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DNA双链断裂（DSB）和链间交叉链接（ICL）的修复，这是最有害的DNA病变。在酵母中，Rad52蛋白在HR中起关键作用。相反，在哺乳动物中，rad52敲除可行，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与BRCA2的功能重叠，而Rad52基因的失活在人BRCA2缺陷型细胞中是致命的（Feng等，PNAS，PNAS，2011）。 BRCA2基因中的突变负责夺取数百万人生命的家族性乳腺癌。我们将通过开发RAD52的小分子抑制剂来利用这一出色的发现，以选择性地杀死BRCA2缺陷型乳腺癌细胞。抑制剂还将为RAD52分析提供有用的工具在单元格中的功能。在体外，RAD52促进了互补ssDNA分子的退火。为了通过高吞吐量筛选（HTS）鉴定RAD52 DNA退火活性的抑制剂，我们开发了一种基于体外的基于FRET的一级测定。该测定在MLPCN化合物库（Z'〜0.85）的试验屏幕上进行了验证，该库产生了五个推定的RAD52抑制剂（hits）。已经开发了强大的二级和三级测定，以评估这些命中的生物学意义。为了消除由于荧光干扰而引起的假阳性，将使用次要测定，该测定法使用与一对荧光团不同的DNA底物，其与主要测定法不同。此外，使用放射性标记的DNA底物和凝胶电泳的正交测定将用于验证“ True”命中。特异性将使用与RAD52无关的人Rad51蛋白来检查选定的抑制剂。确认的RAD52抑制剂对BRCA2缺陷细胞的生存能力（CAPAN-1）的影响。优先抑制剂的结构活动关系（SAR）将是开发以提高其选择性和效力。在继续该赠款的持续下，将使用几种第三次分析，包括RAD52 DNA结合，低聚和SSDNA退火来研究所选化合物的RAD52抑制机理。将使用具有移植的人异种移植物的免疫缺陷型小鼠检查优先化合物的治疗潜力。