Large-Scale Screen for APE1 Inhibitors Figure 7. Screening hits showing significant activity in the MMS cytotoxicity enhancement experiments. qHTS, IC50 (mM) obtained in the initial quantitative high-throughput screen; Gel, percent incision observed in the presence of 100 mM compound using the radiotracer detection or estimated IC50 value (mM) using the fluorescence detection; FP, IC50 (mM) 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). doi:10.1371/journal.pone.0047974.g007 radioassay, 112 displayed at least 50% inhibition of APE1 activity at 100 mM. Given that the radioassay was specifically conducted at a substrate conversion rate approaching 100%, the fact that a majority of the HTS hits (namely, the weakest and most nonspecific) failed to pass this rigorous APE1 inhibition criterion was not unexpected. Of the 204 compounds tested in the fluorescence-based gel assay, 111 displayed reproducible dose- dependent inhibition. A total of 223 positive compounds showing activity in these electrophoretic separation based assays were then subjected to a panel of assays in order to further assess their engagement with the APE1 target in vitro, as well as to evaluate their selectivity. The complete set of results obtained for these 223 compounds in the below tests is provided within Table S1. To detect screening hits that inhibit APE1 activity through non- specific DNA interactions, we employed a previously established miniaturized ThO dye displacement assay [21]. Forty-three compounds were active in the DNA-binding counter-screen; the majority of these compounds were weak DNA binders (Table S1). Most of the DNA binders possessed the typical chemical features associated with DNA binding: (i) extended conjugated unsaturated ring systems, which would allow them to intercalate between the stacked bases, and/or (ii) accumulation of positively-charged nitrogens, which would permit nonspecific electrostatic interac- tions with DNA. Of note, classic DNA binders, such as daunorubicin- and tetracycline-like compounds (for example NCGC00093976, NCGC00024246, and NCGC00163605, Table S1), exhibited a strong dose-dependent fluorescence signal increase due to their autofluorescent properties, which interfered with the ThO signal and produced distorted dose-response curves. One of the most potent DNA binders among the tested HTS hits was phthalocyanine tetrasulfonate hydrate (NCGC00165867), with a ThO dye displacement IC50 of 0.631 mM, which is a very similar potency to that seen in the primary screening assay (Figure 4B). We note that the ThO assay reports primarily on DNA binders acting through intercalation, while potentially missing other types of DNA binders, such as those acting through the minor groove. However, promiscuous DNA binders which may have been missed by the ThO assay are likely to be flagged during the next profiling step, i.e. the test for inhibition of E. coli EndoIV, described below. To probe the selectivity of the candidate APE1 inhibitors, we tested them against E. coli EndoIV [36], employing the same assay as used in the HTS. E. coli EndoIV, while exhibiting similar biochemical activities to APE1, such as AP site incision, has no sequence or structural homology to human APE1 [37], and thus serves as a tool for identifying broad-acting endonuclease inhibitors. Twenty-one compounds yielded full or partial concen- tration response curves against EndoIV (Table S1). Of these 21 compounds, 15 were already revealed as DNA binders by the ThO assay, and thus, were anticipated to represent non-specific inhibitors. Of the EndoIV-positive hits, only six (compounds NCGC00024246, NCGC00094813, NCGC00159344, NCGC00161415, NCGC00165867, NCGC00166058, Table S1) possessed complete concentration response curves for EndoIV, and the IC50 values of these compounds were significantly higher than the corresponding APE1 potencies. These results indicate that the majority of qHTS-output compounds more selectively inhibit APE1 than EndoIV, and the six compounds above represent potential starting points for the design of EndoIV- specific inhibitors. To gain insight into the mode of action of the hits, we employed a displacement assay combined with fluorescence polarization (FP) detection to test the small molecule’s effect on the binding of APE1 to a version of the fluorogenic AP site-containing oligonucleotide substrate that was devoid of the quencher functionality. FP is a PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47974