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BioAssay: AID 623895

HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set6

The HDL uptake primary assay is looking for a decrease in fluorescence. It is possible that a compound will cause cells to decrease HDL uptake due to non-specific consequences of cellular toxicity. Therefore, we would like to make sure that lead compounds are not toxic in the 3 hour time period used in the primary assay. Cells are treated with compounds for 24 hours and then cell viability is more ..
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 Tested Compounds
 Tested Compounds
All(107)
 
 
Active(16)
 
 
Inactive(91)
 
 
 Tested Substances
 Tested Substances
All(107)
 
 
Active(16)
 
 
Inactive(91)
 
 
AID: 623895
Data Source: Broad Institute (2085-02_Inhibitor_Dose_DryPowder_Activity_Set6)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-03-28
Hold-until Date: 2013-03-15
Modify Date: 2013-03-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 16
Related Experiments
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AIDNameTypeComment
488952Broad Institute Inihibitors of the HDL Receptor, SR-BI Inhibitors Probe ProjectSummarydepositor-specified cross reference: Summary assay
488896HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
493194HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
540246HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
540326DiI Quencher Assay Measured in Biochemical System Using Plate Reader - 2085-08_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
540354HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588392HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
588403DiI Quencher Assay Measured in Biochemical System Using Plate Reader - 2085-08_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588548HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
588553HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
588754HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatorysame project related to Summary assay
588777HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588810HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
588818HDL MOA Assay: Efflux Assay Measured in Cell-Based System Using Plate Reader - 2085-06_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
588825HDL Uptake counterscreen Measured in Cell-Based System Using Plate Reader - 2085-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588826HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
588828HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatorysame project related to Summary assay
588829HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
588830HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-11_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588831HDL Inhib: Reversibility Measured in Cell-Based System Using Plate Reader - 2085-14_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588833HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
588836HDL Inhib: Activity in wild type cell line Measured in Cell-Based System Using Scintillation - 2085-12_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
588837HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Scintillation - 2085-13_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
588842HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
588843HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
602126HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatorysame project related to Summary assay
602134HDL Counter Assay: Tf Endo Assay Measured in Cell-Based System Using Plate Reader - 2085-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602138HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
602152HDL MOA Assay: Efflux Assay Measured in Cell-Based System Using Plate Reader - 2085-06_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
602154HDL Inhib: Reversibility Measured in Cell-Based System Using Plate Reader - 2085-15_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602155Liposome Uptake Assay Measured in Biochemical System Using Scintillation - 2085-16_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
602285HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set8Confirmatorysame project related to Summary assay
623880HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set9Confirmatorysame project related to Summary assay
624007HDL Uptake counterscreen Measured in Cell-Based System Using Plate Reader - 2085-07_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624010HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Scintillation - 2085-13_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624015HDL Inhib: Activity in mutant cell line Measured in Cell-Based System Using Plate Reader - 2085-10_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624017HDL MOA Assay: Efflux Assay Measured in Cell-Based System Using Plate Reader - 2085-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624022HDL Inhib: Activity in wild type cell line Measured in Cell-Based System Using Scintillation - 2085-12_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624024HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
624026HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
624029HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_DryPowder_Activity_Set10Confirmatorysame project related to Summary assay
Description:
Keywords: cytotoxicity, Cell titer glo, ldlA[SRB1] cells


Assay Overview:

The HDL uptake primary assay is looking for a decrease in fluorescence. It is possible that a compound will cause cells to decrease HDL uptake due to non-specific consequences of cellular toxicity. Therefore, we would like to make sure that lead compounds are not toxic in the 3 hour time period used in the primary assay. Cells are treated with compounds for 24 hours and then cell viability is measured using the Cell Titer Glo Assay which is a luciferase-based reagent that measures cellular ATP levels. Compounds will be tested at 8 different doses to determine IC50 values. Compounds that are toxic after 3 hours at an IC50 less than 30 uM will be excluded from additional studies. Compounds that are active in the primary assay but toxic below 30 uM at 24 hours will still be pursued.

Expected Outcome:

Compounds that are cytotoxic will decrease cellular ATP levels resulting in a decrease in luminescence.
Protocol
Protocol:
Day 0:
Plate 4,000 cells ldlA[mSR-BI] 30microl/ well in Ham's F12/10% FCS/PSG
Day 1:
1)Pin transfer 100 nl compounds and positive control (sentinel plate).
2)Incubate 24 hours @ 37 degrees C
3)Add 20 uL Promega Cell titer glo per well
4)Shake plate for 15 seconds
5)Incubate 10 minutes
6)Read luminescence with Perkin-Elmer Envision plate reader
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
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 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: 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.
DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
From ChEMBL:
Assay Type: Functional
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
4ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
5ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
6ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.String
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_0.000060uM_(%)_TEST1 (6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.00012uM_(%)_TEST1 (0.00012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.00026uM_(%)_TEST1 (0.00026μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.0005uM_(%)_TEST1 (0.0005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.001uM_(%)_TEST1 (0.001μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.0021uM_(%)_TEST1 (0.0021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.0042uM_(%)_TEST1 (0.0042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.008uM_(%)_TEST1 (0.008μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.016uM_(%)_TEST1 (0.016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_0.03uM_(%)_TEST1 (0.03μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_0.068uM_(%)_TEST1 (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_0.135uM_(%)_TEST1 (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_0.26uM_(%)_TEST1 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_0.5uM_(%)_TEST1 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_1uM_(%)_TEST1 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_2.1uM_(%)_TEST1 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_4.2uM_(%)_TEST1 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_8uM_(%)_TEST1 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_16uM_(%)_TEST1 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_19.5uM_(%)_TEST1 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37Activity_at_35uM_(%)_TEST1 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
38ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
39ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
40ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
41AC50_Qualifier_TEST2'>','=', or '<'String
42AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
43pAC50_M_TEST2Equal to -1*log10(AC50)String
44Hill_Slope_TEST2The slope at AC50Float
45S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
46Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
47Num_Points_TEST2The number of data points used to generate the plotInteger
48Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
49Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
50Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
51Activity_at_0.015uM_(%)_TEST2 (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
52Activity_at_0.016uM_(%)_TEST2 (0.016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
53Activity_at_0.046uM_(%)_TEST2 (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
54Activity_at_0.05uM_(%)_TEST2 (0.05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
55Activity_at_0.135uM_(%)_TEST2 (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
56Activity_at_0.15uM_(%)_TEST2 (0.15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
57Activity_at_0.42uM_(%)_TEST2 (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
58Activity_at_1.2uM_(%)_TEST2 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
59Activity_at_1.35uM_(%)_TEST2 (1.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
60Activity_at_3.8uM_(%)_TEST2 (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
61Activity_at_11uM_(%)_TEST2 (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
62Activity_at_12uM_(%)_TEST2 (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
63Activity_at_35uM_(%)_TEST2 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 2 R01 HL052212-11

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