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BioAssay: AID 2727

Luminescence Microorganism 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
_
   
 Tested Compounds
 Tested Compounds
All(749)
 
 
Active(711)
 
 
Inactive(29)
 
 
Inconclusive(9)
 
 
 Tested Substances
 Tested Substances
All(749)
 
 
Active(711)
 
 
Inactive(29)
 
 
Inconclusive(9)
 
 
AID: 2727
Data Source: Broad Institute (2035-02_INHIBITORS_SINGLE-POINT_MLPCN-CHERRYPICK)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-31

Data Table ( Complete ):           Active    All
Targets
BioActive Compounds: 711
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,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
Single-Point Cherrypick 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.

The final PUBCHEM_ACTIVITY_SCORE was calculated by multiplying the mean of all valid replicate values (which can be described in units of negative percent activity) by -1, resulting in a score between 0 and 100 (in units of 'percent inhibition').

The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an activity threshold of -75%:

Activity_Outcome = 1 (inactive)
<50% of replicates fall outside threshold.

Activity_Outcome = 2 (active)
100% of replicates fall outside threshold.

Activity_Outcome = 3 (inconclusive)
>= 50% and <= 75% of replicates fall outside threshold.

Compounds ideal for further study were active in this assay, and inactive in 2035-02_INHIBITORS_SINGLE-POINT_MLPCN-CHERRYPICK_VIABILITY.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two
samples. Computed as the absolute value of the cosine between the "replicate vector"
(ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility.
NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid
Float
2BROAD_SCREENING_RUNIDSThis is a comma separated list of unique IDs given to each screening run at the Broad Institute.String
3REPLICATE_A_ACTIVITY_SCOREThe calculated activity score of the indicated replicate.Float%
4REPLICATE_B_ACTIVITY_SCOREThe calculated activity score of the indicated replicate.Float%
5REPLICATE_C_ACTIVITY_SCOREThe calculated activity score of the indicated replicate.Float%
6REPLICATE_D_ACTIVITY_SCOREThe calculated activity score of the indicated replicate.Float%
7DATE_REPORTEDDate data reported internallyString
Additional Information
Grant Number: 1 R03 MH086452-01

Data Table (Concise)
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