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BioAssay: AID 2089

Luminescence Cell-Based Confirmation at Dose to Identify Transcriptional Activators of Hypoxia-Inducible Factor Pathway

Keywords: Hypoxia, Hypoxia Inducible Factor (HIF), Hypoxia Responsive Element (HRE), luciferase, transcriptional activation, tissue regeneration, ischemia ..more
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 Tested Compounds
 Tested Compounds
All(1169)
 
 
Active(313)
 
 
Inactive(856)
 
 
 Tested Substances
 Tested Substances
All(1169)
 
 
Active(313)
 
 
Inactive(856)
 
 
 Related BioAssays
 Related BioAssays
AID: 2089
Data Source: Broad Institute (2030-01_ACTIVATORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-10-30

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 313
Related Experiments
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AIDNameTypeComment
1910Luminescence Cell-Based Primary HTS to Identify Transcriptional Activators of Hypoxia-Inducible Factor PathwayScreeningdepositor-specified cross reference: Primary HTS
1971Broad Institute MLPCN Hypoxia Inducible Factor Activation ProjectSummarydepositor-specified cross reference: Project Summary
2486Luminescent Cell-Based Dose Titration Retest Counterscreen to Identify Proteasome InhibitorsConfirmatorysame project related to Summary assay
434951Fluorescent Cell-Based Secondary Screen to Identify Activators of the Hypoxia Factor PathwayConfirmatorysame project related to Summary assay
488804Proteasome Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
488805Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
488820Hypoxia Inducible pathway Measured in Cell-Based System Using Imaging - 2030-03_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
488843HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493180Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493193Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493195Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set6Confirmatorysame project related to Summary assay
493213Counterscreen Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493225Hypoxia inducible pathway Measured in Cell-Based System Using Imaging - 2030-03_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493227Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set8Confirmatorysame project related to Summary assay
493228Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493230HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493234Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493235Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
493236Counterscreen Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493237Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493238HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493239Hypoxia inducible pathway Measured in Cell-Based System Using Imaging - 2030-03_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493249Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Toxicity_Set7Confirmatorysame project related to Summary assay
504323Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_ToxicityConfirmatorysame project related to Summary assay
504324Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
504331Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_ToxicityConfirmatorysame project related to Summary assay
504588Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set10Confirmatorysame project related to Summary assay
504597Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set9Confirmatorysame project related to Summary assay
Description:
Keywords: Hypoxia, Hypoxia Inducible Factor (HIF), Hypoxia Responsive Element (HRE), luciferase, transcriptional activation, tissue regeneration, ischemia

Assay:
Luciferase assay (Steady-Glo, Promega).
Confirmation at dose screen using human osteosarcoma U2OS cells stably over-expressing a plasmid containing 3 copies of the Hypoxia Responsive Element linked to luciferase gene (U2OS HRE-luciferase cells) to identify small molecules inducing an increased luciferase activity in these cells. The small molecules inducing expression of luciferase under HRE promoter will be measured by the conversion of luciferin to oxyluciferin and light (luminescence) mediated by the luciferase.

