL H1299-p53A138V or H1299-neo cell cells/well
2. Time; overnight; 37C incubation
3. Compounds; 23 nL; 5.9 nM to 46 uM
4. Controls; 23 nL; Tamoxifen 100 uM
5. Time; 24 hr; 37C incubation
6. Reagent; 3 uL; ATP_Lite reagent
7. Time; 20 min; Room Temperature
8. Detection; Luminescence; Viewlux plate reader


Keywords: p53, synthetic lethal, cancer, cell viability, cytotoxicity, H1299 cell line, luminescence, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation (calculated based on molar units).Float
7Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0006000000 uM (0.0006μM**)% Activity at given concentration.Float%
15Activity at 0.00130 uM (0.0013μM**)% Activity at given concentration.Float%
16Activity at 0.00295 uM (0.00295023μM**)% Activity at given concentration.Float%
17Activity at 0.00300 uM (0.003μM**)% Activity at given concentration.Float%
18Activity at 0.00660 uM (0.0066μM**)% Activity at given concentration.Float%
19Activity at 0.015 uM (0.0148μM**)% Activity at given concentration.Float%
20Activity at 0.033 uM (0.033μM**)% Activity at given concentration.Float%
21Activity at 0.074 uM (0.0738μM**)% Activity at given concentration.Float%
22Activity at 0.165 uM (0.1649μM**)% Activity at given concentration.Float%
23Activity at 0.369 uM (0.3687μM**)% Activity at given concentration.Float%
24Activity at 0.825 uM (0.8245μM**)% Activity at given concentration.Float%
25Activity at 1.844 uM (1.8435μM**)% Activity at given concentration.Float%
26Activity at 4.122 uM (4.1222μM**)% Activity at given concentration.Float%
27Activity at 9.217 uM (9.2172μM**)% Activity at given concentration.Float%
28Activity at 20.61 uM (20.6095μM**)% Activity at given concentration.Float%
29Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
30Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Classification
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