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BioAssay: AID 2741

Counterscreen for APE1 Inhibitors: Confirmatory Fluorescent Dye Displacement Assay

In order to distinguish true inhibitors of DNA-processing enzymes from promiscuous DNA-binding compounds among screening assay hits, we developed a homogeneous and miniaturized assay based on fluorescent dye displacement test as originally described by Tse and Boger (Accounts of Chemical Research (2004) 37: 61-69). The assay is based on the strong enhancement of fluorescence when Thiazole Orange (ThO) binds to double-stranded DNA: conversely, in the presence of a DNA-binding compound, Thiazole Orange is displaced from the DNA and its fluorescence is reduced. ..more
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 Tested Compounds
 Tested Compounds
All(389)
 
 
Active(20)
 
 
Inactive(273)
 
 
Inconclusive(96)
 
 
 Tested Substances
 Tested Substances
All(389)
 
 
Active(20)
 
 
Inactive(273)
 
 
Inconclusive(96)
 
 
 Related BioAssays
 Related BioAssays
AID: 2741
Data Source: NCGC (THO1705)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-04-01

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 20
Related Experiments
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AIDNameTypeComment
1705qHTS Validation Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)Confirmatorydepositor-specified cross reference: qHTS Validation Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
1707Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement Validation AssayConfirmatorydepositor-specified cross reference: Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement Validation Assay
1708Counterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IVConfirmatorydepositor-specified cross reference: Counterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IV
2324Probe Development Summary of Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)Summarydepositor-specified cross reference: Probe Development Summary of Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
2517qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
2565Counterscreen for APE1 Inhibitors: qHTS Assay for Inhibitors of Endonuclease IVConfirmatorydepositor-specified cross reference: Counterscreen for APE1 Inhibitors: qHTS Assay for Inhibitors of Endonuclease IV
2572Confirmation qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)Confirmatorydepositor-specified cross reference: Confirmation qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
488940Radiotracer Incision Assay (RIA) for Inhibitors of Human Apurinic/apyrimidinic Endonuclease 1 (APE1)Confirmatorydepositor-specified cross reference
2564Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement AssayConfirmatorysame project related to Summary assay
2573qHTS FP-Based Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)Confirmatorysame project related to Summary assay
504595Inhibitors of APE1: Aqueous Solubility ProfilingOthersame project related to Summary assay
504603Inhibitors of APE1: Caco-2 Cell Permeability ProfilingOthersame project related to Summary assay
504618Inhibitors of APE1: Efflux Ratio ProfilingOthersame project related to Summary assay
504624Inhibitors of APE1: Mouse Plasma Stability ProfilingOthersame project related to Summary assay
504643Inhibitors of APE1: Metabolic Stability ProfilingOthersame project related to Summary assay
Description:
In order to distinguish true inhibitors of DNA-processing enzymes from promiscuous DNA-binding compounds among screening assay hits, we developed a homogeneous and miniaturized assay based on fluorescent dye displacement test as originally described by Tse and Boger (Accounts of Chemical Research (2004) 37: 61-69). The assay is based on the strong enhancement of fluorescence when Thiazole Orange (ThO) binds to double-stranded DNA: conversely, in the presence of a DNA-binding compound, Thiazole Orange is displaced from the DNA and its fluorescence is reduced.
Protocol
Buffer. 50 mM Tris-HCl (pH7.5) containing 50mM NaCl, 2 mM MgCl2, and 0.01% Tween-20.

Reagents/Controls:
Buffer containing 250 nM ThO in columns 3 and 4 as negative control (no dsDNA).
Reaction mixture: 250 nM ThO and 50 nM dsDNA at final concentration dispensed throughout the plate: in columns 1, 2, 5-48. Column 1 is neutral (100% activity).
dsDNA is:
5''TAMRA- TC ACC FTC GTA CGA CTC
3'' AG TGG GAG CAT GCT GAG
where F denotes tetrahydrofuran.

Control: Pintool transfer of control compound WB64 (W4761, Sigma-Aldrich) to column 2 of all assay plates as a two-fold 16-point dilution in duplicate (concentration range 5.75 uM - 0.175 nM).

Assay Steps:
Four uL of reaction mixtures were dispensed to 1536-well Greiner black solid bottom plates. Compounds (23 nL) were transferred into ViewLux High-throughput CCD imager (Perkin-Elmer) in order to measure the endpoint fluorescence using 480 nm excitation and 540 nm emission fluorescence protocol.
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 Description". 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. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_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
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_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 significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_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
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0001608376 uM (0.000160838μM**)% Activity at given concentration.Float%
15Activity at 0.0003225731 uM (0.000322573μM**)% Activity at given concentration.Float%
16Activity at 0.0004825129 uM (0.000482513μM**)% Activity at given concentration.Float%
17Activity at 0.0009677193 uM (0.000967719μM**)% Activity at given concentration.Float%
18Activity at 0.00145 uM (0.00144754μM**)% Activity at given concentration.Float%
19Activity at 0.00290 uM (0.00290316μM**)% Activity at given concentration.Float%
20Activity at 0.00434 uM (0.00434262μM**)% Activity at given concentration.Float%
21Activity at 0.00871 uM (0.00870947μM**)% Activity at given concentration.Float%
22Activity at 0.013 uM (0.0130278μM**)% Activity at given concentration.Float%
23Activity at 0.037 uM (0.0367119μM**)% Activity at given concentration.Float%
24Activity at 0.081 uM (0.0811717μM**)% Activity at given concentration.Float%
25Activity at 0.118 uM (0.117578μM**)% Activity at given concentration.Float%
26Activity at 0.244 uM (0.243515μM**)% Activity at given concentration.Float%
27Activity at 0.353 uM (0.352734μM**)% Activity at given concentration.Float%
28Activity at 0.705 uM (0.705467μM**)% Activity at given concentration.Float%
29Activity at 1.055 uM (1.05526μM**)% Activity at given concentration.Float%
30Activity at 2.116 uM (2.1164μM**)% Activity at given concentration.Float%
31Activity at 3.166 uM (3.16577μM**)% Activity at given concentration.Float%
32Activity at 6.349 uM (6.34921μM**)% Activity at given concentration.Float%
33Activity at 9.497 uM (9.4973μM**)% Activity at given concentration.Float%
34Activity at 19.05 uM (19.0476μM**)% Activity at given concentration.Float%
35Activity at 28.49 uM (28.4919μM**)% Activity at given concentration.Float%
36Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH086444-01

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