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
BioActive Compounds: 313
Keywords: Hypoxia, Hypoxia Inducible Factor (HIF), Hypoxia Responsive Element (HRE), luciferase, transcriptional activation, tissue regeneration, ischemia
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.
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).
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.
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.
Inactive compounds = 0
Active compounds = -10*Log(EC50)
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%
The fit was not valid due to poor fit quality
* Activity Concentration. ** Test Concentration.
Data Table (Concise)