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BioAssay: AID 488940

Radiotracer Incision Assay (RIA) for Inhibitors of 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(13)
 
 
Active(13)
 
 
 Tested Substances
 Tested Substances
All(13)
 
 
Active(13)
 
 
AID: 488940
Data Source: NCGC (APE1794)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-27
Hold-until Date: 2011-04-26
Modify Date: 2013-10-29

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 13
Related Experiments
Show more
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: Summary AID
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)
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)
2741Counterscreen for APE1 Inhibitors: Confirmatory Fluorescent Dye Displacement AssayConfirmatorydepositor-specified cross reference: Counterscreen for APE1 Inhibitors: Confirmatory Fluorescent Dye Displacement Assay
2564Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement AssayConfirmatorysame project related to Summary assay
2565Counterscreen for APE1 Inhibitors: qHTS Assay for Inhibitors of Endonuclease IVConfirmatorysame 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:
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.

This secondary Radiotracer Incision Assay was implemented to validated hits from the primary screen (AID 2517).
Protocol
Recombinant wild type APE1 protein was purified as previously described (Erzberger et al., NAR, 1998). Fifty pg of APE1 (~140 pM) was incubated without (positive control containing 1% DMSO) or with 100 microM of the indicated inhibitor at room temperature in RIA buffer (50 mM Tris pH 7.5, 25 mM NaCl, 1mM MgCl2, 1 mM DTT, 0.01% Tween -20) for 15 min. One-half pmol of 32P 5'-radiolabeled AP-DNA substrate (18 mer) was added to a 10 microL final volume (see Wilson et al., JBC, 1995), and the reactions were incubated at 370C for 5 min and stopped by adding stop buffer (0.05% Bromophenol blue/ Xylene cyanol dissolved in 95% formamide, 20 mM EDTA) and heating at 950C for 10 min. Intact substrate was separated from incised product on a 15% polyacrylamide denaturing gel in tris boric acid EDTA buffer. Following electrophoresis, the gel was subjected to standard phosphoimager analysis using the ImageQuant 5.2 software, and the percent incision activity (amount of substrate converted to product) was calculated. For IC50 determinations (i.e. the concentration of inhibitor at which 50% inactivation was observed), 50 pg of APE1 (~140 pM) was incubated without or with increasing concentrations (1 nM to 100 microM) of the indicated inhibitor as above and the percent incision activity was determined.
Comment
Compound Ranking:
For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given based on -Log[Potency*10^-6]
Assay Provider: David M. Wilson, III, National Institute on Aging, NIH
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Potency*Concentration at which compound exhibits half-maximal efficacy, IC50. Extrapolated IC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM

* Activity Concentration.

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