Large-Scale Screen for APE1 Inhibitors Figure 6. Modest potentiation of the genotoxic effect of MMS exhibited by 16 prioritized hits (designated I 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 vehicle control. doi:10.1371/journal.pone.0047974.g006 indicative of an assay with high test-to-test reproducibility of dose responses) [27,34] (Figure 1D). Unlike traditional HTS, qHTS provides a concentration response curve (CRC) for each compound and allows for calculation of an IC50 value for each compound in the primary screen. Approximately 1,100 compounds with full concentration- response curves and IC50 values of less than 30 mM were identified, and similarity analysis of the hits led to 121 clusters and 154 singletons, representing a wide variety of structural classes (the qHTS results are available in PubChem under Assay Identifier 2517, http://pubchem.ncbi.nlm.nih.gov/). Representa- tive concentration-response curves from 8 hits spanning most of the potency range (double-digit nanomolar to double-digit micromolar IC50) are shown in Figure 2. The progression of hits through the respective steps of cheminformatics analysis, confir- matory testing, and additional profiling, is depicted as a flow chart in Figure 3. After exclusion of heavy metal- and reactive functionality- containing molecules, and after using the real-time kinetic screening data to flag compounds that interfere with the assay signal by contributing excessive amounts of fluorescence [35] (an example of a highly fluorescent hit, the bioactive fluorescent sensor calcein NCGC00094849, is shown in Figure 4A), 745 hits were selected for further characterization based on potencies and concentration-response curve quality. Of the 745 cherry-picked compounds, 595 (80%) exhibited activity upon retesting using the original fluorogenic screening assay. Follow-up Testing of Primary Screening Hits To eliminate false positive hits, all 595 confirmed molecules were tested for their ability to inhibit APE1 incision activity using biochemical assays that involve electrophoretic separation of the substrate and cleavage product. We adopted a two-step approach: (1) hits possessing complete screen-derived concentration response curves were tested at a single concentration in the low-throughput electrophoretic separation assay with radiolabel detection and (2) lower confidence hits possessing either incomplete or noisy concentration response curves were tested as a seven-point dilution series using a higher-throughput electrophoretic separation assay with fluorescence detection. Of the 391 compounds tested in the PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e47974