Counterscreen Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set3
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: 21
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 uM) added to the negative control (DMSO) will be considered as hits. These compounds will not be used for further development as they are not specific to the hypoxia inducible factor pathway.
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# FB5001-H) and 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) at 37 degrees 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# 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 red (Invitrogen, SKU# 11995) as mentioned above (for normal cell propagation) or DMEM medium without phenol red (Cambrex; Cat# 12-917F) with 10% Heat inactivated fetal bovine serum (Invitrogen, 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016). Cell number is counted using a Nexelcom Bioscience cell counter (Cellometer 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 red (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 uL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
2. The assay plates (cell plates) are placed in Liconic Instruments cassettes (22 plates/cassette) in the BL2 automation unit and incubated for 24 hours at 37 degreees C 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 dry powder compound plates (8 different concentrations starting at 16.5 mM and diluted 3 fold on each subsequent) are pulled from Walkup refrigerator. First, the MG132 dose response plates are pinned twice using 384 well pin tool (100 nl) on pin table (BL2) and transferred to assay plates. Pins are washed with methanol and DMSO between each pinning. Second, the dry powder compounds plates are pinned twice into one assay plate (duplicate). Finally, 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 moved back to Liconic CO2 incubator to be incubated for an additional 4 hours. After 3hours 30 minutes, each assay plate is pulled out of the incubator and cooled down at room temperature for 30 minutes. 30 uL/well (384 well) of Proteasome-Glo (Promega, G8661)(Chymotrypsin) reagent 0.5X (diluted in H2O) is dispensed using the the MultiDrop Combi/tubing dispensing cassette from Thermo Scientific. The assay plates are returned to room temperature for 2 hours 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).
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=16) 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 (10)(Max_Concentration).
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 neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)