Large-Scale Screen for APE1 Inhibitors Figure 2. Representative curves observed from 10 screening hits chosen to demonstrate the range of potencies observed in the concentration-response-based screen. Structures and additional data associated with these hits are presented within Figure 7. doi:10.1371/journal.pone.0047974.g002 ThO DNA Displacement and E. coli Endo IV Profiling Assays In order to screen out nonselective compounds, such as non- specific DNA binders and inhibitors of related enzymes, we used a miniaturized ThO DNA displacement assay and a counterscreen against the bacterial endonuclease E. coli Endo IV, respectively, following the previously published protocols [21]. Fluorescence Polarization (FP) Displacement Assay Double-stranded oligodeoxynucleotide containing tetrahydro- furan (THF) abasic site labeled with TAMRA at the 59-end was used as the labeled binding probe. Inhibition of APE1 binding to labeled probe was detected by a decrease in the fluorescence polarization (FP) of the fluorophore. Briefly, 4 mL mixture of 20 nM APE1 and 5 nM probe contained in the enzymatic assay buffer from which Mg2+ was omitted, was dispensed into 1536- Figure 3. Hit progression flow chart. doi:10.1371/journal.pone.0047974.g003 PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47974