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Potent Inhibition of Human Apurinic/Apyrimidinic Endonuclease 1 by Arylstibonic Acids

From the Department of Pharmacology and Molecular Sciences, the Johns Hopkins University School of Medicine, Baltimore, Maryland (L.S. and J.T.S); Screening Technologies Branch, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland (J.H.C.); and Natural Products Support Group, SAIC, Inc., Frederick Cancer Research and Development Center, Frederick, Maryland (R.A.)

Received October 12, 2007; accepted November 26, 2007

	Abstract

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Human apurinic/apyrimidinic endonuclease (Ape1) plays an important role by processing the >10,000 highly toxic abasic sites generated in the genome of each cell every day. Ape1 has recently emerged as a target for inhibition, in that its overexpression in tumors has been linked with poor response to both radiation and chemotherapy and lower overall patient survival. Inhibition of Ape1 using siRNA or the expression of a dominant-negative form of the protein has been shown to sensitize cells to DNA-damaging agents, including various chemotherapeutic agents. However, potent small-molecule inhibitors of Ape1 remain to be found. To this end, we screened Ape1 against the NCI Diversity Set of small molecules and discovered aromatic nitroso, carboxylate, sulfonamide, and arylstibonic acid compounds with micromolar affinities for the protein. A further screen of a 37-compound arylstibonic acid sublibrary identified ligands with IC₅₀ values in the range of 4 to 300 nM. The negatively charged stibonic acids act by a partial-mixed mode and probably serve as DNA phosphate mimics. These compounds provide a useful scaffold for development of chemotherapeutic agents against Ape1.

Both the DNA cleavage and transcription factor binding activities of Ape1 result in increased resilience of cells to proapototic stimuli such as radiation, oxidative stress, and chemotherapy (Fishel and Kelley, 2007). Not surprisingly, the overexpression of Ape1 has led to resistance to DNA-damaging agents in several human tumor cell lines (Silber et al., 2002; Yang et al., 2005b). Conversely, decreasing Ape1 expression using small interfering RNA or a dominant-negative form of the protein leads to hypersensitivity to chemically induced DNA damage in both cell culture and tumor xenograft models (Walker et al., 1994; Silber et al., 2002; Liu et al., 2003; Wang et al., 2004; McNeill and Wilson, 2007). Tumor promotion by Ape1 hasbeen shown not only in the laboratory but also in the clinic, where higher levels of Ape1 expression and altered Ape1 localization have been correlated with tumor progression and poor prognosis for patients with various malignancies including osteosarcomas and breast, lung, cervical, prostate, germ cell, ovarian, and head-and-neck cancers (Xu et al., 1997; Herring et al., 1998; Kelley et al., 2001; Koukourakis et al., 2001; Puglisi et al., 2001, 2002; Robertson et al., 2001; Tanner et al., 2004; Wang et al., 2004; Minisini et al., 2005). Although small molecule inhibitors of the Ape1 endonuclease or redox factor activities have been reported (Luo and Kelley, 2004; Madhusudan et al., 2005; Yang et al., 2005a,b), these inhibitors are either fairly weak or nonspecific or the effects in cell culture have been difficult to reproduce (Fishel and Kelley, 2007). The development of effective small-molecule inhibitors of Ape1 would provide useful research tools to explore its role in DNA repair, cancer, and redox-coupled transcription.

It is difficult to envision the rational design of small molecule inhibitors of Ape1 using the structural information provided by its complex with an abasic DNA substrate (Mol et al., 2000). These structures reveal an active site composed of a small hydrophobic pocket surrounded by residues that form electrostatic interactions with the DNA phosphates flanking the abasic site. Specific interactions with the abasic sugar are lacking (Mol et al., 2000), and, as a result, Ape1 is able to cleave 5' to any abasic analog that is small, hydrophobic, and unbranched, including a simple ethanediol linkage (Wilson et al., 1995). Although we have previously used substrate-fragment tethering strategies to design small molecule inhibitors of several DNA repair enzymes (Jiang et al., 2005; Jiang et al., 2006; Krosky et al., 2006), this approach requires an initial substrate fragment, which is not obvious in the case of Ape1. Accordingly, we turned to high-throughput screening methods to identify and then characterize potent small molecule inhibitors of Ape1 that specifically bind to the enzyme and inhibit its activity at nanomolar concentrations.

