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BioAssay: AID 623951

Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_Activity

Bacteria typically require total iron concentrations in the micromolar 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 more ..
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 Tested Compounds
 Tested Compounds
All(41)
 
 
Active(41)
 
 
 Tested Substances
 Tested Substances
All(41)
 
 
Active(41)
 
 
AID: 623951
Data Source: Broad Institute (2091-02_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-29
Hold-until Date: 2013-03-29
Modify Date: 2013-03-29

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 41
Related Experiments
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AIDNameTypeComment
488968Broad Institute Inhibitors of Pyoverdine Production ProjectSummarydepositor-specified cross reference: Summary assay
488965Fluorescent 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_ActivityScreeningsame project related to Summary assay
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
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 micromolar 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. The PvdQ enzyme is pre-incubated with the compounds 30 minutes before the substrate 4-MU laurate is added.

Expected outcome: Identification of probe(s) inhibiting PvdQ enzymatic activity. Compounds reducing the fluorescence mediated by PvdQ and 4-methylumbelliferone with an IC50 below 10 uM will be considered hits.
Protocol
2091 Pyoverdine Inhibitors (Pre-incubation 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 4mM 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-methylumbelliferone laurate (4-MU laurate) substrate preparation:
162 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 10.8 mg/ml (135.9 uM stock concentration). The PvdQ preparation came as 40 ul droplets. We added 40ul of the PvdQ preparation in 54.3 ml TNT buffer (final concentatration of 0.1 uM) with 250 uL triton X-100 (0.5%).

The PvdQ (1.5 ul) was dispensed using BioRaptrTM FRD microfluid workstation (Beckman Coulter) at a final concentration 0.02uM (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 7.5 nl (8 concentrations, 3 fold dilution (starting final concentration 10 uM)) 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). After 30 minutes pre-incubation, 4-MU laurate (6 ul) was then added and the assay plates incubated for an additional 60 minutes at room temperature.

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 the difference between the read at 60 minutes and 0 minute.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=176) and positive control wells (PC; n=24) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing 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 (2)(Max_Concentration).

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: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
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 >= -70% but <= -30%, 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: Biochemical
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
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_0.003uM_(%) (0.003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.009uM_(%) (0.009μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.0285uM_(%) (0.0285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.08uM_(%) (0.08μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.235uM_(%) (0.235μM**)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_2.1uM_(%) (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_6.8uM_(%) (6.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_19.5uM_(%) (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

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

Data Table (Concise)
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