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BioAssay: AID 488804

Proteasome Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set2

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
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 Tested Compounds
 Tested Compounds
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Active(5)
 
 
Inactive(54)
 
 
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 Tested Substances
All(59)
 
 
Active(5)
 
 
Inactive(54)
 
 
 Related BioAssays
 Related BioAssays
AID: 488804
Data Source: Broad Institute (2030-02_Activator_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-18
Hold-until Date: 2011-10-15
Modify Date: 2011-10-15

Data Table ( Complete ):           Active    All
BioActive Compounds: 5
Depositor Specified Assays
AIDNameTypeComment
1971Broad Institute MLPCN Hypoxia Inducible Factor Activation ProjectsummarySummary assay
Description:
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.
Protocol
Protocol:
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 (Invitrogen, SKU# 11995) as mentioned above (for normal cell propagation) or DMEM medium without phenol (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 (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).
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) 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 (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).


NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Compounds Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate sample compound 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.


PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) 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

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 not valid due to poor fit quality.


PUBCHEM_ACTIVITY_SCORE:

PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)

PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:

120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M

PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3Log_AC50_MThe log of the molar AC50Float
4Log_AC50_M_Standard_ErrorThe standard error for the log of the molar AC50 valueFloat
5Hill_SlopeThe slope at AC50Float
6S0The fitted activity value at zero concentrationFloat%
7SinfThe fitted activity value at infinite concentrationFloat%
8Num_PointsThe number of data points used to generate the plotInteger
9Max_ActivityThe maximum activity value observed, based on mean of replicates per concentrationFloat%
10Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
11Activity_at_0.018uM (0.018μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.056uM (0.056μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.16uM (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.5uM (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_1.5uM (1.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_4.6uM (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_13.5uM (13.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_38uM (38μM**)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)
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