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BioAssay: AID 624315

MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_Activity_Set3

The confirmed modulators of MITF activity will be tested for MITF dependence in SK-MEL-5 melanoma cells which do not proliferate when MITF levels are reduced with RNA interference methods. This is the parental cell line for the reporter cell line used in the primary assay. Cells will be pinned with compounds and cell viability will be measured using cellular ATP levels as a read out. Cells will be treated with multiple doses of the compounds. Cell will be treated for 24 hours and ATP will be measured with Promega's Cell Titer Glo reagent. ..more
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 Tested Compounds
 Tested Compounds
All(105)
 
 
Active(85)
 
 
Inactive(20)
 
 
 Tested Substances
 Tested Substances
All(107)
 
 
Active(87)
 
 
Inactive(20)
 
 
AID: 624315
Data Source: Broad Institute (2084-02_Inhibitor_Dose_DryPowder_Activity_Set3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-06-20
Hold-until Date: 2013-06-19
Modify Date: 2013-06-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 85
Related Experiments
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AIDNameTypeProbeComment
488944Summary of Broad Institute MLPCN Identification of MITF Inhibitors ProjectSummary1 depositor-specified cross reference: Summary assay
488899MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_SinglePoint_HTS_ActivityScreening same project related to Summary assay
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
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
Description:
Keywords: melanoma, MITF, luciferase, cell titer glo

Assay Overview:
The confirmed modulators of MITF activity will be tested for MITF dependence in SK-MEL-5 melanoma cells which do not proliferate when MITF levels are reduced with RNA interference methods. This is the parental cell line for the reporter cell line used in the primary assay. Cells will be pinned with compounds and cell viability will be measured using cellular ATP levels as a read out. Cells will be treated with multiple doses of the compounds. Cell will be treated for 24 hours and ATP will be measured with Promega's Cell Titer Glo reagent.

Expected Outcome: A true inhibitor of MITF activity should decrease proliferation of MITF-dependent cells, such as SK-MEL-5, but not a cell line that is not dependent upon MITF. A candidate probe will decrease cellular viability with an IC50 of less than 10 uM.
Protocol
- On day 1, plate cells 3000 per well in 30 uL phenol red free media
- On day 2, pin 100 nL into 30 uL assay volume in white, opaque 384 plates. (will also require sentinel pinning)
- Incubate 24 hours at 37 degrees C
- On day 3, add 20 uL 100% Promega Cell Titer glo with Thermo Combi fluid transfer apparatus.
- Shake 15 seconds on "big bear" plate shaker.
- Incubate at RT for 5 minutes.
- Read on Envision with standard luminescence settings
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) 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 (2)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

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)

PUBCHEM_ACTIVITY_OUTCOME:
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.

PUBCHEM_ACTIVITY_SCORE:
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.

Note:
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.
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
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.000060uM_(%) (6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.00012uM_(%) (0.00012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.00026uM_(%) (0.00026μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.0005uM_(%) (0.0005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.001uM_(%) (0.001μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.0021uM_(%) (0.0021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.0042uM_(%) (0.0042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.008uM_(%) (0.008μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.016uM_(%) (0.016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.03uM_(%) (0.03μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.068uM_(%) (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.135uM_(%) (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.26uM_(%) (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.5uM_(%) (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_1uM_(%) (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_2.1uM_(%) (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_4.2uM_(%) (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_8uM_(%) (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_16uM_(%) (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_19.5uM_(%) (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_23.5uM_(%) (23.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_35uM_(%) (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** 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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