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]

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.

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.

Grant Number: MH086444-01