BER Y-27632 dihydrochloride inhibitors or activators would provide novel resources, not only for basic science purposes, but for the potential development of high affinity targeted, therapeutics that may improve the efficacy of treatment paradigms by promoting selective sensitization of diseased cells or increasing the protection of normal cells, respectively. Small molecule protein modulators are viewed as the perfect complement to frequently-used techniques such as RNAi and gene knockout. In addition, the scientific and medical communities have become increasingly interested in the design of small molecule inhibitors of DNA repair, with the potential of improving the therapeutic efficacy of clinical DNA-interactive drugs. While there have been reports of small molecules directed at APE1, the utility of these inhibitors has been brought into doubt given the inability to reproduce their effectiveness or the failure of the compounds to elicit a cellular consequence. It is anticipated that the reconfigured APE1 assay described here will serve as a useful guide to future investigations aimed at screening other nucleic acid processing enzymes. The concentration-response screen of the LOPAC collection yielded a number of previously-unreported APE1 inhibitors. The most potent hit was ATA, which inactivated the enzyme consistently in the low nanomolar range. While very effective, ATA has been noted to exist as a stable radical homopolymer of varying length and to act as a strong inhibitor of a large number of DNA- and RNA-processing enzymes. As a means of interrogating the primary screening hits, and to gain further insight into their mechanism of action, we employed a fluorescent dye-displacement assay, substituting the frequently-used ethidium bromide with the more sensitive and robust reporter ThO. In a screen of the LOPAC collection against a ThO-substrate DNA complex, all annotated fluorescent DNA intercalators within the library, e.g. idarubicin, doxorubicin and distamycin, displayed strong displacement activity. Furthermore, the non-fluorescent APE1 screening hits WB 64 and mitoxantrone exhibited dye-displacement IC50 values similar to or better than those displayed in the APE1 enzymatic incision assay. This behavior is reminiscent of an indirect, non-competitive DNA binding effect and is consistent with the multiple fused ring Epoxomicin systems, which have a tendency for DNA intercalation, featured in both molecules. On the other hand, APE1 inhibitor molecules that lacked obvious DNA-binding features, such as thiolactomycin and Tyrphostin AG 538, yielded weak or no displacement activity. These findings support the ThO displacement assay as a convenient counterscreen to exclude DNA binders from further consideration, and the complete results with the LOPAC1280 are available in the corresponding PubChem deposition. To further probe the selectivity of the inhibitors identified in the APE1 qHTS, we tested the LOPAC collection against E. coli EndoIV. AP endonucleases are sub-classified into two major superfamilies based on homology to either E. coli exonuclease III or E. coli EndoIV. While members of the two superfamilies exhibit similar biochemical properties, such as AP endonuclease activity, there exists no sequence or structural homology between the different superfamily constituents. Among the actives shared between the two endonucleases were the nonspecific DNA binders mitoxantrone and WB 64, whereas compounds with selectivity to APE1 included thiolactomycin, 6hydroxy-DL-DOPA, methyl 3,4-dephostatin, myricetin and Reactive Blue 2. The fact that the EndoIV screen did not identify either potent or EndoIV-selective hits may suggest an active site for this enzyme that is hard to access. Our ongoing studies of inhibitors for E. coli EndoIV are outside the scope of the present investigation and will 
be the subject of a separate report. Finally, we tested the top screening hits in a standard radiotracer incision assay.