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BioAssay: AID 2735

Luminescence Microorganism Dose Retest to Identify Inhibitors of the AI-2 Quorum Sensing System

Keywords: Quorum Sensing, Auto-inducer 2 (AI-2), Vibrio harveyi, LuxS,LuxPQ,LuxO,LuxU,LuxR,luciferase,Luminescence ..more
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 Tested Compounds
 Tested Compounds
All(7)
 
 
Active(4)
 
 
Inactive(1)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(4)
 
 
Inactive(1)
 
 
Inconclusive(2)
 
 
AID: 2735
Data Source: Broad Institute (2035-02_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-31

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 4
Related Experiments
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AIDNameTypeComment
2106Broad Institute MLPCN Quorum Sensing ProjectSummarydepositor-specified cross reference
2094Plate Read Microorganism-Based Primary HTS to Identify Modulators of the AI-2 Quorum Sensing SystemScreeningsame project related to Summary assay
2723Absorbance Microorganism Dose Retest to Identify Inhibitors of Vibrio harveyiConfirmatorysame project related to Summary assay
2724Luminescence Microorganism-Based Dose Retest to Identify Modulators of the AI-2 Quorum Sensing SystemConfirmatorysame project related to Summary assay
2725Absorbance Microorganism-Based Dose Retest to Identify Inhibitors of Vibrio harveyiConfirmatorysame project related to Summary assay
2726Absorbance Microorganism-Based Retest to Identify Inhibitors of Vibrio harveyiScreeningsame project related to Summary assay
2727Luminescence Microorganism Retest to Identify Inhibitors of the AI-2 Quorum Sensing SystemScreeningsame project related to Summary assay
2728Absorbance Microorganism-Based Retest to Identify Inhibitors of Vibrio harveyiScreeningsame project related to Summary assay
2736Luminescence Microorganism-Based Retest to Identify Modulators of the AI-2 Quorum Sensing SystemScreeningsame project related to Summary assay
434943Absorbance Microorganism Dose Response to Identify Inhibitors of Vibrio harveyiConfirmatorysame project related to Summary assay
434944Luminescence Cell-Free Homogeneous Dose Response to Identify Inhibitors of Lux-SConfirmatorysame project related to Summary assay
493063Quorum Sensing, LuxS, LuxPQ Measured in Microorganism System Using Plate Reader - 2035-01_Inhibitor_DoseNoFile_DryPowder_ActivityConfirmatorysame project related to Summary assay
493072Quorum Sensing, LuxS, LuxPQ Measured in Microorganism System Using Plate Reader - 2035-01_Inhibitor_DoseNoFile_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493075Quorum Sensing, LuxS, LuxPQ Measured in Microorganism System Using Plate Reader - 2035-01_Inhibitor_DoseNoFile_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493096Quorum Sensing Measured in Microorganism System Using Plate Reader - 2035-02_Inhibitor_DoseNoFile_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493097Quorum Sensing Measured in Microorganism System Using Plate Reader - 2035-02_Inhibitor_DoseNoFile_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493101Quorum Sensing, LuxS, LuxPQ Measured in Microorganism System Using Plate Reader - 2035-01_Inhibitor_DoseNoFile_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493103Quorum Sensing Measured in Microorganism System Using Plate Reader - 2035-02_Inhibitor_DoseNoFile_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493104Quorum Sensing Measured in Microorganism System Using Plate Reader - 2035-02_Inhibitor_DoseNoFile_DryPowder_ActivityConfirmatorysame project related to Summary assay
493116Quorum Sensing LuxPQ Measured in Microorganism System Using Plate Reader - 2035-03_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493117Quorum Sensing LuxPQ Measured in Microorganism System Using Plate Reader - 2035-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493118Quorum Sensing LuxPQ Measured in Microorganism System Using Plate Reader - 2035-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493119Quorum Sensing LuxPQ Measured in Microorganism System Using Plate Reader - 2035-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493167LuxS Measured in Biochemical System Using Plate Reader - 2035-05_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493172LuxS Measured in Biochemical System Using Plate Reader - 2035-05_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
Description:
Keywords: Quorum Sensing, Auto-inducer 2 (AI-2), Vibrio harveyi, LuxS,LuxPQ,LuxO,LuxU,LuxR,luciferase,Luminescence

Assay Overview: A modified strain of Vibrio harveyi with constitutive on quorum sensing system will be exposed to small molecules identified from the HTS screen used to identify LuxS inhibitors and LuxPQ antagonists. Growth of the organism post exposure will be followed using optical density and disruption of down stream quorum sensing elements will be observed based on decreased luminescent signal.

