AID	Name	Type	Comment
1707	Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement Validation Assay	confirmatory	Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement Validation Assay
1708	Counterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IV	confirmatory	Counterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IV
1705	qHTS Validation Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)	confirmatory	qHTS Validation Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
2517	qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)	confirmatory	qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
2572	Confirmation qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)	confirmatory	Confirmation qHTS Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)
2741	Counterscreen for APE1 Inhibitors: Confirmatory Fluorescent Dye Displacement Assay	confirmatory	Counterscreen for APE1 Inhibitors: Confirmatory Fluorescent Dye Displacement Assay

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).

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.

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]

Grant Number: MH086444-01