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BioAssay: AID 2572

Confirmation qHTS 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(745)
 
 
Active(328)
 
 
Inactive(173)
 
 
Inconclusive(245)
 
 
 Tested Substances
 Tested Substances
All(748)
 
 
Active(330)
 
 
Inactive(173)
 
 
Inconclusive(245)
 
 
AID: 2572
Data Source: NCGC (APE1918)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-16

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 328
Depositor Specified Assays
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)
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
1708Counterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IVconfirmatoryCounterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IV
2517qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)confirmatoryqHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
488940Radiotracer Incision Assay (RIA) for Inhibitors of Human Apurinic/apyrimidinic Endonuclease 1 (APE1)confirmatory
2741Counterscreen for APE1 Inhibitors: Confirmatory Fluorescent Dye Displacement Assayconfirmatory
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.

Inhibition of APE1 activity was screened by utilizing double-stranded short substrate containing tetrahydrofuran (THF) abasic site labeled with rhodamine-type fluorophore (TAMRA) at the 5'-end and with non-fluorescent Black Hole Quencher-2 (BHQ-2) at the opposing 3'-end. An increase in the fluorescence intensity due to incision of the abasic site by APE1 was used to measure the enzyme activity.

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 0.0000068120 uM (6.81196e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000136239 uM (1.36239e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000272478 uM (2.72478e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0000544957 uM (5.44957e-05μM**)% Activity at given concentration.Float%
18Activity at 0.0001486573 uM (0.000148657μM**)% Activity at given concentration.Float%
19Activity at 0.0002179827 uM (0.000217983μM**)% Activity at given concentration.Float%
20Activity at 0.0004084469 uM (0.000408447μM**)% Activity at given concentration.Float%
21Activity at 0.0009431843 uM (0.000943184μM**)% Activity at given concentration.Float%
22Activity at 0.00150 uM (0.00150314μM**)% Activity at given concentration.Float%
23Activity at 0.00306 uM (0.00305522μM**)% Activity at given concentration.Float%
24Activity at 0.00480 uM (0.00479965μM**)% Activity at given concentration.Float%
25Activity at 0.011 uM (0.0112648μM**)% Activity at given concentration.Float%
26Activity at 0.026 uM (0.0261284μM**)% Activity at given concentration.Float%
27Activity at 0.037 uM (0.0365067μM**)% Activity at given concentration.Float%
28Activity at 0.072 uM (0.0720902μM**)% Activity at given concentration.Float%
29Activity at 0.116 uM (0.116086μM**)% Activity at given concentration.Float%
30Activity at 0.235 uM (0.234668μM**)% Activity at given concentration.Float%
31Activity at 0.370 uM (0.370314μM**)% Activity at given concentration.Float%
32Activity at 0.886 uM (0.885971μM**)% Activity at given concentration.Float%
33Activity at 2.030 uM (2.02964μM**)% Activity at given concentration.Float%
34Activity at 3.244 uM (3.24397μM**)% Activity at given concentration.Float%
35Activity at 6.536 uM (6.53615μM**)% Activity at given concentration.Float%
36Activity at 10.32 uM (10.3153μM**)% Activity at given concentration.Float%
37Activity at 24.43 uM (24.4347μM**)% Activity at given concentration.Float%
38Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
39Compound QCSource of compound QCString

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

Data Table (Concise)
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