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BioAssay: AID 2723

Absorbance Microorganism Dose Retest to Identify Inhibitors of Vibrio harveyi

Keywords: Quorum Sensing,Auto-inducer 2(AI-2),Vibrio harveyi,LuxS,LuxPQ,LuxO,LuxU,luciferase,Luminescence ..more
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 Tested Compounds
 Tested Compounds
All(7)
 
 
Active(1)
 
 
Inactive(6)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(1)
 
 
Inactive(6)
 
 
AID: 2723
Data Source: Broad Institute (2035-02_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK_VIABILITY)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-31

Data Table ( Complete ):           Active    All
Targets
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
2106Broad Institute MLPCN Quorum Sensing Projectsummary
Description:
Keywords: Quorum Sensing,Auto-inducer 2(AI-2),Vibrio harveyi,LuxS,LuxPQ,LuxO,LuxU,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.
Initial and final absorbance readings were taken for each well. The difference between these two measurements (dABS) was smaller for compounds which resulted in reduced growth of cells in the well. Therefore active compounds result in decreased readout signal.


Analysis used to determine PubChem Activity Score and Outcome

dABS was normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median calculated 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
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ACTIVITY_OUTCOME1=Inactive/2=Active/3=InconclusiveInteger
2ACTIVITY_SCORERelative activity score for PubchemInteger
3EC50_Qualifier'>', '=', or '<'String
4EC50_uM*the concentration at which activity reaches 50% of the maximumFloatμM
5Log_EC50_Mthe log of the EC50 (molar)Float
6Log_EC50_M_Standard_Errorthe standard error for the log of the EC50 value (molar)Float
7Hill_Slopethe slope at EC50Float%
8S0the fitted activity level at zero concentrationFloat%
9Sinfthe fitted activity level at infinite concentrationFloat%
10Num_Pointsthe number of data points included in the plotInteger
11Max_Activitythe maximum activity value observedFloat%
12Activity_at_0.03uM (0.03μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.068uM (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.135uM (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.26uM (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.5uM (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_1uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_2.1uM (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_4.2uM (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_8uM (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_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)
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