	Materials and Methods

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Ape1 Purification and DNA substrates. The plasmid HAP1-pET15b was a gift of Dr. J. B. Alexander Ross (University of Montana, Missoula, MT). Recombinant Ape1 was purified as described previously (Erzberger et al., 1998). The DNA substrates used in this study were synthesized using conventional solid phase synthesis and reagents supplied by Glen Research. The sequences of the substrate DNA strands are 5'-FAM-GAG AA ATA GTC GCG-3' and 3'-dabsyl-CTC TTG TAT CAG CGC-5' (where is a tetrahydrofuran abasic site analog). After synthesis, the oligonucleotides were HPLC purified over a Zorbax column (Phenomenex, Torrance, CA) and desalted using reversed phase columns (PD-10; Bio-Rad Laboratories, Hercules, CA). The sequence of the oligonucleotides was verified using matrix-assisted laser desorption ionization mass spectrometry. The kinetic parameters for Ape1 cleavage of the hybridized DNA were determined using a Fluoromax-3 fluorimeter (Horiba Jobin Yvon, Edison, NJ).

High-Throughput Screening. To perform the screening assay, 5 µl of each library member in DMSO was first spotted to a well of a black 384-well microtiter plate (Costar 3710; Corning Life Sciences, Acton, MA). The addition of protein and substrate was then carried out using a MAP-C2 liquid handling system (Titertek, Huntsville, AL). First, 40 µl of Ape1 in reaction buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, and 0.05% Brij-35) was added to each well, and the plate was incubated at room temperature for 20 min. The reaction was then initiated through the addition of 10 µl of substrate, and the plate was incubated at room temperature for an additional 60 min. After 60 min, the final fluorescence was measured using a FLUOstar Optima plate reader (BMG Labtech GmbH, Offenburg, Germany) at excitation and emission wavelengths of 485 and 520 nm, respectively. The final concentrations of the reagents used in the assay were 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 0.05% Brij-35, 20 µM small molecule, 50 nM abasic-containing DNA substrate, and 5 pM Ape1. Hits were defined as those compounds able to inhibit Ape1 activity (measured as final fluorescence) by 40% or greater. Compounds that appeared highly colored, contained disulfide bonds, or had structures suggestive of DNA intercalation were excluded from further analysis. Hits from the initial screen were analyzed further for inhibitory potency using the fluorescence-based assay and decreasing dilutions of inhibitor. The assay was carried out as before, except that the reactions were initiated by the addition of enzyme rather than substrate, and FAM fluorescence was monitored every 5 min for a total of 30 min to acquire initial rate values. The final concentrations of the reagents used in the assay were 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 0.05% Brij-35, 0.1 to 10 µM small molecule, 50 nM abasic-containing DNA substrate, and 10 pM Ape1. Percentage of inhibition was determined relative to a DMSO-only control.

DNA oligonucleotides for a secondary radioactivity assay were synthesized as described above with the exclusion of the fluorescent groups. The sequences of the DNA strands are 5'-GGG CGC PA GTC GCG-3', where P is 2-aminopurine, and 3'-CCC GCG TAT CAG CGC-5'. The THF-containing strand was labeled on the 5' end using [-³²P]ATP and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) before hybridization. The individual reactions were initiated in the same manner as the fluorescence assay and quenched at 30 or 60 min by adding EDTA and heating for 20 min at 70°C. The final concentration of the reagents used in the assay were 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 0.05% Brij-35, 100 nM inhibitor, 50 nM abasic-containing DNA substrate, and 5 pM Ape1. Samples from each quenched reaction were run on a 20% denaturing polyacrylamide gel and the substrate and product bands were quantified using a Fuji BAS 2500 filmless autoradiographic analyzer.

IC₅₀ Determinations. IC₅₀ analyses were carried out on the best hits by first plating 10-fold dilutions (10 pM-10 µM) of each inhibitor onto black 384-well plates. The reactions were set up in the same manner as the initial screen except that FAM fluorescence was measured every 5 min after reaction initiation for a total of 60 min. Using the initial rate values from the assay, percentage activity was calculated for each sample relative to a negative (DMSO only) control. The data were fitted to a sigmoidal dose-response model using Prism software (GraphPad Software, San Diego, CA), and IC₅₀ values were determined using the equation % Activity = 100/(1 + 10^{(log[I] - logic₅₀)}).

