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BioAssay: AID 2573

qHTS FP-Based Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)

The apurinic/apyrimidinic endonuclease APE1 is the primary mammalian enzyme responsible for the removal of abasic (or AP) sites in DNA and functions centrally in the base excision DNA repair (BER) pathway. Recent studies suggested a link between an overexpression of APE1 in many cancers and resistance of these tumor cells to radio- and chemotherapy. Thus, targeting APE1 could improve the efficacy of current treatment paradigms by promoting selective sensitization or protection of diseased and normal cells, respectively. ..more
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 Tested Compounds
 Tested Compounds
All(474)
 
 
Active(34)
 
 
Inactive(325)
 
 
Inconclusive(115)
 
 
 Tested Substances
 Tested Substances
All(475)
 
 
Active(34)
 
 
Inactive(326)
 
 
Inconclusive(115)
 
 
AID: 2573
Data Source: NCGC (APE1608)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-16
Hold-until Date: 2011-03-01
Modify Date: 2011-03-01

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 34
Depositor Specified Assays
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AIDNameTypeComment
2324Probe Development Summary of Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)summaryProbe Development Summary of Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
2517qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)confirmatoryqHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
1705qHTS Validation Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)confirmatoryqHTS Validation Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
1707Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement Validation AssayconfirmatoryCounterscreen for APE1 Inhibitors: Fluorescent Dye Displacement Validation Assay
2564Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement AssayconfirmatoryCounterscreen for APE1 Inhibitors: Fluorescent Dye Displacement Assay
2565Counterscreen for APE1 Inhibitors: qHTS Assay for Inhibitors of Endonuclease IVconfirmatoryCounterscreen for APE1 Inhibitors: qHTS Assay for Inhibitors of Endonuclease IV
2572Confirmation qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)confirmatoryConfirmation qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
1708Counterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IVconfirmatoryCounterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IV
504618Inhibitors of APE1: Efflux Ratio Profilingother
504603Inhibitors of APE1: Caco-2 Cell Permeability Profilingother
504595Inhibitors of APE1: Aqueous Solubility Profilingother
504624Inhibitors of APE1: Mouse Plasma Stability Profilingother
504643Inhibitors of APE1: Metabolic Stability Profilingother
Description:
The apurinic/apyrimidinic endonuclease APE1 is the primary mammalian enzyme responsible for the removal of abasic (or AP) sites in DNA and functions centrally in the base excision DNA repair (BER) pathway. Recent studies suggested a link between an overexpression of APE1 in many cancers and resistance of these tumor cells to radio- and chemotherapy. Thus, targeting APE1 could improve the efficacy of current treatment paradigms by promoting selective sensitization or protection of diseased and normal cells, respectively.

In order to gain insight into the mode of action of confirmed APE1 inhibitors, we implemented a miniaturized fluorescence polarization assay designed to report on compounds which competitively displace ds-DNA substrate. The fluorescence polarization assay has been implemented in 384- and 1536-well format for binding of TAMRA-labeled double-stranded DNA probe not containing a quencher. The binding of protein to DNA is inhibited by ATA, a general inhibitor of APE1 activity.

See also related primary fluorogenic assay (AID 2517).

Assay Provider: David M. Wilson, III, National Institute on Aging, NIH
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
Protocol
Reagents/Controls:
1. Substrate: 50 nM final concentration of TAMRA/BHQ-2 substrate dispensed throughout the plate.
2. Enzyme: 0.75 nM APE1 final concentration in columns 1, 2, 5-48. Column 1 is neutral (100% activity)
3. Control: Pintool transfer of control inhibitor NSC-13755 to column 2 of all assay plates. Two-fold, 16 pt dilution in duplicate to produce final concentrations in the 5.75 uM - 0.175 nM range.
3. Buffer columns in columns 3 and 4 were used as negative control (no enzyme).

Assay Steps:
Three uL of enzyme were dispensed to 1536-well Greiner black solid bottom plates. Compounds (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. The plates were immediately transferred into ViewLux High-throughput CCD imager (Perkin-Elmer) in order to measure the reaction progress in kinetic mode (three reads every 60 seconds) using 525 nm excitation and 598 nm emission fluorescence protocol. The fluorescence intensity difference between the third and the first time points was used to compute reaction progress.
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%
36Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
37Compound QCSource of compound QCString

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

Data Table (Concise)
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