Diverse small molecule inhibitors of human apurinic/apyrimidinic endonuclease APE1 identified from a screen of a large public collection.

The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of base damage via participation in the DNA base excision repair (BER) pathway. Increased activity of APE1, often observed in tumor cells, is thought to contribute to resistance to various anticancer drugs, whereas down-regulation of APE1 sensitizes cells to DNA damaging agents. Thus, inhibiting APE1 repair endonuclease function in cancer cells is considered a promising strategy to overcome therapeutic agent resistance. Despite ongoing efforts, inhibitors of APE1 with adequate drug-like properties have yet to be discovered. Using a kinetic fluorescence assay, we conducted a fully-automated high-throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR), as well as additional public collections, with each compound tested as a 7-concentration series in a 4 µL reaction volume. Actives identified from the screen were subjected to a panel of confirmatory and counterscreen tests. Several active molecules were identified that inhibited APE1 in two independent assay formats and exhibited potentiation of the genotoxic effect of methyl methanesulfonate with a concomitant increase in AP sites, a hallmark of intracellular APE1 inhibition; a number of these chemotypes could be good starting points for further medicinal chemistry optimization. To our knowledge, this represents the largest-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment

High-throughput screen.

<p>A) Assay principle. APE1 catalyzes and incision 5′ relative to the abasic site analog (THF) to liberate a short 5′-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.</p

Screening hits showing significant activity in the MMS cytotoxicity enhancement experiments.

<p>qHTS, IC₅₀ (µM) obtained in the initial quantitative high-throughput screen; Gel, percent incision observed in the presence of 100 µM compound using the radiotracer detection or estimated IC₅₀ value (µM) using the fluorescence detection; FP, IC₅₀ (µM) 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).</p

Representative curves observed from 10 screening hits chosen to demonstrate the range of potencies observed in the concentration-response-based screen.

<p>Structures and additional data associated with these hits are presented within <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047974#pone-0047974-g007" target="_blank">Figure 7</a>.</p

Modest potentiation of the genotoxic effect of MMS exhibited by 16 prioritized hits (designated I in Table S1).

<p>HeLa cells were exposed to a dilution series of each compound shown in the absence (empty squares) and presence of 400 µM MMS (filled squares), and after a 24-hour incubation the cell viability was measured by ATP-content detection using CellTiter Glo. Results are presented as averages and standard deviations from duplicate samples, normalized against vehicle control.</p

Synthesis, Biological Evaluation, and Structure–Activity Relationships of a Novel Class of Apurinic/Apyrimidinic Endonuclease 1 Inhibitors

APE1 is an essential protein that operates in the base excision repair (BER) pathway and is responsible for ≥95% of the total apurinic/apyrimidinic (AP) endonuclease activity in human cells. BER is a major pathway that copes with DNA damage induced by several anticancer agents, including ionizing radiation and temozolomide. Overexpression of APE1 and enhanced AP endonuclease activity have been linked to increased resistance of tumor cells to treatment with monofunctional alkylators, implicating inhibition of APE1 as a valid strategy for cancer therapy. We report herein the results of a focused medicinal chemistry effort around a novel APE1 inhibitor, <i>N</i>-(3-(benzo­[<i>d</i>]­thiazol-2-yl)-6-isopropyl-4,5,6,7-tetrahydrothieno­[2,3-<i>c</i>]­pyridin-2-yl)­acetamide (<b>3</b>). Compound <b>3</b> and related analogues exhibit single-digit micromolar activity against the purified APE1 enzyme and comparable activity in HeLa whole cell extract assays and potentiate the cytotoxicity of the alkylating agents methylmethane sulfonate and temozolomide. Moreover, this class of compounds possesses a generally favorable in vitro ADME profile, along with good exposure levels in plasma and brain following intraperitoneal dosing (30 mg/kg body weight) in mice

Significant potentiation of the genotoxic effect of MMS by 12 prioritized hits (designated P in Table S1).

<p>HeLa cells were exposed to a dilution series of each compound shown in the absence (empty squares) and presence of 400 µM MMS (filled squares), and after a 24-hour incubation the cell viability was measured by ATP-content detection using CellTiter Glo. Results are presented as averages and standard deviations from duplicate samples, normalized against control.</p

Hit progression flow chart.

<p>Hit progression flow chart.</p

Elimination of assay artifacts and promiscuous hits.

<p>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).</p