DNA Intercalation Assay. An ethidium bromide-based DNA binding assay was carried out essentially as described previously (Boger et al., 2001). In brief, 10-fold dilutions of hit compounds in DMSO were spotted onto a 384-well black microtiter plate in 2.5-µl aliquots. To each well was added 47.5 µl of a mixture of ethidium bromide and DNA substrate in Ape1 reaction buffer. Ethidium bromide fluorescence was monitored at excitation and emission wavelengths of 544 and 612 nm, respectively. The final concentrations of the reagents used in the assay were 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 0.05% Brij-35, 100 pM-100 µM inhibitor, 1 µM ethidium bromide, and 133 nM DNA substrate to maintain a ratio of 1:2 of ethidium bromide to DNA base pairs. Percentage fluorescence was calculated relative to a negative (DMSO only) control.

Mode of Inhibition Analysis. A mode of inhibition analysis was carried out on the best arylstibonate inhibitors by first plating dilutions of each inhibitor onto white nonbinding 96-well microtiter plates (Corning 3600). Dilutions of substrate in Ape1 buffer were added to each well, followed by the addition of Ape1 protein to initiate the reactions. FAM fluorescence was then monitored every 5 min for a total of 30 min. The final concentration of the reagents used in the assay were the same as the screening conditions except that the NaCl concentration was increased to 75 mM to increase K_m and allow rate measurements at substrate concentrations much less than K_m. A range of inhibitor concentrations between 5 nM and 10 µM and substrate concentrations in the range between 62.5 nM and 2 µM were used. The data were analyzed using Prism software, and initial rate values were used for global discrimination fitting by the computer simulation program Dynafit v.3.28 (BioKin, Pullman, WA) (Kuzmic, 1996).

View larger version (10K): [in this window] [in a new window]   Fig. 1. Molecular beacon assay for Ape1. A, an oligonucleotide containing a THF abasic site mimic was used in the Ape1 molecular beacon substrate. The top strand was labeled on the 5' end with a FAM fluorophore, the bottom on the 3'end with a dabsyl (DAB) quench. B, upon cleavage by Ape1, the fluorophore dissociates from the dabsyl quench, causing an increase in fluorescence. C, an approximate 6-fold increase in fluorescence is observed upon DNA cleavage by Ape1.

	Results

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High-Throughput Screening. A robust molecular beacon assay was used to identify inhibitors of Ape1. In brief, a double-stranded DNA substrate containing a tetrahydrofuran (THF) abasic site mimic was synthesized, purified, and annealed to its complementary strand. The strand containing the THF site was labeled on the 5' end using FAM, whereas the 3' end of the opposite strand was labeled with a dabsyl quench (Fig. 1A, DAB). Upon DNA backbone cleavage by Ape1, a small seven-base oligonucleotide labeled with the FAM group spontaneously dissociates from the remaining DNA (Fig. 1B), causing fluorophore emission to increase by approximately 6-fold under the assay conditions used (Fig. 1C). The k_cat and K_m of Ape1 for this substrate in a buffer containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, and 0.05% Brij-35 are 8 ± 1 s^-1 and 90 ± 30 nM, respectively (Supplemental Fig. 1). The turnover number k_cat was not significantly dependent on the NaCl concentration in the range 50 to 150 mM, but K_m increased to 570 ± 100 nM at = 100 mM NaCl (data not shown). Based on these findings, we chose to carry out the screening studies at 50 mM NaCl, where K_m was fairly low (to conserve substrate), and to use a substrate concentration equal to the K_m, so that both competitive and uncompetitive inhibitors could be detected.

