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BioAssay: AID 686928

Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Inhibitor_Dose_DryPowder_Activity

Assay Overview: A probe candidate for this project could potentially be tested in animal models of the disease. Therefore, the candidate should have activity in Vibrio cholerae but no liabilities such as cytoxicity in the mammalian host. One means of assessing cytotoxicity is the CellTiter-Glo luciferase assay which measures cellular ATP as a surrogate marker of cell viability. Cells are more ..
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AID: 686928
Data Source: Broad Institute (2132-02_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-04-10
Hold-until Date: 2013-08-06
Modify Date: 2013-08-06

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 5
Related Experiments
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AIDNameTypeProbeComment
588521Broad Institute Quorum Sensing in Vibrio cholera: CqsS/LuxQ Agonist Probe ProjectSummary1 depositor-specified cross reference: summary
686945Broad Institute Quorum Sensing in Vibrio cholera: LuxO Inhibitor Probe ProjectSummary depositor-specified cross reference
588436Cholera Quorum: HTS for inducers of light production in the absence ofautoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_SinglePoint_HTS_ActivityScreening same project related to Summary assay
602243Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
624140Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624254Quorum LuxO: Assay for inducers of light production in the absence of#autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
624269Quorum LuxO: Assay for inducers of light production in the absence of#autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624270Cholera Quorum: HTS for inducers of light production in the absence of#autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624275Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of#autoinducers using WN1103 (delta luxQ) Measured in Microorganism System Using Plate Reader - 2132-05_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624281Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence#of autoinducers using DH231 (delta cqsS) Measured in Microorganism System Using Plate Reader - 2132-04_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651774Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Agonist_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651808Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using DH231 (delta cqsS) Measured in Microorganism System Using Plate Reader - 2132-04_Agonist_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651809Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
651816Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
651841Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using WN1103 (delta luxQ) Measured in Microorganism System Using Plate Reader - 2132-05_Agonist_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651847Quorum LuxO: Assay for inducers of light production in the absence of autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
651854Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651864Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Agonist_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
651905CqsS In vitro Kinase Assay Measured in Biochemical System Using Imaging - 2132-10_Agonist_Dose_DryPowder_ActivityOther same project related to Summary assay
652219Quorum LuxO: Assay for change of GFP production in the absence of autoinducers using SL353 (luxOD47E, qrr4-gp) Measured in Microorganism System Using Plate Reader - 2132-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
652239Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
686929Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
686930Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using WN1103 (delta luxQ) Measured in Microorganism System Using Plate Reader - 2132-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
686931Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using DH231 (delta cqsS) Measured in Microorganism System Using Plate Reader - 2132-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
686936In vitro LuxO ATPase Assay Measured in Biochemical System Using Plate Reader - 2132-11_Inhibitor_Dose_DryPowder_ActivityOther same project related to Summary assay
686939Counterscreen for activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM): Luminescence-based cell-based high throughput screening assay to identify non-selective compounds using the VP16 reporter assayScreening same project related to Summary assay
686942Quorum LuxO: Assay for change of GFP production in the absence of autoinducers using SL353 (luxOD47E, qrr4-gp) Measured in Microorganism System Using Plate Reader - 2132-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
588436Cholera Quorum: HTS for inducers of light production in the absence ofautoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_SinglePoint_HTS_ActivityScreening same project related to Summary assay
602243Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
624140Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624254Quorum LuxO: Assay for inducers of light production in the absence of#autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
624269Quorum LuxO: Assay for inducers of light production in the absence of#autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651847Quorum LuxO: Assay for inducers of light production in the absence of autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
651864Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Agonist_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
652219Quorum LuxO: Assay for change of GFP production in the absence of autoinducers using SL353 (luxOD47E, qrr4-gp) Measured in Microorganism System Using Plate Reader - 2132-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
652239Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
652289Quorum LuxO: Assay for inducers of light production in the absence of autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
686929Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
686936In vitro LuxO ATPase Assay Measured in Biochemical System Using Plate Reader - 2132-11_Inhibitor_Dose_DryPowder_ActivityOther same project related to Summary assay
686942Quorum LuxO: Assay for change of GFP production in the absence of autoinducers using SL353 (luxOD47E, qrr4-gp) Measured in Microorganism System Using Plate Reader - 2132-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
Description:
Keywords: CellTiter-Glo, HeLa, cytotoxicity, ATP

