Abstract 3689: Identification of DNA ligase IV inhibitors as possible drug and probe candidates for enhancement of radiation treatment and chemotherapy

Abstract

DNA double strand breaks (DSBs) are highly lethal DNA lesions, as one DSB is sufficient to trigger cell death. Ionizing radiation and some chemotherapeutic drugs are designed to generate DSBs in cancer cells that in turn induce cell death. However, mammalian cells have multiple DNA repair mechanisms to defend against DSBs, primarily large protein complexes that conduct non-homologous end joining (NHEJ) and homologous recombination (HR) to repair the breaks. While HR primarily functions in the S phase of cell cycle, NHEJ is active in all cell-cycle stages and plays a predominant role in mammals. Thus disruption of the NHEJ process may be an efficient way to make cancer cells more sensitive to chemotherapy and radiotherapy. There are two fundamental steps in NHEJ: end-bridging by the DNA-PK complex and then end-ligation by the DNA ligase IV/XRCC4 complex. Because of the restricted function of the DNA ligase IV/XRCC4 complex to NHEJ, this enzyme is an ideal target for inhibitor development. Our study aimed to develop inhibitors of the DNA ligase IV/XRCC4 complex to stop the final step in NHEJ. DNA ligation is initiated by formation of a covalent ligase-adenylate intermediate followed by transfer of AMP onto DNA ends. An assay for DNA ligase IV suitable for high throughput screening (HTS) has not been reported. As the majority of endogenous DNA ligase IV is adenylated in living cells, our experimental design is focused on the AMP transfer step to identify inhibitors for inhibiting transfer of AMP onto DNA ends by DNA ligase IV. An assay was established to screen for small molecules that block the transfer of P33-AMP to 50-bp double-stranded DNAs and miniaturized to 70 ul for application to large scale HTS. A library including 5280 known compounds was screened as a pilot run to validate the HTS assay and potentially to identify inhibitors of human DNA ligase IV suitable for re-purposing. Surprisingly, 28 compounds, including antifungals, chelates, cell cycle inhibitors and ion transport inhibitors were active in the pilot assay and further evaluated by re-screening, dose titration, and SDS-gel assay. Four compounds had low micromolar potency in these assays. In an in vitro AMP transfer assay, these compounds specifically blocked AMP transfer from DNA ligase IV-adenylate intermediates onto double-strand DNA ends. Ongoing studies of these potential inhibitors will determine their effects on DNA repair, and possible enhancement of radiation treatment and chemotherapy. This study serves as a proof-of-concept for full scale HTS with our 370,000 compound library and has already led to potential inhibitors for biologic study of the enzyme complex and its role in DSB repair and therapeutic approaches to cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3689.