High-throughput screening for inhibitors of Ape1 was carried out using the 2000-compound Diversity Set obtained from the National Cancer Institute. Initial hits were defined as those compounds that inhibited protein activity by 40% or greater. The initial screen identified approximately 140 molecules as inhibitors of Ape1, giving a hit rate of 7%. All but 15 of these compounds reconfirmed as hits in a secondary fluorescence assay. To cull the remaining 125 confirmed hits, 86 molecules with weak inhibition (<75% inhibition at 20 µM compound) were eliminated. For these 125 compounds, those that contained disulfide bonds or with structures suggestive of DNA intercalation were also not investigated further, as were nine weakly inhibitory and intensely colored dye-like compounds. Ten of the remaining 32 hits were found to inhibit Ape1 activity by 50% at concentrations in the range of 0.1 to 10 µM (Table 1). The inhibitory activities of four representative compounds (13778, P7810, 28620, and 15596) were then verified using a radioactive assay (Supplemental Fig. 2).

From the initial screen, two of the best Ape1 inhibitors, 15596 and 13778, were arylstibonic acids, suggesting that such compounds may serve as useful scaffolds for further diversification. Accordingly, we screened an additional 37-member arylstibonic acid library that we obtained from the Developmental Therapeutics Program at the National Cancer Institute. This effort identified two more inhibitors (13755 and 13793) with increased potency over the initially screened compounds (Fig. 2). The IC₅₀ values for these arylstibonic acids ranged from 4 to 17 nM using the screening buffer that contains 50 mM NaCl (Table 2).

View larger version (12K): [in this window] [in a new window]   Fig. 2. IC50 analyses for the most potent aryl-stibonic acid inhibitors. The five best antimony-containing inhibitors have IC50 values in the low to mid-nanomolar range (Table 2).

View larger version (7K): [in this window] [in a new window]   Scheme 1. Partial mixed-type inhibition mechanism for arylstibonic acids.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Representative substrate saturation profiles of Ape1 in the presence of increasing concentrations of 13755 and simulations of the inhibition data. A, Michaelis-Menten curves in the presence of increasing concentrations of 13755. B, Dynafit simulations for 13755 using a partial mixed-type model for the inhibition.

Arylstibonates Showed Specificity for Ape1 and Did Not Intercalate DNA. As a test for whether these compounds were general protein poisons, we checked whether 13778 inhibited uracil DNA glycosylase (UNG), the enzyme that acts just before Ape1 in the base excision repair pathway. Compared with Ape1, a 333-fold higher concentration of 13778 was needed to reduce UNG activity by 50% [IC₅₀ (UNG) 100 µM; Supplemental Fig. 3]. Because 13778 inhibited Ape1 with an IC₅₀ of 300 nM, we concluded that 13778, and its related inhibitors, showed considerable specificity for Ape1 and were not general protein poisons. In addition, an ethidium bromide competition assay was used to examine the possibility that these compounds inhibited Ape1 activity by binding to DNA rather than the enzyme. In this assay, ethidium bromide is prebound to the DNA substrate and increasing concentrations of inhibitor are added. If the inhibitor were able to bind DNA by displacing ethidium bromide, a decrease in ethidium bromide (EtBr) fluorescence would be observed (Boger et al., 2001). As shown in Fig. 4, the five strongest Ape1 inhibitors did not displace bound EtBr at concentrations up to 100 µM. In contrast, the known intercalator distamycin A produced a 50% decrease in ethidium bromide fluorescence in the same concentration range.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Ethidium bromide inhibitor-DNA intercalation assay. An ethidium bromide competition assay was used to evaluate the ability of the arylstibonate Ape1 inhibitors to bind DNA. No decrease in ethidium bromide fluorescence was observed upon inhibitor addition, indicating that these compounds do not bind DNA.

Cell Culture Studies. Inhibition of Ape1 in cell culture through the use of small molecules, RNA interference, or the expression of a dominant-negative form of the protein has been shown to increase sensitivity of human cells to the DNA alkylating agent methyl methanesulfonate (MMS) (Luo and Kelley, 2004; Wang et al., 2004; Madhusudan et al., 2005; Fishel and Kelley, 2007; McNeill and Wilson, 2007). In this type of experiment, cells are treated with inhibitor, MMS, or both, and cell survival is determined by counting or clonogenic survival. Using this approach, we examined the activity of the arylstibonate inhibitors in human cell culture. Despite their potent in vitro inhibitory activity, we were unable to detect any decreased survival when cells were treated with 5 µM 25857, 10470, 13755, or 13793 in the presence of 0, 25, or 50 µM MMS (Supplemental Fig. 4). As a positive control, small interfering RNA knockdown of Ape1 expression by 80% produced a 2-fold decrease in cell survival in the presence of 100 µM MMS (Supplemental Fig. 5). It should be noted that none of the tested compounds displayed any cytotoxicity up to concentrations of 5 µM with the exception of 10470, which was highly toxic as a single agent to HOS, MCF-7 and PC-3 cells at this concentration (Supplemental Fig. 4).

