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BioAssay: AID 488899

MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_SinglePoint_HTS_Activity

The primary HTS will be performed using SK-MEL-5 cells stably expressing the TRPM1 promoter upstream of the luciferase gene. TRPM-1 is a known target of the transcription factor, MITF. The assay in its current format utilizes plating of 2000 cells per well in 384 well plates using 30 ul total volume (cells plus media) per well. This will allow us to use a volume of 100 nl for pinning of the compounds 24 hours later. The cells will be incubated with test molecules for 24 hours and the luciferase activity will be detected using 20 ul per well of the Steady Glo Reagent from Perkin Elmer. ..more
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 Tested Compounds
 Tested Compounds
All(331348)
 
 
Active(2760)
 
 
Inactive(328427)
 
 
Inconclusive(163)
 
 
 Tested Substances
 Tested Substances
All(331578)
 
 
Active(2767)
 
 
Inactive(328648)
 
 
Inconclusive(163)
 
 
AID: 488899
Data Source: Broad Institute (2084-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2760
Related Experiments
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AIDNameTypeProbeComment
488944Summary of Broad Institute MLPCN Identification of MITF Inhibitors ProjectSummary1 depositor-specified cross reference
540328Broad Institute Identification of MITF small molecule inhibitors Activator Probe ProjectSummary depositor-specified cross reference
493073MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_DoseNoFile_CherryPick_Activity_Set3Confirmatory same project related to Summary assay
493102MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_DoseNoFile_CherryPick_Activity_Set4Confirmatory same project related to Summary assay
493177MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_DoseNoFile_CherryPick_Activity_Set2Confirmatory same project related to Summary assay
493191MITF Orthogonal Assay: MALME3 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-04_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
493240MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_CherryPick_Activity_Set3Confirmatory same project related to Summary assay
540335MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_CherryPick_Activity_Set2Confirmatory same project related to Summary assay
540339MITF Orthogonal Assay: MALME3 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
540346MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
540347MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
540348MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624259MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
624289MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
624290MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
624299MITF Orthogonal Assay: MALME3 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
624315MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
624316MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
624324MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
624362MITF Orthogonal Assay: MALME3 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-04_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
624363MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatory same project related to Summary assay
624364MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
624366MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
624368MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatory same project related to Summary assay
624426MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatory same project related to Summary assay
624427MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatory same project related to Summary assay
624428MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatory same project related to Summary assay
624429MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
624430MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set8Confirmatory same project related to Summary assay
624440MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
624488MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
624489MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
624490MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatory same project related to Summary assay
624492MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_DryPowder_Activity_Set8Confirmatory same project related to Summary assay
651584MITF Orthogonal Assay: MALME3 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-04_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatory same project related to Summary assay
651585MITF Orthogonal Assay: MALME3 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-04_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
651586MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_Activity_Set8Confirmatory same project related to Summary assay
651588MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set9Confirmatory same project related to Summary assay
651591MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_DryPowder_Activity_Set9Confirmatory same project related to Summary assay
651753MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set10Confirmatory same project related to Summary assay
651770qPCR Assay for TRPM1 Expression Measured in Cell-Based System Using RT-PCR - 2084-09_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651771qPCR Assay for DCT Expression Measured in Cell-Based System Using RT-PCR - 2084-12_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651772qPCR Assay for CDK2 Expression Measured in Cell-Based System Using RT-PCR - 2084-11_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651773qPCR Assay for MITF Expression Measured in Cell-Based System Using RT-PCR - 2084-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651795qPCR Assay for MLANA Expression Measured in Cell-Based System Using RT-PCR - 2084-13_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651920Primary human melanocyte cytotoxicity assay Measured in Cell-Based System Using Plate Reader - 2084-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
540258MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Activator_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
540259MITF Act Counter Assay: HeLa CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-08_Activator_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
588334MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Activator_SinglePoint_HTS_ActivityScreening same project related to Summary assay
651775MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Activator_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
Description:
Keywords: MITF, TRPM-1, melanoma, SK-MEL-5, luciferase reporter

Assay Overview:

The primary HTS will be performed using SK-MEL-5 cells stably expressing the TRPM1 promoter upstream of the luciferase gene. TRPM-1 is a known target of the transcription factor, MITF. The assay in its current format utilizes plating of 2000 cells per well in 384 well plates using 30 ul total volume (cells plus media) per well. This will allow us to use a volume of 100 nl for pinning of the compounds 24 hours later. The cells will be incubated with test molecules for 24 hours and the luciferase activity will be detected using 20 ul per well of the Steady Glo Reagent from Perkin Elmer.
Protocol
MITF Primary Screening Protocol
(TRPM-1 Promoter//Luciferase reporter assay)
On day 1, plate cells 2000 per well in 30 uL media (phenol red free DMEM/10% iFBS/Pen/Strep/L-Glutamine)
On day 2, pin 100 nL into 30 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates. (will also require sentinel pinning with the positive control, parthenolide)
Incubate 24 hours at 37 degrees C in Liconic incubator
On day 3, add 20 uL 100% Promega Steady glo with Thermo Combi fluid transfer apparatus.
Shake 15 seconds on 'big bear' plate shaker.
Incubate at RT for 5 minutes.
Read on Perkin-Elmer Envision with US LUM settings for 0.5 sec per well
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.
NORMALIZATION:
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 'Assay Pattern (multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 60.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 60.
PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: SK-MEL-5
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was InvalidFloat
2REPLICATE_A_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%
3REPLICATE_B_ACTIVITY_SCORE (12.5μM**)The calculated percent activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1 R03 DA031089-01

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