Elimination of assay artifacts and promiscuous hits.

<p>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 promiscuous 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).</p

High-throughput screen.

<p>A) Assay principle. APE1 catalyzes and incision 5′ relative to the abasic site analog (THF) to liberate a short 5′-fluorophore donor F-labeled deoxyoligonucleotide, causing increased fluorescence signal. F represents TAMRA fluorophore and Q represents Black Hole Quencher 2. The APE1 incision site is indicated by the arrow. B) High-speed data collection allows monitoring of the reaction progress in kinetic mode as shown in the main panel (3 data points collected over the course of 2 min, shown for 7 wells representing the serial dilution of library compound MLS000090966); the changes in fluorescence signal for each well over the two-minute period are normalized against no-enzyme and no-inhibitor controls to produce the concentration response curve for the sample as shown in the inset. C) A stable Z’ screening factor was maintained throughout the screen. D) A dilution series of the previously reported arylstibonic acid inhibitor NSC-13755 applied to every assay plate yielded a near-constant IC₅₀ of 35 nM.</p

Significant potentiation of the genotoxic effect of MMS by 12 prioritized hits (designated P in Table S1).

<p>HeLa cells were exposed to a dilution series of each compound shown in the absence (empty squares) and presence of 400 µM 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.</p

Modest potentiation of the genotoxic effect of MMS exhibited by 16 prioritized hits (designated I in Table S1).

<p>HeLa cells were exposed to a dilution series of each compound shown in the absence (empty squares) and presence of 400 µM 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.</p

Representative curves observed from 10 screening hits chosen to demonstrate the range of potencies observed in the concentration-response-based screen.

<p>Structures and additional data associated with these hits are presented within <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047974#pone-0047974-g007" target="_blank">Figure 7</a>.</p

Intracellular localization of APE1 protein variants.

<p>(<b>A</b>) Representative microscopy images of the mCherry APE1 fusion proteins following plasmid transfection into HeLa cells. Shown are the DAPI nuclear staining, mCherry fusion protein fluorescence and the merged images. (<b>B</b>) Comparative cytoplasm to nuclear distribution for the different mCherry APE1 proteins. Using densitometry, the ratio of exogenous cytoplasmic mCherry-tagged wild-type (WT) APE1 protein to endogenous cytoplasmic protein was divided by the ratio of exogenous nuclear mCherry-tagged WT APE1 protein to endogenous nuclear protein, and this value was designated as 1. The identical ratio was then determined for each of the APE1 variant proteins, and plotted relative to the WT value. Shown is the average and standard deviation of results from 3 separate extract preparations and western blot experiments.</p

Screening hits showing significant activity in the MMS cytotoxicity enhancement experiments.

<p>qHTS, IC₅₀ (µM) obtained in the initial quantitative high-throughput screen; Gel, percent incision observed in the presence of 100 µM compound using the radiotracer detection or estimated IC₅₀ value (µM) using the fluorescence detection; FP, IC₅₀ (µM) or annotation of response (N.A., no activity observed; P.C., partial concentration response curve) obtained in the fluorescence polarization displacement assay; MMS, potentiation of the genotoxic effect of methylmethane sulfonate (P, positive).</p

Hit progression flow chart.

<p>Hit progression flow chart.</p

REF-1 assay.

<p>(<b>A</b>) HCT116 nuclear extract was incubated with ³²P-labeled consensus (CON) or mutant (MUT) AP-1 oligonucleotide substrates, and binding reactions were resolved on a non-denaturing polyacrylamide gel. Control reactions without nuclear extract (no extract) are shown. The arrow designates the position of the AP-1-specific consensus binding complex, not seen with the MUT double-stranded DNA. Higher molecular weight non-specific complexes are observed. (<b>B</b>) Reduced wild-type (WT) or variant APE1 protein was incubated with HCT116 nuclear extract in the presence of the ³²P-labeled AP-1 CON DNA substrate. Shown is the AP-1-specific complex in the absence (no protein) or presence of the indicated reduced APE1 protein after phosphorimager analysis. Plotted is the relative AP-1 DNA binding activity, in comparison with reduced WT protein. Values represent the average and standard deviation of 3 independent experimental points.</p

APE1 protein variants and oligonucleotide substrates.

<p>(<b>A</b>) Linear schematic of the 318 residue APE1 protein, including several reported amino acid substitutions. NLS = nuclear localization sequence; REF-1 = redox regulatory portion of the protein; italics = polymorphic variants; * = unique disease-associated variants; red text = variants with reduced AP endonuclease activity. The repair nuclease domain and several functionally important amino acids (C65, E96, D210, D283 and H309) are indicated. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065922#pone-0065922-t001" target="_blank">Table 1</a> for additional details. (<b>B</b>) The 18F NMR and 18G NMR oligonucleotides were used to design the double-stranded AP endonuclease substrate. (<b>C</b>) The 15P or 17P oligonucleotide was annealed to the 34G oligonucleotide to generate a 3′-recessed exonuclease/repair substrate, whereas the 34U oligonucleotide was annealed to 34G to create the uracil-containing duplex for the reconstitution assay. Oligonucleotides are written 5′ to 3′, with the non-labeled strands written upside-down. F = the AP site analog, tetrahydrofuran.</p