Expected Outcome:
Identification of probe(s) activating the transcription factor hypoxia inducible factor (HIF) pathway. More specifically, EC50 determination of cherry-pick compounds from primary screen inducing a luminescent response (RLU) greater than 10% of the signal obtained from the in-plate positive control (100 microM Desferrioxamine) added to the negative control (DMSO).
Protocol
U2OS HRE-luciferase assay:
Summary of ELN assay protocols taken from Cbip ID runs: 2030-01-A01-04-01 to 2030-01-A01-11-04.
The U2OS-HRE-luc cell line was originally obtained from Dr. Margaret Ashcroft's lab and kindly provided by Dr. Shawn Gilbert.
The U2OS-HRE-luc cell line is propagated in DMEM media (Invitrogen, SKU#11995) supplemented with 10% heat inactivated fetal bovine serum (Invitrogen, Cat. No. 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) and 0.1 mg/ml Hygromycin B (Invitrogen, 10687-01) at 37'C in CO2 incubators (Thermo Scientific) with 5% CO2, 21% O2, and 95% humidity. For High-Throughput Screening assays, cells are grown in T175 flask (BD Falcon, Ref# 353112) or Hyperflasks (Corning, Cat# 10010), harvested at more than 80% confluence using 7 or 75 ml Trypsin-EDTA 0.25% (Cellgro, Cat. No. 25-053-CI) for 5 minutes and then the trypsin is inactivated with 7 or 75 ml of complete medium respectively. Cells should not be trypsinized more than 10 minutes because massive cell death will start to occur. Cells are centrifuged at 1000rpm/5min and resuspended in fresh complete DMEM media with phenol (Invitrogen, SKU#11995) as mentioned above (for normal cell propagation) or DMEM medium without phenol (Cambrex; Cat. No 12-917F or Invitrogen; cat. no.31053) with 10% Heat inactivated fetal bovine serum (Invitrogen, Cat. No. 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016), sodium pyruvate (only used with DMEM without phenol from Invitrogen (cat# 11306-070)) and without hygromycin B (for compound screening). Cell number is counted using a Nexelcom Bioscience cell counter (Cellometer(r) Auto M10) and viability is measured by mixing one volume of cells with one same volume of Trypan Blue solution (0.4%)(dilution 1/2). Only cultures of >94% viability are utilized for HTS.
Compound Screening is carried out on the Broad Institute/Chemical Biology Platform General system (GS) automation unit:
Day 1 (Cell plating):
1. U2OS-HRE-luc cells are harvested and resuspended in DMEM without phenol (Cambrex, 12-917F) with 10% Heat inactivated fetal bovine serum (Invitrogen, Cat. No. 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen,10378-016) without hygromycin B. U2OS HRE-luciferase cells (from an initial cell suspension of 120,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning, Cat.No. 8867BC) at a final density of 6,000 cells per well in final volume of 50 μL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
2. The assay plate (cell plate) are placed in Liconic Instruments cassettes (22 plates/cassette) and incubated for 24 hours at 37'C in the Liconic CO2 incubator 9 (Model STX 220IC)(General automation system (GS)) (Liconic Instruments) calibrated at 5% CO2, 21% O2, and 95% humidity.
Day 2 (Compound pinning into assay plate):
3. The MLPCN test compounds plates are transferred from the Compound Management incubators STX1000#1 and #2 system (Liconic Instruments) on the GS to the Liconic CO2 incubator 8 (Model STX 220HR) before the initiation of the pinning run. The In-plate positive control compound plate (sentinel)(100 μM DFX (Desferrioxamine), Sigma-Aldrich D9533, BRD-K09821361-066-08-4), dose response plate (DFX) (12 different concentrations starting at 160 μM and diluted 2 fold on each subsequent well) or vehicle (Base plate, DMSO) are already present in the Liconic incubator 8 at the beginning of the pinning. Both STX and Liconic 8 CO2 incubator temperature are kept at 20'C. First, the base plate and the dose response plate are pinned once using 384 well pin tool (100 nl) on pin table (GS) and transferred to assay plate. Pins are washed with methanol and DMSO between each pinning. Second, the MLPCN test compounds plates are pinned as well as the in-plate positive control (32 wells, 100 μM DFX) are pinned consecutively one after the other and transferred into one assay plate. Each compound plate is pinned twice in 2 different assay plates (duplicate). The final concentration for the MLPCN test compounds is 7.5μM with final concentration no more than 1% DMSO. For the Cherry picks retest (confirmation at dose), the MLPCN test compounds retest plates were pinned once (100 nl) with Cherry picks 1170 compounds at 8 concentrations with a dilution factor of 3 (starting concentration 33 microM). Finally, a second round of the base plate and the dose response plate pinning is achieved at the end of the run.
4. After the pinning has occurred, the In-plate positive control, dose response and base plates are returning into the Liconic CO2 incubator 8 and the MLPCN compounds plates are placed back into the STX1000 systems. The assay plates treated with compounds are moving back to Liconic CO2 incubator 9 to be incubated for an additional 24 hours.
Day 3 (Reading luminescence from assay plates with Envision):
5. The assay plates are physically transferred from Liconic CO2 incubator 9 to Liconic CO2 incubator 7 (Model STX 220IC). Each assay plate is pulled out of the incubator and cooled down at room temperature for 30 minutes on Liconic Carousel 1. 30 μL/well (384 well) of Steady-Glo luciferase reagent 0.5X (diluted in H2O) (Promega, E2550) is dispensed using the the MultiDrop Combi/long tubing dispensing cassette from Thermo Scientific. The assay plate returned to Liconic Carousel 1 for 30 minutes to allow a complete cellular lysis.
6. Luminescence is measured (0.2 second/well) using the ultra sensitive luminescence detector (384-well aperture, 0.5 mm height) in each well using the Envision plate reader (Perkin Elmer)(Corning plate setting).
7. HRE activation is calculated based on the following equation using mean luminescence (RLU) values:
Fold Activation = #Treatment - Background No treatment - Background
Compounds inducing a luminescent response (RLU) greater than 10% of the value obtained with in-plate positive (100 microM Desferrioxamine) added to the negative control (DMSO) background in both assay plates will be considered as active hits. Compounds inducing a luminescent response (RLU) greater than 10% of the value obtained with in-plate positive (100 microM Desferrioxamine) added to the negative control (DMSO) background in only one out of 2 assay plates will be considered inconclusives and the compounds inducing a luminescent response (RLU) lower than 10% of the value obtained with in-plate positive (100 microM Desferrioxamine) added to the negative control (DMSO) background in both assay plates will be considered not actives.
Comment
Negative control: DMSO only
Positive control: Desferrioxamine, 100 micromolar
Negative control wells (n=32) and positive controls well (n=32) were included on every plate.
Normalization of all wells tested was performed by
1) subtracting background using the median of the negative control wells on the same plate (0% activity), and
2) scaling using the median value of the positive control wells on the same plate (100% activity).
Pattern correction of systemic patterns in the data was performed using the Genedata Screener (v7.0.3) Assay Analyzer module's "Runwise Pattern (Multiplicative)" algorithm.
EC50 values were calculated using the curve fitting algorithms of Genedata Screener (v7.0.3) Condoseo module. EC50 values were extrapolated up to 1 log over the highest tested concentration.
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 activity >30% but <70%
or
The fit was not valid due to poor fit quality
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: U-2 OS
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3EC50 Standard Errorthe standard error for the calculated EC50 valueFloatμM
4S0the fitted activity level at zero concentrationFloat%
5SInfthe fitted activity level at infinite concentrationFloat%
6Hill Slopethe slope at EC50Float
7Num. Pointsthe number of data points included in the plotInteger
8Max. Activitythe maximum activity value observedFloat%
9Activity at 0.01uM (0.01μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.05uM (0.05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.14uM (0.14μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 0.42uM (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 1.20uM (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 3.80uM (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 11.00uM (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 35.00uM (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH082355-01A2

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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