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BioAssay: AID 603

qHTS Assay for Inhibitors of DNA Polymerase III Holoenzyme System

E. coli DNA polymerase III holoenzyme complex was assayed for DNA production by fluorescent detection of the double-stranded DNA product with PicoGreen dye. The holoenzyme complex was reconstituted from the following purified protein components: DNA Polymerase III, beta subunit processivity factor, SSB single-stranded DNA binding protein, and Primase. Single-stranded, circular M13Gori was used as more ..
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 Tested Compounds
 Tested Compounds
All(69666)
 
 
Active(169)
 
 
Inactive(69130)
 
 
Inconclusive(379)
 
 
Unspecified(1)
 
 
 Tested Substances
 Tested Substances
All(70699)
 
 
Active(176)
 
 
Inactive(70142)
 
 
Inconclusive(380)
 
 
Unspecified(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 603
Data Source: NCGC (DNAR301)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-02-23
Modify Date: 2007-03-01

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 169
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

MLSCN Grant: 1 X01 MH077636-01
Assay Provider: Charles McHenry, University of Colorado

NCGC Assay Overview:
E. coli DNA polymerase III holoenzyme complex was assayed for DNA production by fluorescent detection of the double-stranded DNA product with PicoGreen dye. The holoenzyme complex was reconstituted from the following purified protein components: DNA Polymerase III, beta subunit processivity factor, SSB single-stranded DNA binding protein, and Primase. Single-stranded, circular M13Gori was used as a substrate with dNTPs and NTPs also supplied to the substrate reagent. Compounds from the NCGC collection were incubated with the holoenzyme mixture for 15 min at room temperature to allow binding to occur, and then the reaction was started by the addition of substrate. After 30 min incubation at room temperature, PicoGreen solution containing EDTA was added to stop the reaction and to visualize the double-stranded DNA produced. Reaction progress was measured in a ViewLux CCD imager in fluorescence intensity mode by using 480 nm excitation and 540 nm emission filter set.
Protocol
NCGC Assay Protocol Summary:
Buffer. 50 mM Hepes-KOH, pH 7.5, 5 % Glycerol, 0.02% NP40, 30 mM potassium;
glutamate, 1.5 mM Magnesium Acetate, 16 uM TCEP.

Reagents/Controls:
Buffer in columns 3 and 4 as negative control (no enzyme).
Substrate solution: 5 uM each dATP, dGTP, dCTP and TTP, 50 uM each GTP, CTP, UTP and 200 uM ATP, 1.4 nM M13gori (final concentrations).
Enzyme: 154 nM SSB, 2 nM Primase, 1 nM Beta, 0.35 nM Pol III (final concentrations).
PicoGreen/EDTA: 10 mM Tris-HCl, 10 mM EDTA, 5 uM PicoGreen (final concentrations).
Control titration: ChemBridge 5805060 (from 10 mM, then 1:2, in duplicate) was pin-transferred to upper half of column 2.

Assay Steps:
Three uL of holoenzyme mixture were dispensed to 1536-well Greiner black solid bottom plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then 1 uL of substrate solution was added to start the reaction. After room temperature incubation for 35 minutes, 1 uL of PicoGreen solution was added to each well and the plates were read 480 nm excitation and 540 nm emission using ViewLux (Perkin-Elmer) High-throughput CCD imager and fluorescence protocol settings.

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, DNA Polymerase III, DNAPol3, DNAPoly3, DNA replication targets
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 Class". 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 Excluded PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
9Curve ClassNumerical encoding of curve description for the fitted Hill equation.Float
10Activity at 0.732nM% Activity at given concentration.Float%
11Activity at 1.636nM% Activity at given concentration.Float%
12Activity at 3.658nM% Activity at given concentration.Float%
13Activity at 8.18nM% Activity at given concentration.Float%
14Activity at 18.291nM% Activity at given concentration.Float%
15Activity at 0.041uM% Activity at given concentration.Float%
16Activity at 0.091uM% Activity at given concentration.Float%
17Activity at 0.204uM% Activity at given concentration.Float%
18Activity at 0.457uM% Activity at given concentration.Float%
19Activity at 1.022uM% Activity at given concentration.Float%
20Activity at 2.286uM% Activity at given concentration.Float%
21Activity at 5.111uM% Activity at given concentration.Float%
22Activity at 11.429uM% Activity at given concentration.Float%
23Activity at 25.556uM% Activity at given concentration.Float%
24Activity at 57.143uM% Activity at given concentration.Float%
25Compound 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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