qHTS Screen for Compounds that Selectively Target Cancer Cells with p53 Mutations: Cytotoxicity of p53ts Cells at the Nonpermissive 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 ..
BioActive Compounds: 1890
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.
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; 39C 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
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)