Abstract 2130: DNA repair biochip-based screening to identify Glycosylase/AP endonuclease inhibitors: from the concept to the identification of effective molecules

Abstract

Abstract Base Excision Repair (BER) is the predominant repair pathway responsible for removal of small base/sugar lesions. Three BER activities are predominant in cells: correction of AP sites by APE1, removal of uracil (U) by uracil DNA glycosylase (UNG) and removal of thymine glycols (Tg) by human Endonuclease III homolog (NTH1) or by NEIL1. Despite the tremendous progress accomplished these last years, a lot of uncertainties remain regarding the involvement of other redundant activities able to act as a backup system for repair of these latter lesions. Many studies link chemoresistance and overexpression of DNA repair enzymes. Enzymes from BER pathways are attractive targets for anticancer drug development since therapeutic modulation of repair proteins could potentiate effects of genotoxic drugs. Identification of effective BER inhibitors is thus an emerging promising approach. Here, we used a multiplexed fluorescent oligonucleotide (ODN) cleavage assay on support to the screen of a small compound (SM) library, using HeLa extracts containing the various DNA repair components. Five fluorescent ODN duplexes containing tetrahydrofuran (AP site), U (paired either with A or with G), Tg and normal base pairs were addressed at specific sites on coated slides in a 96-well plate array format adapted for robotic handling. After (1) automated screening of a chemical library of small molecules at 200µM, and (2) establishment of dose/response inhibition curves using the cell extracts and 10 selected compounds, we eventually retained 4 molecules possessing different repair inhibition spectra. Using the same assay, we then performed inhibitory studies in the presence of different pure human BER enzymes in order to identify the putative inhibitors’ targets. Interesting inhibitory properties were found against Smug1, hAAG, hNEIL1 and hAPE1 (IC50 around 10µM). We subsequently characterized the ability of the 4 SM used at non toxic doses (range 2.5µM) to potentiate the cytoxicity effect of genotoxic drugs using MMR-proficient or MMR -deficient cell lines. For some effective combinations, the reduction of BER activities was further confirmed by the in vitro testing of extracts prepared from the treated cell lines. However as the potentiating properties were detected even in the presence of drugs known to induce the formation of bulky adducts, the rationale behind these effects were complex to establish. These results suggest that our knowledge on drug-induced lesions and on the intricacy of repair pathways needs to be refined. In conclusion, we demonstrated that our strategy is a fast and efficient mean to identify SM able to target specific BER activities. The identified compounds may be used to potentiate the effect of genotoxic drugs, but also more widely for chemical genetic strategies, to turn off specific functions in order to dissect and elucidate the DNA repair mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2130. doi:1538-7445.AM2012-2130