Large-Scale Screen for APE1 Inhibitors Figure 8. AP-site accumulation. HeLa cells were exposed to each compound shown in the absence and presence of 275 mM MMS, and total genomic AP sites were measured as described in Methods. The number of AP sites per 106 base pairs of genomic DNA are presented as the average and standard deviation of two independent measurements. P-values (*P,0.05, **P,0.01) were calculated to evaluate the significance of the enhancement of AP site levels produced by the combined treatment versus MMS alone. N.S., not significant (P$0.05); P-value designations for MLS000587064 and MLS000115025 shown in parentheses are derived from the repeat testing reported in Figure S1. doi:10.1371/journal.pone.0047974.g008 convenient technique for providing a basic characterization of macromolecular associations, such as protein-DNA [38] and protein-protein interactions [39]. 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 DNA- specific, as APE1 binds with higher affinity to duplex DNA containing an AP site than to an undamaged counterpart [36]. 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. Potentiation of the Genotoxic Effect of MMS in HeLa Cells To examine the biological prospects of the top compounds, we tested their ability to inhibit APE1 DNA repair activity by assessing enhancement of MMS toxicity in mammalian cells. PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47974