	Discussion

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Small-Molecule Inhibitors of Ape1. Several classes of inhibitors of Ape1 have been described previously. One unusually simple compound used to inhibit AP endonuclease activity is methoxyamine (MX), which attacks the ring-opened form of abasic sites to form an oxime linkage. Thus, rather than targeting the enzyme directly, MX blocks Ape1 from accessing the lesion site (Liuzzi and Talpaert-Borlé, 1985). Because of this unusual mechanism, MX is not specific for Ape1, and other off-target effects would be expected (Fishel and Kelley, 2007). A second compound, lucanthone, inhibits Ape1 repair in vitro but is also a very potent topoisomerase II poison, which probably accounts for its toxicity with cancer cell lines (Bases and Mendez, 1997; Luo and Kelley, 2004). The small-molecule 7-nitroindole-2-carboxylic acid has recently been shown to inhibit Ape1 in vitro with an IC₅₀ of 3 µM and was also reported to sensitize cells to MMS and several other DNA-damaging agents (Madhusudan et al., 2005). However, subsequent studies have not been able to reproduce the sensitizing effects of 7-nitroindole-2-carboxylic acid (Fishel and Kelley, 2007). Similar controversy surrounds the natural product resveratrol, which was originally reported to inhibit the redox activity of Ape1 (Yang et al., 2005b); once again, this effect was not reproducible by another group (Fishel and Kelley, 2007). The present compounds inhibit Ape1 at low nanomolar concentrations in vitro (Table 2), making them the most potent Ape1 inhibitors yet reported and promising candidates for further development.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Structural and electrostatic comparisons of phosphorus and antimony acids. A, models of phenylstibonic acid (pink) and phenylphosphonic acid (green) calculated using the PM3 semiempirical method (Spartan '04; Wavefunction, Inc., Irvine, CA). The Sb-O, Sb-C, and Sb = O bondlengths are all much longer than the corresponding distances in the phosphorus compound (Sb-O = 1.99 Å, Sb-C = 2.10 Å, Sb = O = 1.95 Å; P-O = 1.69 Å, P-C = 1.77 Å, P = O = 1.46 Å.) The electrostatic potential is plotted on the Van der Waals surface of each compound. B, superposition of the antimony and carboxylate atoms of 13755 with the phosphodiester groups of the abasic site extracted from the complex of Ape1 with abasic DNA (Protein Data Bank code 1DEW).

The three most potent aryl-stibonic acid inhibitors of Ape1 share a common partial mixed-type inhibition pattern (Scheme 1). Thus, these negatively charged compounds can bind to both the free enzyme and the ES complex. The dissociation constants of both the substrate and inhibitor EI and ES complex are approximately 2- to 3-fold greater than their respective constants for binding to the free enzyme, indicating energetic cross-talk between the binding sites (Table 3, Scheme 1). The 10 to 20% residual enzymatic activity in the presence of saturating concentrations of substrate and inhibitor indicates that the ESI complex is in someway perturbed from its optimal state. Plausible physical interpretations for this inhibition pattern are suggested below.

