Luminescent Cell-Based Dose Titration Retest Counterscreen to Identify Proteasome Inhibitors
Assay Overview: Counterscreen using human osteosarcoma U2OS cells to identify inhibitor of the proteasome pathway. The small molecules reducing the cleavage of peptide linked with luciferin and measured by the conversion of luciferin to oxyluciferin and light (luminescence) mediated by the luciferase will be considered as an inhibitor. ..more
BioActive Compounds: 258
Keywords: Proteasome, MG132
Assay Overview: Counterscreen using human osteosarcoma U2OS cells to identify inhibitor of the proteasome pathway. The small molecules reducing the cleavage of peptide linked with luciferin and measured by the conversion of luciferin to oxyluciferin and light (luminescence) mediated by the luciferase will be considered as an inhibitor.
Expected Outcome: Identification of compounds inhibiting the proteasome pathway. More specifically, compounds diminishing the luminescent response (RLU) greater than 50% of the signal in a dose response obtained from the in-plate positive control (MG132, 1 microM) added to the negative control (DMSO) will be considered as hits.
Proteasome inhibition (Chymotrypsin-GLO, Promega) Counterscreen assay in U2OS cells:
The U2OS (ATCC, HTB-96) cell line is propagated in DMEM media (Invitrogen, SKU 11995) supplemented with 10% heat inactivated fetal bovine serum (Denville Scientific Inc, Cat. No. FB5001-H) and 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) at 37C 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) with 10% Heat inactivated fetal bovine serum (Invitrogen, Cat. No. 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016). 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 BL2 automation unit:
Day 1 (Cell plating):
1. U2OS cells are harvested and resuspended in DMEM without phenol (Cambrex, 12-917F) with 10% Heat inactivated fetal bovine serum (Denville Scientific Inc, Cat. No. FB5001-H) and 1% penicillin/streptomycin/glutamine (Invitrogen,10378-016). U2OS cells (from an initial cell suspension of 50,000 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 2,500 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) BL2 automation unit and incubated for 24 hours at 37C in the Liconic CO2 incubator (Liconic Instruments) calibrated at 5% CO2, 21% O2, and 95% humidity.
Day 2 (Compound pinning into assay plate and reading on Envision):
3.The dose plate containing the positive control compound (MG132, BRD-K60230970-001-05-0) (12 different concentrations starting at 0.25 mM and diluted 2 fold on each subsequent well), vehicle (Base plate, DMSO) and the Cherry pick plate (20 plates total) (5 different concentrations starting at 16.5 mM and diluted 3 fold on each subsequent) are pulled from Walkup refrigerator.
First, the base plate and the dose response plate are pinned twice using 384 well pin tool (100 nl) on pin table (BL2) and transferred to assay plate. Pins are washed with methanol and DMSO between each pinning. Second, the Cherry Pick compounds plates are pinned twice into one assay plate (duplicate). 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 assay plates treated with compounds are moving back to Liconic CO2 incubator to be incubated for an additional 4 hours. After 3h30, each assay plate is pulled out of the incubator and cooled down at room temperature for 30 minutes. 30 μL/well (384 well) of Proteasome-Glo (Chymotrypsin) reagent 0.5X (diluted in H2O) (Promega, G8661) is dispensed using the the MultiDrop Combi/tubing dispensing cassette from Thermo Scientific. The assay plate returned to room temperature for 2hrs to allow a complete cellular permeabilization and maximum signal.
5. Luminescence is measured in each well (0.1 second/well) using the Envision plate reader (Perkin Elmer)(Corning plate setting) (Standard luminescence).
Dose Data Analysis
Neutral control (NC) wells and positive control (PC) wells were included on every plate.
Active inhibitor compounds result in decreased signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
The plate pattern correction algorithm 'Runwise Pattern (Mulitiplicative)' (Genedata v7.0.3) was applied.
IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
Inactive compounds = 0
Active compounds = -10*Log(IC50)
Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
IC50 <= 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.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: U-2 OS
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)