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Development of RAD52 Inhibitors to Induce Lethality of BRCA2-Deficient Cells

开发 RAD52 抑制剂以诱导 BRCA2 缺陷细胞致死

基本信息

批准号：
8441581
负责人：
ALEXANDER V MAZIN
金额：
$ 3.75万
依托单位：
DREXEL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-12 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8441581
关键词：
Affect Biochemical Biological Biological Assay Biological Models Breast Cancer Cell Cancer Patient Cell Line Cell physiology Cells Chemistry Collaborations DNA DNA Binding DNA Double Strand Break DNA Repair DNA annealing DNA lesion Development Fluorescein Fluorescence Fluorescence Resonance Energy Transfer Future Genes Genetic Recombination Grant Hereditary Breast Carcinoma Human In Vitro Institutes Knock-out Label Letters Libraries Malignant Neoplasms Malignant neoplasm of ovary Mammals Morphologic artifacts Mus Mutate Mutation Normal Cell Pathway interactions Phase Phenotype Play Proteins RAD52 homolog (S. cerevisiae) protein, human Rad51 recombinase Rad52 protein Reporting Specificity Structure-Activity Relationship System Testing Therapeutic Transplantation United States National Institutes of Health Universities Woman Xenograft procedure Yeasts base cancer cell crosslink cyanine dye 5 fluorophore gel electrophoresis genetically modified cells high throughput screening homologous recombination inhibitor/antagonist killings lifetime risk malignant breast neoplasm member recombinational repair repaired repository small molecule tool tumor

项目摘要

DESCRIPTION (provided by applicant): The system of homologous recombination (HR) is responsible for the repair of DNA double-stranded breaks (DSB) and inter-strand cross-links (ICL), the most harmful DNA lesions. In yeast, Rad52 protein plays a key role in HR. In contrast, in mammals Rad52 knockouts are viable and show no distinct DNA repair and recombination phenotype. However, recently it was discovered that in mammals the function of RAD52 overlaps with that of BRCA2 and that inactivation of the RAD52 gene is lethal in human BRCA2-deficient cells (Feng et al., PNAS, 2011). Mutations in the BRCA2 gene are responsible for familial breast cancer that claims millions of lives. We will take advantage of this remarkable discovery by developing small-molecule inhibitors of RAD52 in order to selectively kill BRCA2-deficient breast cancer cells. The inhibitors will also present a useful tool for analysis of RAD52 function in the cell. In vitro, RAD52 promotes annealing of complementary ssDNA molecules. In order to identify inhibitors of the RAD52 DNA annealing activity by high throughput screening (HTS) we developed an in vitro FRET- based primary assay. The assay was validated in a pilot screen of the MLPCN compound library (Z' ~0.85) that yielded five putative RAD52 inhibitors (hits). Robust secondary and tertiary assays have been developed to evaluate the biological significance of these hits. To eliminate false positives due to fluorescence interference, the secondary assay will be used that employs DNA substrates with a pair of fluorophores that are different than in the primary assay. Additionally, an orthogonal assay using radioactively-labeled DNA substrates and gel-electrophoresis will be used to validate "true" hits. The specificity of the selected inhibitors will be examined using human RAD51 protein that is structurally unrelated to RAD52. The effect of confirmed RAD52 inhibitors on viability of BRCA2-deficient cells (Capan-1) will be tested. The Structure Activity Relationships (SAR) of the prioritized inhibitors will be developed to increase their selectivity and potency. In continuation of this grant, the mechanisms of RAD52 inhibition by the selected compounds will be investigated using several tertiary assays including RAD52 DNA binding, oligomerization, and ssDNA annealing. The therapeutic potential of the prioritized compounds will be examined using immuno-deficient mice with transplanted human xenografts.

描述（由申请人提供）：同源重组（HR）系统负责修复DNA双链断裂（DSB）和链间交联（ICL），这是最有害的DNA损伤。在酵母中，Rad52蛋白在HR中起关键作用。相比之下，在哺乳动物中，Rad52基因敲除是可行的，并且没有明显的DNA修复和重组表型。然而，最近发现，在哺乳动物中RAD52的功能与BRCA2的功能重叠，RAD52基因失活对人类BRCA2缺陷细胞是致命的（Feng et al.， PNAS, 2011）。BRCA2基因的突变是导致家族性乳腺癌的原因，它夺去了数百万人的生命。我们将利用这一非凡的发现，开发RAD52的小分子抑制剂，以选择性地杀死brca2缺失的乳腺癌细胞。这些抑制剂也将为RAD52的分析提供一个有用的工具