High-throughput Screen
The screen was performed following the previously published protocol [21]. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) [28] with library plates screened proceeding from the lowest to the highest concentration to minimize compound carryover [26]. Vehicleonly plates, with DMSO being pin-transferred to the entire column 5?8 compound area, were included regularly throughout the screen in order to record any systematic shifts in assay signal.Figure 1. High-throughput screen. A) Assay principle. APE1 catalyzes and incision 59 relative to the abasic site analog (THF) to liberate a short 59fluorophore 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 noinhibitor 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.
During the screen, reagent bottles were kept at 4uC and all liquid lines were covered with aluminum foil to minimize degradation. Screening data were corrected and normalized, and concentrationffect relationships was derived using in-house developed algorithms [25]. Percent activity was computed after normalization using the median values of the uninhibited enzyme control (32 wells located in column 1) and the no-enzyme, or 100% inhibited, control (64 wells, entire columns 3 and 4), respectively, and concentration-response data were fitted using a four parameter Hill equation [29] by minimizing the residual error between the modeled and observed responses. After curve fitting, active compounds were analyzed based on their potency and concentration-response curve characteristics, taking into consideration the presence of asymptotes, efficacy of response, and confidence of curve fit [25]. After preliminary clustering of actives based on structural similarity analysis using Leadscope software (Leadscope Inc., Columbus, OH) [30] selected hits were procured for retesting in the primary screening assay and potential follow-up studies.Figure 2. Representative curves observed from 10 screening hits chosen to demonstrate the range of potencies observed in the concentration-response-based screen. Structures and additional data associated with these hits are presented within Figure 7.ThO DNA Displacement and E. coli Endo IV Profiling Assays
In order to screen out nonselective compounds, such as nonspecific DNA binders and inhibitors of related enzymes, we used a miniaturized ThO DNA displacement assay and a counterscreen against the bacterial endonuclease E. coli Endo IV, respectively, following the previously published protocols [21].
Fluorescence Polarization (FP) Displacement Assay
Double-stranded oligodeoxynucleotide containing tetrahydrofuran (THF) abasic site labeled with TAMRA at the 59-end was used as the labeled binding probe. Inhibition of APE1 binding to labeled probe was detected by a decrease in the fluorescence polarization (FP) of the fluorophore.Figure 3. Hit progression flow chart.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 polyacrylamide 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 (overlapping dose-response curves); inconclusive (I), if the shift in the cell viability dose-response upon MMS inclusion relative to compound-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.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 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).
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?0 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).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 ViewLux High-throughput CCD imager (480 nm excitation and 540 nm emission) to determine the degree of probe displacement caused by the test compounds.
