Figure 4. Elimination of assay artifacts and promiscuous hits. A) An autofluorescent compound contributes fluorescence intensity well in excess of the assay reaction’s average leading to the computation of aberrant concentration response curve. B) An example of a promiscu- ous hit acting by strong DNA binding as evidenced by the highly similar concentration responses observed in the primary screen (empty squares) and the ThO counterscreen (filled squares). doi:10.1371/journal.pone.0047974.g004 well Greiner black solid bottom plates. Compounds (23 nL) were transferred via pintool, and after a 15-minute incubation at room temperature, fluorescence polarization was measured in a View- Lux High-throughput CCD imager (480 nm excitation and 540 nm emission) to determine the degree of probe displacement caused by the test compounds. Gel-based APE1 Assays To test the compounds’ inhibitory activity after the primary screen, two assays designed to monitor the cleavage of substrate to product through electrophoretic separation were utilized. The radiotracer incision assay, utilizing a 32P-labeled substrate, was performed essentially as described [21,31,32]. Incision reactions utilizing 32P-labeled substrate were carried out for 5 min at 37uC. After the addition of an equal volume of stop buffer (0.05% bromophenol blue and xylene cyanol, 20 mM EDTA, 95% formamide), the substrate and product were separated on a standard polyacrylamide denaturing gel and quantified by PhosphorImager analysis [32]. The fluorescence-based assay utilized a 59-TAMRA labeled 32-mer oligonucleotide containing a THF moiety at position 18 (59-TAMRA- TTTTTTTTTTTTTCACCFTCGTACGACTCCGT-39) an- nealed with a complementary 20-mer oligonucleotide (59-ACG- Large-Scale Screen for APE1 Inhibitors GAGTCGTACGAGGGTGA-39) was used in this assay. APE1 cleavage of the substrate results in a 18-mer TAMRA-labeled fragment which was separated by a non-denaturing polyacryl- amide gel electrophoresis and quantitated in a Gel Doc XR imaging system, BioRad (Hercules, CA). The advantage of this assay is that it can be used in a relatively high throughput mode with the inhibitors tested at a range of concentrations (0.17 mM to 125 mM was used here). APE1 (0.15 nM) was added to a reaction mixture of 20 mL containing assay buffer (40 mM HEPES pH 7.5, 50 mM NaCl, 1 mM MgCl2 and 2 mM DTT), and labeled ds DNA substrate (final 75 nM). The mixture was incubated at RT for 15 min, and the reaction was stopped by the addition 20 mL of 2X dye stop solution (10% glycerol, 20 mM EDTA and bromophenol blue). Then 10 mL of the mixture was resolved on 20% polyacrylamide gel in 1X Tris-borate EDTA running buffer at 250 V for 40 min. MMS Potentiation Assay HeLa cells were plated at 6000 cells/well in DMEM culture medium containing 10% FBS into white solid bottom 384-well cell culture plates. The plates were incubated at 37uC overnight for cell attachment, followed by media replacement. The fresh medium contained serial dilutions of compounds of interest in the presence (0.4 mM final) or absence of MMS. The plates were incubated for 24 h at 37uC. Cell viability was determined by luminescence detection after an addition of 15 mL of CellTiter Glo reagent (Promega, Madison, WI). Percent viability was calculated for each concentration of compound tested in duplicate by relating the corresponding luminescence to that of a negative DMSO vehicle control. MMS potentiation trends were defined as follows: negative (-), if cell viability in the compound-plus-MMS treatment was the same as that of the DMSO-plus-MMS control (overlap- ping dose-response curves); inconclusive (I), if the shift in the cell viability dose-response upon MMS inclusion relative to com- pound-alone was less than 30%; and positive (P), if the difference between the compound-plus-MMS and compound-alone curves was at least 30%, and maintained within at least a two-fold compound concentration span. AP Site Measurement HeLa cells at 80% confluency (66105 cells) were subjected to the following treatments in a 6-well plate for 24 hours at 37uC: 1) DMSO alone, 2) 275 mM MMS, 3) APE1 inhibitors dissolved in DMSO (5–20 mM final compound concentration), and 4) the inhibitors as in treatment 3 in combination with 275 mM MMS. Cells were then harvested and processed using a Qiagen Genomic DNA isolation kit (Germantown, MD). After DNA quantitation, AP sites were measured using the DNA Damage Quantification kit from Dojindo Molecular Technologies (Rockville, MD). Results and Discussion Quantitative High-throughput Screen To screen for inhibitors of APE1 incision activity, we employed a purified-enzyme biochemical assay with a model substrate featuring a red-shifted fluorescent reporter in combination with a dark non-emitting quencher. These moieties were incorporated as end labels onto a 17-mer double-stranded DNA fragment containing a centrally-located tetrahydrofuran AP site sugar mimetic, which serves as the APE1 cleavage site. Upon APE1- catalyzed strand scission immediately 59 to the AP site analog, the shortened oligodeoxynucleotide carrying the fluorescent reporter spontaneously dissociates from the rest of the substrate, leading to an increase in fluorescence due to its spatial separation from the PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47974