A High-Throughput Assay for DNA Repair Activity in the Presence of AberrantBRCA1

描述（由申请人提供）：当DNA损伤超过DNA修复时，肿瘤发生。DNA损伤最常见的形式是内源性氧化损伤，可通过碱基切除修复（BER）、核苷酸切除修复（NER）、同源重组（HR）和/或非同源末端连接（NHEJ）修复。BRCA1是一种220kD的肿瘤抑制蛋白，通过其防止DNA氧化损伤和促进DNA修复（包括BER、NER、HR和NHEJ）的能力来维持遗传稳定性，与预防乳腺癌有关。BRCA1突变或失活后，DNA过度损伤导致突变积累和遗传不稳定，最终形成BRCA1相关的恶性肿瘤。BRCA1相关的恶性肿瘤包括由于BRCA1突变而导致的遗传性乳腺癌和卵巢癌，由于BRCA1的表观遗传沉默而导致的散发性乳腺癌，以及具有基底样表型（即ER/PR/HER2三阴性）的乳腺癌亚群，其表达谱与BRCA1突变的癌症相似。它们占乳腺癌病例的30-50%。这些恶性肿瘤通常对目前的化学预防药物没有反应，这表明需要有效的预防策略。这项研究将开启一种新的、有针对性的方法来预防brca1相关的恶性肿瘤。假设认为：高通量（HT）筛选化合物文库可以识别在存在异常BRCA1的情况下激活DNA氧化损伤修复的小分子。然后，先导化合物可用于识别brca1相关恶性肿瘤的潜在化学预防剂或作为基础科学研究的工具。以下具体目标将被解决：(1)验证高通量格式的DNA修复测定。(2)使用DNA修复试验进行定量高通量先导筛选，(3)使用氧化DNA损伤反应试验进行定量高通量计数筛选。首先，将开发一种基于细胞的检测方法，该方法使用在GFP报告基因编码区内含有氧化DNA损伤的腺病毒来监测宿主细胞再激活对DNA修复的激活，用于ht筛选。因此，将确定最佳细胞生长和处理条件，并以384孔格式进行DNA修复试验的板验收。其次，一个小的、多样化的化合物文库和定量高通量筛选格式将用于进行试点筛选和确定试验的可重复性。最后，我们将适应并验证高通量格式的H2O2敏感性分析，在定量高通量先导筛选中测试该分析，并使用DNA修复和H2O2敏感性分析比较从先导筛选中获得的命中值。总之，DNA修复试验将被开发、调整和验证，用于高通量筛选化合物文库，以识别brca1相关恶性肿瘤的潜在化学预防药物。

DESCRIPTION (provided by applicant): Tumorigenesis arises when DNA damage exceeds DNA repair. The most common form of DNA damage is endogenous oxidative damage, which is repaired by base excision repair (BER), and in some cases nucleotide-excision repair (NER), homologous recombination (HR) and/or non- homologous end-joining (NHEJ). BRCA1, a 220kD tumor suppressor protein, has been implicated in preventing breast cancer by maintaining genetic stability through its ability to prevent oxidative DNA damage and promote DNA repair, including BER, NER, HR, and NHEJ. Upon mutation or inactivation of BRCA1, excessive DNA damage leads to the accumulation of mutations and genetic instability, and ultimately formation of BRCA1-associated malignancies. BRCA1-associated malignancies include hereditary breast and ovarian cancers due to mutations in BRCA1, sporadic breast cancers due to epigenetic silencing of BRCA1, and a subset of breast cancers with the basal-like phenotype (i.e. triple-negative for ER/PR/HER2), which have a similar expression profile to cancers with mutations in BRCA1. They comprise 30-50% of breast cancer cases. These malignancies often do not respond to current chemoprevention agents, suggesting a need for effective prevention strategies. This study will initiate a novel, targeted approach to preventing BRCA1-associated malignancies. The hypothesis states: high-throughput (HT) screening of a compound library may identify small molecules that activate DNA repair of oxidative DNA damage in the presence of aberrant BRCA1. Lead compounds can then be used to identify potential chemoprevention agents for BRCA1-associated malignancies or as tools in basic science research. The following specific aims will be addressed: (1) validate a DNA repair assay for high- throughput format., (2) carry out a quantitative high-throughput pilot screen using a DNA repair assay, and (3) carry out a quantitative high-throughput counter screen using an oxidative DNA damage response assay. First, a cell-based assay that uses adenovirus containing oxidative DNA damage within the coding region of a GFP reporter gene to monitor for activation of DNA repair by host-cell reactivation will be developed for HT-screening. Therefore, optimal cell growth and treatment conditions will be determined, and plate acceptance for the DNA repair assay will be carried out in 384-well format. Second, a small, diverse compound library and the quantitative high-throughput screening format will be used to carry out a pilot screen and to determine assay reproducibility. Finally, we will adapt and validate an H2O2 sensitivity assay for high-throughput format, test the assay in a quantitative high-throughput pilot screen, and compare the hits obtained from the pilot screens using the DNA repair and H2O2 sensitivity assays. Overall, a DNA repair assay will be developed, adapted, and validated for high-throughput screening of a compound library to identify potential chemoprevention agents for BRCA1-associated malignancies. PUBLIC HEALTH RELEVANCE: We will initiate the discovery of potential drugs for preventing a subset of breast cancer known as BRCA1-associated malignancies. Furthermore, this study may lead to advances in treating other diseases that result from oxidative DNA damage (e.g. repair-deficient cancers, degenerative diseases, and various sclerotic diseases), and may generate tools for basic science research.