Structure-Activity Relationships for Arylstibonates. The 37-compound arylstibonate library provides some useful structure-activity relationships (Supplemental Table 1). All of the inhibitory compounds have an anionic pentavalent stibonic acid moiety attached to a benzene ring, whereas those containing the reduced (neutral) trivalent antimony show no inhibitory activity. The five most potent inhibitors in Table 2 all have carboxylate or nitro substituents on the benzene ring placed meta or para to the stibonate group, indicating that negatively charged or polar substituents at these positions are important. Furthermore, several molecules in the library are structurally similar to our most potent inhibitor, 13755 (K_i = 19 nM), yet have no inhibitory activity. Compound 13755 is substituted at the para and meta positions with a carboxylate and a nitro group, respectively, but compounds 13744, 13743, and 13760, which possess one but not both of these groups, have no inhibitory activity. Thus, the stibonate, nitro and carboxylate functional groups must all be present and in the appropriate positions for maximal inhibition. In addition, P6966 is structurally similar to 13755, with a second carboxylate substituting for the nitro group, but it is also inactive. The second most potent inhibitor, 13793 (K_i = 36 nM), is distinct from 13755 because of its esterified butanoic acid moiety attached para to the antimony group. The length of this chain seems essential because compounds with shorter carboxylate containing chains attached in the same position are not inhibitory (P6982, P6965, and P6971; Supplemental Table 1). Structure-activity relationships are also apparent for compound 13778, which possesses a key ethylene linker connecting a carboxylate group to the benzene ring. If this ethylene linker is changed to ethyl (P6949) or the length of the linker is reduced by one or two carbon units (P6970, P6953, or 13759), inhibition is completely lost.

Did These Arylstibonate Compounds Mimic the Phosphate Esters in DNA? Antimony is a group V-A element of the periodic table with properties similar to phosphorus and arsenic. Compared with phosphorus, antimony has a larger ionic radius and lower charge density, which allows Sb(V) to form an octahedrally coordinated stibonic acid species H[Sb(OH)₆], which dissociates to the stibonate anion [Sb(OH)₆]^- in weak acid or neutral aqueous solution (Filellaa et al., 2002). This property is in contrast with P(V), which is tetrahedrally coordinated with oxygen. The aqueous equilibria of these arylstibonates are not known, but it is possible that they exist in equilibrium between a tetrahedral form (Table 2), or a pentavalent hydrated form in which four hydroxyl groups and one carbon atom are coordinated to the antimony center. In either case, the lower electronegativity of Sb(V) compared with P(V) would be expected to increase the Sb-O bond length compared with P-O, and thus, place a larger negative charge density on the oxygen atoms than the corresponding phosphonate analog. This expectation is confirmed by the electrostatic potential surfaces shown in Fig. 5A, which were calculated using semiempirical (PM3) methods for the model compounds phenyl phosphonic acid and phenyl stibonic acid. Greater charge density on the antimonial oxygens could facilitate stronger coulombic interactions with enzyme groups. Given these properties of arylstibonates, and their similarity with phosphate, we speculate that these anionic molecules may mimic the phosphate backbone groups of DNA. Based on the structure-activity relationships described above, an important requirement of the observed inhibition may be positioning of essential carboxylate and stibonate groups in anion binding pockets originally intended for adjacent 3' and 5' DNA phosphates of a DNA strand. The crystal structure of the Ape1-abasic DNA complex shows several such binding interactions with both DNA strands. To support our proposal, we extracted the abasic site from this structure and superimposed the stibonate and carboxylate groups of 13755 onto the phosphates (Fig. 5B). The alignment of the antimony and phosphate centers is excellent, with deviations of only tenths of an angstrom. A mechanism of inhibition involving binding to adjacent phosphate sites on the enzyme would be consistent with the observed partial mixed-type mechanism, because the DNA could still bind and react even if the inhibitor were blocking one or more phosphate binding sites. Consistent with the proposal that these compounds mimic DNA, 13778 has also been shown to inhibit poxvirus type I topoisomerase but not the closely related human type I topoisomerase (Bond et al., 2006). Although much remains to be understood about the selectivity of these compounds, it seems that selectivity for a given target may be provided by the positions of phosphate binding sites on the target and how well the site accommodates the aromatic functional group of the compound. These data indicate that arylstibonates may be especially useful for targeting enzymes that interact with nucleic acids.

	Acknowledgements

 
We thank the Developmental Therapeutics Program at the National Cancer Institute for the small molecule libraries.

	Footnotes

 
This work was supported by National Institutes of Health grant GM56834 (to J.T.S.) and in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400.

ABBREVIATIONS: Ape1, apurinic/apyrimidinic endonuclease; FAM, 6-carboxyfluorescein; Ref-1, redox factor-1; DMSO, dimethyl sulfoxide; HOS, human osteosarcoma; THF, tetrahydrofuran; UNG, uracil DNA glycosylase; MMS, methyl methanesulfonate; MX, methoxyamine.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: James T. Stivers: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore MD 21205-2185. E-mail: jstivers{at}jhmi.edu

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