Expected Outcome: Identification of AI-2 quorum sensing system inhibitors with modes of action which either antagonize the LuxPQ receptor or inhibit LuxS synthase. Such inhibitors should not perturb growth (observed by optical density) nor the constitutive on quorum sensing (observed by luminescence).
Protocol
Protocol

Add 45 nL 10 mM sample / well to a sterile 384 well black clear bottom assay plate (Greiner microclear). Add 60 uL / well screening culture using a combi dispenser (Thermo) and read OD600 on Envision plate reader (Perkin-Elmer), incubate plate 9 h 30C in a humid incubator, read OD600 and luminescence on Envision plate reader.

Quorum Sensing Reagents

BB721 Vibrio harveyi (Constitutive On Quorum Sensing )

Overnight Culture
5 mL LM medium plus one colony BB721, 30C 250 rpm

Screening Culture
Overnight Culture diluted to OD600 0.0005 in AB medium

LM Medium (Rich medium for overnights and agar plates)
20 g NaCl (JT Baker, 3624-19)
10 g bactotryptone (BD, 211705)
5 g yeast extract (EMD, 1.03753)
brought to 1 liter, 0.22 u sterile filter
For plates, add 7.5 g agar (BD, 28130) / 500 ml LM autoclave 15 min, plate and flame

AB Medium (Autoinducer Bioassay Medium)
100 ml 10X AB salt
2 g casamino acids (BD, 223050)
10 mL 1 M Phosphate Buffer pH 7
10 mL 0.1 M Arginine
100 uL 1 M Borate
brought to 1 liter, 0.22 u sterile filter

Where:
10X AB salt (3M NaCl, 0.5 M MgSO4)
175 g NaCl (JT Baker, 3624-19)+ 123 g MgSO4 * 7 H20 (Sigma, 230391) brought to 1 liter, 0.22 u sterile filter
1 M Phosphate Buffer pH 7
61.5 mL 1 M K2HPO4 (Sigma, P3786 ) + 38.5 mL 1 M KH2PO4 (Sigma, P0662)
1 M Borate
15.5 g Boric Acid (Sigma, B1934) brought to 500 mL 0.22 u sterile filter
100 mM Arginine
8.71 g Arginine (free base) (Calbiochem, 1820) brought to 500 mL 0.22 u sterile filter
Comment
Dose Data Analysis

Negative control wells (NC) and positive controls wells (PC) were included on every plate.
Active compounds result in decreased readout signal.


Analysis used to determine PubChem Activity Score and Outcome

The luminescent raw signal was 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).
Therefore a negative activity value is expected when signal decreases relative to NC.

No plate pattern correction algorithm was used.

IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 values were extrapolated up to 1 log
over the highest tested concentration.


PubChem Activity Score and Outcome

PUBCHEM_ACTIVITY_SCORE:

Inactive compounds = 0
Active compounds = -10*Log(IC50)

PUBCHEM_ACTIVITY_OUTCOME:

Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
IC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
or
The fit was not valid due to poor fit quality.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_Qualifier'>', '=', or '<'String
2EC50_uM*the concentration at which activity reaches 50% of the maximumFloatμM
3Log_EC50_Mthe log of the EC50 (molar)Float
4Log_EC50_M_Standard_Errorthe standard error for the log of the EC50 valueFloat
5Hill_Slopethe slope at EC50Float
6S0the fitted activity level at zero concentrationFloat%
7Sinfthe fitted activity level at infinite concentrationFloat%
8Num_Pointsthe number of data points included in the plotInteger
9Max_Activitythe maximum activity value observedFloat%
10Activity_at_0.03uM (0.03μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.068uM (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.135uM (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.26uM (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.5uM (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_1uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_2.1uM (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_4.2uM (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_8uM (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_16uM (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH086452-01

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