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BioAssay: AID 488965

Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader and Imaging Combination - 2091-01_Inhibitor_SinglePoint_HTS_Activity

Bacteria typically require total iron concentrations in the uM range to support growth. Many pathogens such as P. aeruginosa produce siderophores (e.g. pyoverdine) with molecular weights below 1500 Da that bind to iron with remarkably high affinities. The PvdQ acylase is responsible for removal of the fatty acid on pyoverdine. Interestingly, mutant strains of P. aeruginosa with a deletion in the more ..
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 Tested Compounds
 Tested Compounds
All(337488)
 
 
Active(213)
 
 
Inactive(337077)
 
 
Inconclusive(198)
 
 
 Tested Substances
 Tested Substances
All(337881)
 
 
Active(213)
 
 
Inactive(337470)
 
 
Inconclusive(198)
 
 
AID: 488965
Data Source: Broad Institute (2091-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-28
Modify Date: 2010-11-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 213
Related Experiments
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AIDNameTypeComment
488968Broad Institute Inhibitors of Pyoverdine Production ProjectSummarydepositor-specified cross reference
493231Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader and Imaging Combination - 2091-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
504942Mammalian cell toxicity screen in HeLa cells 48h Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
504944Mammalian cell toxicity screen in HeLa cells 48h Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_CherryPick_Activity_Set2Confirmatorysame project related to Summary assay
602389Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
623950Counterscreen measuring mammalian cell toxicity screen in HeLa cells of pyoverdine inhibitors Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
623951Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
623985Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623987Counterscreen measuring mammalian cell toxicity screen in HeLa cells of pyoverdine inhibitors Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
623990Counterscreen measuring mammalian cell toxicity screen in HeLa cells of pyoverdine inhibitors Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623991Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
623993Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
624011Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624016Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
624018Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624020Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
624033Whole cell secondary assay to identify compounds reducing the production of iron chelator pyoverdine using Chromeazurol S dye in Pseudomonas Aeruginosa Measured in Whole Organism System Using Imaging - 2091-11_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624058Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 600 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_CherryPick_Activity_Set2Confirmatorysame project related to Summary assay
624064Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 600 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
624070Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatorysame project related to Summary assay
624072Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK pump mutant mexAB-OprM strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-10_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
624073Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-08_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
624074Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK pump mutant mexAB-OprM strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-10_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
624075Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-08_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
624102Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set8Confirmatorysame project related to Summary assay
624104Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK pump mutant mexAB-OprM strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-10_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624105Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK pump mutant mexAB-OprM strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-10_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624106Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK strain (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-08_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624107Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK strain (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-08_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624109Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatorysame project related to Summary assay
Description:
Keywords: Pyoverdine, laurate, PvdQ

Assay Overview: Fluorometric assay (4-methylumbelliferone).
Bacteria typically require total iron concentrations in the uM range to support growth. Many pathogens such as P. aeruginosa produce siderophores (e.g. pyoverdine) with molecular weights below 1500 Da that bind to iron with remarkably high affinities. The PvdQ acylase is responsible for removal of the fatty acid on pyoverdine. Interestingly, mutant strains of P. aeruginosa with a deletion in the PvdQ gene do not make pyoverdine and cannot grow on iron limiting media. This primary screen aims to selectively identify small molecules inhibitors of the PvdQ enzymatic activity. The small molecules bind to PvdQ and block the deceylation of 4-methylumbelliferone laurate. The enzymatic activity of PvdQ will be measured through fluorescence emission mediated by the dissociation of 4-methylumbellerone to the fatty acid laurate.

Expected outcome: Identification of probe(s) inhibiting PvdQ enzymatic activity. Compounds (10 uM final concentration) reducing the fluorescence mediated by PvdQ and 4-methylumbelliferone by 20% in duplicate will be considered hits.
Protocol
2091 Pyoverdine Inhibitors (PvdQ Primary assay PROTOCOL)

TNT assay buffer preparation:

- Tris 50 mM (pH8.0)
- NaCl 50 mM
- Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) 0.2 mM (Sigma, C4706)

We diluted 50 mL of Tris 1M pH8.0 (stock solution), 10 mL of NaCl 5M and 50 ml TCEP 4 mM with 890 mL water. The TNT assay buffer was filtered using a filtering unit (Nalgene/Nunc 0.9 mm filter unit, catalog number 151-4020).

4-methylumbelliferyl laurate (4-MU laurate) substrate preparation (volume of substrate required to fill up about 30 1536-well black square well assay plates):
144 mg (16 mM) of 4-MU laurate (Research Organics, 0183M, MW 358.5 g/mol) was dissolved in 25 ml isopropanol and was mixed with 2.5 ml of Triton X-100(Sigma, T8787)(0.1 volume), vortexed for 10 sec and gently mixed with 375 ml TNT buffer (15 volumes).

Enzyme preparation:
The P. aeruginaosa PvdQ was provided by Dr. Andrew Gulick, Hauptman-Woodward Medical Research Institute, Buffalo, NY at a concentration of 10.06 mg/ml (126.6 uM stock concentration). The PvdQ preparation came as 40 ul droplets. We added 80 ul of the PvdQ preparation in 50.2 ml TNT buffer (final concentatration of 0.2 uM).

The PvdQ (0.75 ul) and 4-MU laurate (6.75 ul) solutions were dispensed using BioRaptrTM FRD microfluid workstation (Beckman Coulter) at a final concentration 0.02 uM (PvdQ) and 0.8 mM (4-MU laurate) at room temperature in 1536-well high base black barcoded plate square well assay plate (Aurora Biotechnologies, cat number 00019180BK) where 0.75 nl (10 mM compound concentration) of the MLPCN compound library was already dispensed in the assay plate. The positive control Isopropyl Dodecylfluorophosphonate (IDFP)(Cayman chemicals, cat. number 10215) was added in 24 selective wells (1.7 mM stock solution in TNT buffer to a 200 uM final concentration).

The assay plates were read at time 0 and 60 min using the Viewlux (Perkin Elmer) with 303-367 nm excitation filter and 440-460 nm emission filter. The fluorescence mediated by the enzymatic reaction was calculated as the difference between the read at 60 minutes and 0 minute.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 30.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:

Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
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
2REPLICATE_A_ACTIVITY_SCORE (10μM**)The calculated percent activity for the indicated sampleFloat%
3REPLICATE_B_ACTIVITY_SCORE (10μM**)The calculated percent activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH092076-01

Data Table (Concise)
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