Luminescence Cell-Based Primary HTS to identify inhibitors of the oncoprotein EWS/Fli transcriptional activity Measured in Cell-Based System Using Plate Reader - 7014-01_Inhibitor_Dose_CherryPick_Activity
Ewing sarcoma is a pediatric and young adult cancer that results from a translocation that fuses the DNA binding domain of the Friend leukemia virus integration 1 (FLI1) transcription factor to the transactivation domain of the EWS protein, forming the EWS/FLI oncoprotein. This chromosomal translocation produce a mutant transcription factor which directly alters the expression of genes more ..
BioActive Compounds: 559
Keywords: Ewing sarcoma, transcription factor, chromosomal translocation
Assay Overview: Luciferase reporter assay using NR0B1 promoter in human A673 rhabdosarcoma cells.
Ewing sarcoma is a pediatric and young adult cancer that results from a translocation that fuses the DNA binding domain of the Friend leukemia virus integration 1 (FLI1) transcription factor to the transactivation domain of the EWS protein, forming the EWS/FLI oncoprotein. This chromosomal translocation produce a mutant transcription factor which directly alters the expression of genes responsible for cellular transformation and tumorigenesis. In this assay, we aim to discover small molecules inhibiting the transcriptional induction of a reporter gene (luciferase) under a specific promoter activated by the EWS/FLi oncoprotein in human A673 rharbosarcoma cells. The small molecules reducing the expression of firefly luciferase under NR0B1 (EWS/Fli-induced) promoter will be measured by the conversion of luciferin to oxyluciferin and light (luminescence) mediated by the firefly luciferase.
Expected outcome: Identify small molecules inhibiting the luminescence produced by the expression of luciferase under the NR0B1 promoter. Compounds reducing the relative luminescent response (RLU) by at least 35% (AbsAC-35) compared to the neutral control treatment and normalized by taking account of the cell viability (measured with Alamar Blue (relative fluorescence unit (RFU)) below the highest concentration tested (68uM final concentration) will be considered as active hits.
Protocol for A673 cells NR0B1-Luc/UbC-RL luciferase reporter assay (Changmin Chen, Ph.D., Dr. Andrew Kung's Lab)
Human A673 rhabdosarcoma (RMS) or Ewing tumor (ET) cells have been engineered with a stably integrated firefly luciferase reporter driven by the NR0B1 promoter (NR0B1-Luc) and a stably integrated internal control with the ubiquitin C promoter driving Renilla luciferase expression (UbC-RL)(kindly provided by Dr. Changmin Chen from Dr. Andrew Kung's laboratory at Dana-Farber Cancer Insitute (DFCI) in Boston MA). The screening cell line was derived from a single cell clone, and has been fully validated for dependency on EWS/FLI using shRNA knock-down.
Prior to perform the luciferase reporter assay experiments, the cells are maintained in DMEM medium (Invitrogen, 11965) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) (Gibco, 16140), 1X antibiotic (Penicillin/Streptamycin/Glutamine) (Gibco, Ref. no. 10378-016), 2 microg/ml of puromycin (Invitrogen, A1113802) and 200 microg/ml of G418 (Invitrogen, 10131035) using large flasks (Nunclon Delta surface (triple flasks), Cat. No. 132920) and grown in a 5% CO2 incubator at 37oC (Liconic instruments).
Day 1 (Cell plating):
The A673 NR0B1-Luc/UbC-RL cells are trypsinized using 0.25% trypsin with EDTA (Cellgro, 25-053-CL) and resuspended in phenol red-free DMEM (Invitrogen, 31053-028) 10% Heat inactivated FBS (Gibco, 16140), 1X FBS Penicillin/Streptamycin/Glutamine (Gibco in absence of puromycin and G418. A673 cells are then plated in 384-well format cell culture treated solid white assay plates at a density of 3,000 cells/well using the multidrop dispenser (standard cassette)(Thermo Scientific) in a final volume of 30 ul. The assay plates are placed in 5% CO2 incubator where they are incubated overnight at 37oC.
Day 2 (Compound pinning)
The next day, the assay plates are pulled out of the incubator and are placed side by side on a pinning table adjacent to compound plates containing the MLPCN library and a sentinel plate containing 32 wells containing the positive control cytarabine (Sigma, C1768) at a concentration of 25 uM. 100 nl of the compounds and the positive control cytarabine are then collected on metal pins from these compound plates and transferred to the assay plate. The pins are washed with DMSO and methanol between each transfer. The assay plates are then moved back to the 5% CO2 incubator and incubated for an additional 24h.
Day 3 (Add reagents and read fluorescence/luminescence on Envision)
The assay plates are coming out of the incubator and the fluorescence (Emission 488 nm, Excitation 590 nm) is read using an Envision (Perkin Elmer). Then, 10 ul of a 5% Alamar Blue (Invitrogen, DAL1100) solution in PBS is dispensed in each well of the assay plates using a multidrop dispenser (Thermo Scientific). The cells are incubated for 2h at 37oC. The fluorescence is read again using the Envision. After the reading, 20 ul of Steady-Glo reagent solution (Promega, X1006) diluted 1:1 with water is added in each well. The plates are incubated for 30 minutes at room temperature and the luminescence is read on the Envision. Alamar Blue is used as a cell viability marker and is utilized to normalize the luminescence signal associated with the cell number present in the well. The ratio Steady-Glo (luminescence, compound vs. DMSO treated)/Alamar Blue (fluorescence, compound vs. DMSO treated) is then calculated to reflect whether or not the loss of luminescent signal is due to cell toxicity or not. A compound giving a ratio of 1 (100%) will mean that the compound has no effect on the luminescent signal and viability or the compound will inhibit the luminescent signal and decreases the viability with the same factor. A compound with a ratio of 0.5 (50%) will inhibits the luminescent signal by 50% or more and will affect the viability by a factor of half or less compared to the luminescent signal.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold -35.0
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: A673
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)