Of the 204 compounds tested in the Bortezomib Proteasome inhibitor fluorescence-based gel assay, 111 displayed reproducible dosedependent inhibition. A total of 223 positive compounds showing activity in these electrophoretic separation based assays were then subjected to a panel of assays in order to further assess their engagement with the APE1 target in vitro, as well as to evaluate their selectivity. The complete set of results obtained for these 223 compounds in the below tests is provided within Table S1. To detect screening hits that inhibit APE1 activity through nonspecific DNA interactions, we employed a previously established miniaturized ThO dye displacement assay. Forty-three compounds were active in the DNA-binding counter-screen; the majority of these compounds were weak DNA binders. Most of the DNA binders possessed the typical chemical features associated with DNA binding: extended conjugated unsaturated ring systems, which would allow them to intercalate between the stacked bases, and/or accumulation of PR-171 positively-charged nitrogens, which would permit nonspecific electrostatic interactions with DNA. To gain insight into the mode of action of the hits, we employed a displacement assay combined with fluorescence polarization detection to test the small molecule��s effect on the binding of APE1 to a version of the fluorogenic AP site-containing oligonucleotide substrate that was devoid of the quencher functionality. FP is a convenient technique for providing a basic characterization of macromolecular associations, such as protein-DNA and protein-protein interactions. Following incubation of the compound, APE1, and DNA substrate together, inhibition of APE1 binding to AP-DNA would be revealed as a decrease in FP of the fluorophore. The assay, which is performed in the absence of magnesium to prevent enzymatic turnover, is target DNAspecific, as APE1 binds with higher affinity to duplex DNA containing an AP site than to an undamaged counterpart. Of the 223 hits validated in the electrophoretic separation assays, 70 compounds showed a concentration-dependent decrease in FP in the displacement assay, indicating an inhibition of APE1 AP-DNA binding. These results provide an early indication with respect to the mechanism of action of each compound, although the sensitivity of the displacement assay is lower than that of the enzymatic assay, making it difficult to reveal relevant, but weak, protein binders. From the 223 compounds found positive in the gel-based APE1 assays, exclusion of the most promiscuous hits, that is, those that were both DNA binders and EndoIV-inhibitory, yielded a set of 170 compounds. This shortened group of hits was tested in the cell-based potentiation assay for their ability to dosedependently enhance sensitivity of HeLa cells to the alkylating agent MMS. Of the 170 compounds tested, 12 enhanced the cytotoxicity of MMS significantly, while 16 compounds produced a modest response. This technique measures the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells.