Functional analysis of novel analogues of E3330 that block the redox signaling activity of the multifunctional AP endonuclease/redox signaling enzyme APE1/Ref-1

Abstract

APE1 is a multifunctional protein possessing DNA repair and redox activation of transcription factors. Blocking these functions leads to apoptosis, antiangiogenesis, cell-growth inhibition, and other effects, depending on which function is blocked. Because a selective inhibitor of the APE redox function has potential as a novel anticancer therapeutic, new analogues of E3330 were synthesized. Mass spectrometry was used to characterize the interactions of the analogues (RN8-51, 10-52, and 7-60) with APE1. RN10-52 and RN7-60 were found to react rapidly with APE1, forming covalent adducts, whereas RN8-51 reacted reversibly. Median inhibitory concentration (IC(50) values of all three compounds were significantly lower than that of E3330. EMSA, transactivation assays, and endothelial tube growth-inhibition analysis demonstrated the specificity of E3330 and its analogues in blocking the APE1 redox function and demonstrated that the analogues had up to a sixfold greater effect than did E3330. Studies using cancer cell lines demonstrated that E3330 and one analogue, RN8-51, decreased the cell line growth with little apoptosis, whereas the third, RN7-60, caused a dramatic effect. RN8-51 shows particular promise for further anticancer therapeutic development. This progress in synthesizing and isolating biologically active novel E3330 analogues that effectively inhibit the APE1 redox function validates the utility of further translational anticancer therapeutic development.

FIG. 1.

Small-molecule inhibition of APE1 redox signaling activity. (A) Structure of E3330. (B) Space-filling model of E3330 was produced by using the software Vida 3.0.0 from OpenEye Scientific Software, Inc. (Santa Fe, NM; www.eyesopen.com). (C) APE1 interacts with downstream transcription factors (TFs) such as NF-κB, HIF-1α, CREB, FOS, JUN, and so on, converting them from oxidized to reduced states, allowing them to bind to their target promoters and switch on the transcription of genes. However, E3330 and analogues presented in this article interfere with this redox signaling by blocking the APE1 ability to convert the oxidized TF to a reduced TF, thereby keeping the target gene transcription turned off. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 2.

E3330 analogues blocked APE1 redox function in EMSA assay. Increasing amounts of E3330 or its analogues (RN8-51, RN10-52, and RN7-60) were incubated for 30 min with 2 μl purified hAPE1 (reduced with 1.0 mM DTT, then diluted to 2 μg/ul with 0.2 mM DTT in PBS) in EMSA reaction buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 5% (vol/vol) glycerol] with a total volume 10 μl. EMSA was performed with reduced APE1 and 0.02 mM DTT, which was the amount of DTT carried over from the reduction of APE1 as control. (A) RN8-51; (B) RN10-52, and B) RN7-60 inhibited AP-1 DNA-binding enhanced by APE1 in a dose-dependent manner, with a much lower IC₅₀ (0.5, 0.75, and 1.5 μM, respectively) than E3330 (20 μM). Increasing amounts of resveratrol (C) or analogue RN7-58 (D) were incubated for 30 min with 2 μl purified human APE1 (reduced with 1.0 mM DTT, and then diluted to 2 μg/ul with 0.2 mM DTT in PBS) in EMSA reaction buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 5% (vol/vol) glycerol] with a total volume 10 μl. EMSA was performed with unreduced APE1 and 0.02 mM DTT, which was the amount of DTT carried over from the reduction of APE1 as control. Neither compound showed any inhibitory effects. Experiments were repeated 3 times with similar results, as shown.

FIG. 3.

Effect of E3330 analogues on APE1 and thioredoxin redox function. Increasing amounts of E3330 or its analogues (RN8-51, RN10-52, and RN7-60) were incubated for 30 min with 2 μl purified human APE1 or thioredoxin (reduced with 1.0 mM DTT, then diluted to 2 μg/μl with 0.2 mM DTT in PBS) in EMSA reaction buffer with total volume of 10 μl. The EMSA assay was performed as described in Methods. E3330 (A–C), RN8-51 (A), and RN10-52 (B) blocked the redox activity of APE1 but not thioredoxin. RN7-60 (C) blocked the redox activity of both, but affected APE1 more than thioredoxin.

FIG. 4.

