Abstract A168: Quantitative high-throughput screening for inhibitors of human apurinic/apyrimidinic endonuclease APE1

Abstract

Abstract The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of spontaneous hydrolytic, oxidative, and non-enzymatic alkylation base damage via the DNA base excision repair (BER) pathway. The increased activity of APE1, often observed in tumor cells, is thought to contribute to the development of resistance to various anticancer drugs; conversely, its down-regulation sensitizes tumor cells to DNA damaging agents via induction of apoptosis. Therefore, inhibiting APE1 within the BER pathway in cancer cells is an attractive strategy in the attempts to overcome chemotherapeutic resistance. Despite ongoing efforts, potent specific inhibitors of APE1 have yet to be discovered. We developed a fluorogenic substrate operating in the red-shifted fluorescence spectral region to configure a highly robust kinetic assay for use in 1536-well based automated high-throughput screening (HTS). The assay was used in a titration-based screen of the LOPAC1280 library to identify potential APE1 inhibitors which were further validated and profiled in a panel of assays including radiotracer-based incision assay, Thiazole Orange fluorophore displacement test for promiscuous DNA binders, and E. coli Endo IV counterscreen. Select compounds which passed the above validation steps were progressed to studies of increased biochemical complexity and were shown to inhibit abasic-site incision activity in whole cell protein extracts and to potentiate the genotoxic effect of methyl methanesulfonate (MMS), consistent with suppression of BER on a cellular level. Subsequently, a fully automated HTS was conducted against a collection of 241,291 diverse compounds tested as 7-concentration series at a 4 uL reaction volume in 1536-well plate format. To our knowledge, this represents the first large-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A168.