Large-Scale Screen for APE1 Inhibitors Figure 1. High-throughput screen. A) Assay principle. APE1 catalyzes and incision 59 relative to the abasic site analog (THF) to liberate a short 59- 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. doi:10.1371/journal.pone.0047974.g001 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 concen- tration–effect relationships was derived using in-house developed algorithms [25]. Percent activity was computed after normaliza- tion 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 concen- tration-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 re- testing in the primary screening assay and potential follow-up studies. PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47974