Effect of E3330 analogues on APE1 endonuclease activity. Oligonucleotide gel-based APE1 endonuclease activity assays were performed as described in Methods (8,15). The upper band (26 mer) represents uncleaved AP oligonucleotides, whereas the lower band (14 mer) is the reacted oligonucleotide. E3330, RN8-51, RN10-52, and RN7-60 had no effect on APE1 endonuclease activity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 5.

E3330 analogues inhibited APE1 redox activity in transactivation assay. Increasing amounts of E3330 (A) or its analogues (RN8-51 (B), RN10-52 (C), and RN7-60 (D)) were added to SKOV-3X ovarian cancer cells with pNF-κB-Luc. After 40 h of treatment, luciferase activity was measured, and MTT assays were performed to measure cell numbers. The ratio of luciferase activity to MTT activity was determined to measure NF-κB activity. Data are expressed as the mean ± SEM of three independent experiments performed in duplicate and are presented as percentage transactivation compared with control of no E3330 or E3330 analogues.

FIG. 6.

Effect of E3330 analogues on cell growth/survival in two ovarian cancer cell lines. MTT assays were performed as described in Methods. Increasing amounts of E3330 and analogues resulted in decreased cell growth and decreased cell numbers in both cell lines. Analogues RN7-60 and RN10-52 had a greater effect than E3330 did; RN8-51 had an effect similar to that of E3330. Data are expressed as the mean ± SEM of three independent experiments performed in triplicate.

FIG. 7.

In vitro Matrigel tube formation and TUNEL apoptosis analysis of ECFCs. (A) E3330 and analogues impair the ability of ECFCs to form tubes in a dose-dependent manner. A significant decrease (**p < 0.001) in closed network tube formation was observed at 7.5 μM for E3330, 25 μM for RN8-51, 5 μM for RN10-52, and 10 μM for RN7-60. (B) Representative pictures of dose-dependent tube-formation ablation in ECFCs treated with E3330. (C) ECFCs were treated with E3330, RN8-51, RN10-52, and RN7-60, stained and analyzed with flow analysis for TUNEL-positive cells. Cells were treated with 1 mM H₂O₂ as a positive control. The vehicle control-treated cells were 0.5% EBM-2 cells treated only with media and DMSO. No increase in apoptosis was noted with E3330- and RN8-51–treated cells. A dose-dependent increase in apoptosis was observed in the RN10-52–treated cells, but the increase at 5 and 10 μM did not differ significantly from the vehicle control (DMSO)-treated cells. ECFCs treated with as little as 2.5 μM RN7-60 were almost entirely TUNEL positive. Statistical analysis was performed as described in Methods. (**p < 0.01 in C).

FIG. 8.

ESI-MS analysis of APE1 with E3330 and analogues. (A) ESI spectra of FL-Ape1 and a mixture of ligands (RN7-60, RN10-52, RN8-51, and E3330) after 1-min reaction, showing modifications by RN7-60 and RN10-52. P, FL-Ape1; *the water adducts of the protein and ligand complexes. (B) Mechanism of covalent vs. reversible inhibition of APE1 redox activity.

FIG. 9.

ESI-MS analysis of adduct formation. (A) ESI spectra of FL-APE1 and RN7-60 after 0.5-h reaction, showing multiple (, , , , , , and 7) modifications. (B) ESI spectra of FL-APE1 and RN10-52 after 0.5-h reaction, showing multiple (, , , , , , and 7) modifications. (C) ESI spectra of FL-APE1 and RN8-51 after 1-h reaction, showing 1 and 2 modifications. P, FL-APE1; *the water adducts of the protein and ligand complexes; ^Unidentified peaks. (D) ESI spectra of FL-APE1 and E3330 after 2-h reaction, showing no modifications by E3330. P, FL-APE1; *the water adducts of the protein and ligand complexes; #contamination peaks due to the carryovers on the C18 column. ^Unidentified peaks. #Peaks correspond to a −44 from the adduct peak. This is the same loss as that going from NH(CH₂)2OH to O^-, suggesting amide hydrolysis.

FIG. 10.

ESI mass spectra of ApeΔ40 and its mutants C99A, C138A, and C99/138A after reaction with compound RN8-51. Mass spectra were collected on a Bruker MaXis UHR-TOF instrument, and deconvolution was done with the MaxEnt1 algorithm.