Assay Overview: A probe candidate for this project could potentially be tested in animal models of the disease. Therefore, the candidate should have activity in Vibrio cholerae but no liabilities such as cytoxicity in the mammalian host. One means of assessing cytotoxicity is the CellTiter-Glo luciferase assay which measures cellular ATP as a surrogate marker of cell viability. Cells are treated with compounds for 24 hours and then cell viability is measured using the CellTiter-Glo reagent. Compounds will be tested at multiple doses to determine IC50 values. Compounds that are toxic after 24 hours at IC50<30 uM will be excluded from additional studies.

Expected Outcome: A small percentage of compounds identified as hits will be toxic to cells at less than 10 uM. This will lead to a reduction in cellular ATP levels which correlates with a decreased luminescence signal and increased cytotoxicity. Compounds that exhibit cytotoxicity at an IC50 below 30 uM will not be considered for additional studies.
Protocol
HeLa cells are seeded at 3,000 cells per well in 30 uL phenol-red free DMEM/10% fetal bovine serum to opaque, white Corning 8867BC 384 well plates. The following day, 100 nL of compound is added per well with the CyBio Vario pinning apparatus. HeLa cells are incubated for 24 hours at 37 degrees C. 20 uL of Cell Titer Glo (Promega) is added per well, shaken for 2 minutes, incubated at room temperature for 8 minutes and then read on the Perkin Elmer EnVision plate reader with standard luminescence parameters. Compounds which exhibit no cytotoxicity (IC50 >30 uM) in mammalian cells will be prioritized for follow-up.
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 (v10.0.2):
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.10.0.2) 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: HeLa
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_0uM_(%) (0μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.000001uM_(%) (1e-06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.000002uM_(%) (2e-06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.000003uM_(%) (3e-06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.000006uM_(%) (6e-06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.000007uM_(%) (7e-06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.000010uM_(%) (1e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.000018uM_(%) (1.8e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.000021uM_(%) (2.1e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.000030uM_(%) (3e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.000050uM_(%) (5e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.000060uM_(%) (6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.000090uM_(%) (9e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.00016uM_(%) (0.00016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.000195uM_(%) (0.000195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_0.000285uM_(%) (0.000285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_0.00046uM_(%) (0.00046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_0.00056uM_(%) (0.00056μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_0.0008uM_(%) (0.0008μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_0.00135uM_(%) (0.00135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_0.0016uM_(%) (0.0016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_0.0026uM_(%) (0.0026μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_0.0042uM_(%) (0.0042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_0.005uM_(%) (0.005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_0.0075uM_(%) (0.0075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_0.012uM_(%) (0.012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37Activity_at_0.015uM_(%) (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
38Activity_at_0.0235uM_(%) (0.0235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_0.038uM_(%) (0.038μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_0.046uM_(%) (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_0.068uM_(%) (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_0.11uM_(%) (0.11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_0.135uM_(%) (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_0.21uM_(%) (0.21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_0.35uM_(%) (0.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46Activity_at_0.42uM_(%) (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
47Activity_at_0.6uM_(%) (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
48Activity_at_1uM_(%) (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
49Activity_at_1.2uM_(%) (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
50Activity_at_1.8uM_(%) (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
51Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
52Activity_at_3.8uM_(%) (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
53Activity_at_5.6uM_(%) (5.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
54Activity_at_9uM_(%) (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
55Activity_at_11uM_(%) (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
56Activity_at_16uM_(%) (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
57Activity_at_28.5uM_(%) (28.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
58Activity_at_35uM_(%) (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
59Activity_at_38uM_(%) (38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
60Activity_at_50uM_(%) (50μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
61Activity_at_60uM_(%) (60μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
62Activity_at_80uM_(%) (80μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
63Activity_at_100uM_(%) (100μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03 MH094166-01

Data Table (Concise)
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