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BioAssay: AID 605

qHTS Assay for Inhibitors of YjeE

YjeE is an essential E.coli protein of unknown function that binds adenosine diphosphate (ADP). A complex of YjeE and BODIPY Texas Red-labeled ADP probe (Invitrogen, catalog number A22359) is incubated with library members. Inhibitors of YjeE-ADP binding are detected by a decrease in the fluorescence polarization (FP) of the fluorophore. Purified YjeE was supplied by Chand Mangat and Eric Brown, McMaster University. ..more
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 Tested Compounds
 Tested Compounds
All(69666)
 
 
Active(34)
 
 
Inactive(69431)
 
 
Inconclusive(215)
 
 
 Tested Substances
 Tested Substances
All(70699)
 
 
Active(34)
 
 
Inactive(70447)
 
 
Inconclusive(218)
 
 
 Related BioAssays
 Related BioAssays
AID: 605
Data Source: NCGC (YJEE978)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-02-27
Modify Date: 2007-03-16

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 34
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

Assay Provider: Eric Brown, McMaster University

NCGC Assay Overview:
YjeE is an essential E.coli protein of unknown function that binds adenosine diphosphate (ADP). A complex of YjeE and BODIPY Texas Red-labeled ADP probe (Invitrogen, catalog number A22359) is incubated with library members. Inhibitors of YjeE-ADP binding are detected by a decrease in the fluorescence polarization (FP) of the fluorophore. Purified YjeE was supplied by Chand Mangat and Eric Brown, McMaster University.
Protocol
NCGC Assay Protocol Summary:
Buffer:
50mM HEPES pH 7.5, 50mM NaCl, 10mM MgCl2, 0.01% tween 20
Reagents/Controls:
2 uM probe/10 uM YjeE plus high concentration ADP (60 mM) pinned into columns 3 and 4 to generate negative control (full inhibition of binding, -100 % activity).
2 uM probe/10 uM YjeE in columns 1, 2, 5-48. Titration of ADP control (from 40 mM, then 1:2) pin-transferred to top half of column 2. Column 1 and bottom half of column 2 are neutral.
Assay Steps:
Two and a half uL of reagents were dispensed to 1536-well Greiner black plates. Compounds and controls (20 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then read on ViewLux (Perkin-Elmer) High-throughput CCD imager using BODIPY polarization filter set and 30 seconds exposure time. During dispense, reagent bottle was kept submerged into 4 deg C water bath and all liquid lines were covered with aluminum foil to minimize probe and protein degradation.
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, YjeE
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Classification". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity DirectionIndicates direction of observed activity: inactive, decreasing, increasing.String
2Curve DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control.String
3Log of AC50Log of AC50.Float
4Hill CoefficientHill slope of fitted curve.Float
5Curve R2R^2 fit value of curve. Closer to 1.0 equates to better Hill equation fit.Float
6Curve Infinite ActivityHill Equation Infinite ActivityFloat
7Curve Zero ActivityHill Equation Zero ActivityFloat
8Curve ClassNumerical encoding of curve description for the fitted Hill equation.Float
9Compound TypeNCGC designation for compound stage: 'qHTS ECL (Exploratory)', 'NIHSMR (MLSCN)', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String

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