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BioAssay: AID 624254

Quorum 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_Activity

Assay Overview: This assay using Vibrio cholerae strain BH1651(luxOD47E) will help to define the mechanism of action for each compound. LuxOD47E is a constitutive allele of LuxO that locks the protein into a form mimicking LuxO~P. The LuxO~P mimetic activates transcription of the genes encoding the small regulatory RNAs (Qrr1-4). This, in turn prevents translation of HapR which is required for more ..
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 Tested Compounds
 Tested Compounds
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Active(94)
 
 
Inactive(586)
 
 
Inconclusive(10)
 
 
 Tested Substances
 Tested Substances
All(690)
 
 
Active(94)
 
 
Inactive(586)
 
 
Inconclusive(10)
 
 
 Related BioAssays
 Related BioAssays
AID: 624254
Data Source: Broad Institute (2132-03_Agonist_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-06-04

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 94
Related Experiments
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AIDNameTypeProbeComment
588521Broad Institute Quorum Sensing in Vibrio cholera: CqsS/LuxQ Agonist Probe ProjectSummary1 depositor-specified cross reference: Summary assay
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
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
686928Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_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
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
686928Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_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: Vibrio cholerae, quorum sensing, luxO, luciferase


Assay Overview: This assay using Vibrio cholerae strain BH1651(luxOD47E) will help to define the mechanism of action for each compound. LuxOD47E is a constitutive allele of LuxO that locks the protein into a form mimicking LuxO~P. The LuxO~P mimetic activates transcription of the genes encoding the small regulatory RNAs (Qrr1-4). This, in turn prevents translation of HapR which is required for activation of luciferase. Therefore, this strain is naturally luciferase negative and only if a compound acts on luxO or downstream of luxO will there be light production, measured as an increase in signal.


Expected Outcome: Compounds that act on LuxO or downstream of LuxO (e.g. on Hfq) will induce light production in BH1651 and will lead to an increase in signal (i.e. light production). Since this assay is used to define mechanism of action, any outcome is acceptable for a probe candidate.
Protocol
Reporter strain:
1.For LuxO inhibitors: BH1651 (V. cholerae luxOD47E carrying pBB1 cosmid)
Materials:
LB Medium: Dissolve 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
Tetracycline (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL 100% ethanol, store at -20 oC, protect from light
LB/tet: add 1 mL tetracycline (10 mg/mL) to 1L of LB medium. Final concentration of tetracycline is 10 microg/mL. Use it fresh everytime.
CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20C
Procedures:
1.Grow up reporter strain in 50 mL LB/Tet at 30 oC for >16 hours with shaking (200 rpm). The final OD600 of each culture should be > 3.0
2.Dilute culture to a final OD600 of 0.3. (Note: avoid biofilm aggregates in the culture, a low speed centrifugation (200 rpm for 1 min) should remove most aggregates)
3.Dispense 20 uL of LB/Tet per well of the 384 well plate (Black with clear bottom, such as Greiner 781096 plates).
4.Pin transfer compound (100 nL volume).
5.Dispense 10 microL of diluted culture into each well of a 384 well plate
6.Incubate the plates at 30 oC without shaking for 6 hours.
7.Measure bioluminescence (USLum(384)) and OD600 in a Perkin-Elmer EnVision Multilabel Plate Reader.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing 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 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): absACnn, the concentration at which the curve crosses threshold 50.0
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 >= 30% but <= 70%, 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: Organism-based
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AbsAC50_Qualifier<, =, or >String
2AbsAC50_uM*The concentration at which the fitted curve passes activity threshold 50.FloatμM
3pAbsAC50_MEqual to -1*log10(AbsAC50).Float
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_0.0235uM_(%) (0.0235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.068uM_(%) (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.21uM_(%) (0.21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.6uM_(%) (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.75uM_(%) (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_1.5uM_(%) (1.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_1.6uM_(%) (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_1.8uM_(%) (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_5.6uM_(%) (5.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_6uM_(%) (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_16uM_(%) (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_23.5uM_(%) (23.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_26uM_(%) (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_50uM_(%) (50μM**)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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