Large-Scale Screen for APE1 Inhibitors Figure 5. Significant potentiation of the genotoxic effect of MMS by 12 prioritized hits (designated P in Table S1). HeLa cells were exposed to a dilution series of each compound shown in the absence (empty squares) and presence of 400 mM MMS (filled squares), and after a 24- hour incubation the cell viability was measured by ATP-content detection using CellTiter Glo. Results are presented as averages and standard deviations from duplicate samples, normalized against control. doi:10.1371/journal.pone.0047974.g005 dark quencher (Figure 1A). Further details on the generation and validation of this substrate in fully-integrated robotic miniaturized assays have been reported elsewhere [21]. Prior to the full- collection screen, the assay was tested and found to perform reproducibly by screening the LOPAC1280 library of pharmaco- logically active compounds in triplicate using a fully-integrated robotic system (data not shown). The assay was applied to screen a diverse 352,498-compound library that contained the National Institutes of Health public collection, the MLSMR. The compounds tested were arrayed as seven-point titrations, at final concentrations of 57 mM, 11.4 mM, 2.3 mM, 457 nM, 91 nM, 18 nM, and to 3.7 nM. Using a high- speed whole-plate fluorescence imager, the assay data for the entire screen was conducted in kinetic mode, with the APE1 incision reaction being monitored over the initial linear time frame of 2 min (Figure 1B). Thus, any inhibition associated with each sample was computed from the alteration in fluorescence intensity over the time-course measurement period, after normalization against the appropriate controls (Figure 1B, inset). The assay performed well during the entire course of the screen: the Z’ statistical factor remained consistent without fluctuation, at an average of 0.79 (the maximum Z’ factor possible is 1.0, with values of greater than 0.5 being considered an indication of a highly stable screening assay) [33] (Figure 1C). In addition, the intra-plate control titration of the arylstibonic inhibitor NSC-13755 [20] yielded a near-constant concentration-response curve with an average IC50 of 35 nM and a minimum significant ratio of 1.9 (the best possible ratio is 1.0, while ratios of less than 4 are generally PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47974