Identification of inhibitors of RAD54, an important DNA repair protein

重要 DNA 修复蛋白 RAD54 抑制剂的鉴定

基本信息

批准号：
8262294
负责人：
ALEXANDER V MAZIN
金额：
$ 3.86万
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：
2012-02-15 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8262294
关键词：
ATP Hydrolysis Apoptosis Biochemical Biological Biological Assay Biological Models Cell Survival Cell physiology Cells Chemicals Chemistry Cisplatin Collaborations Cruciform DNA DNA DNA Binding DNA Damage DNA Double Strand Break DNA Repair DNA lesion DNA repair protein Development Eukaryota Event Fluorescence Fluorescence Resonance Energy Transfer Future Genes Genetic Genomic Instability Goals Grant Human Human Activities Immune In Vitro Institutes Ionizing radiation Knock-out Knockout Mice Label Lead Letters Libraries Malignant Neoplasms Mammals Morphologic artifacts Mus Mutation Normal Cell Pathway interactions Phase Phenotype Predisposition Process Property Proteins Rad51 recombinase Reporting Small Interfering RNA Specificity Structure-Activity Relationship System Testing Therapeutic Transplantation Treatment Efficacy United States National Institutes of Health Xenograft procedure Yeasts base cancer cell cancer therapy crosslink fluorophore gel electrophoresis high throughput screening homologous recombination inhibitor/antagonist migration mutant repaired repository small molecule small molecule libraries tool

ATP Hydrolysis Apoptosis Biochemical Biological Biological Assay Biological Models Cell Survival Cell physiology Cells Chemicals Chemistry Cisplatin Collaborations Cruciform DNA DNA DNA Binding DNA Damage DNA Double Strand Break DNA Repair DNA lesion DNA repair protein Development Eukaryota Event Fluorescence Fluorescence Resonance Energy Transfer Future Genes Genetic Genomic Instability Goals Grant Human Human Activities Immune In Vitro Institutes Ionizing radiation Knock-out Knockout Mice Label Lead Letters Libraries Malignant Neoplasms Mammals Morphologic artifacts Mus Mutation Normal Cell Pathway interactions Phase Phenotype Predisposition Process Property Proteins Rad51 recombinase Reporting Small Interfering RNA Specificity Structure-Activity Relationship System Testing Therapeutic Transplantation Treatment Efficacy United States National Institutes of Health Xenograft procedure Yeasts base cancer cell cancer therapy crosslink fluorophore gel electrophoresis high throughput screening homologous recombination inhibitor/antagonist migration mutant repaired repository small molecule small molecule libraries tool

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项目摘要

DESCRIPTION (provided by applicant): Our long-term goal is to understand the process of DNA repair promoted by the system of homologous recombination in human cells. The system of homologous recombination is responsible for the repair of DNA double-stranded breaks (DSB) and inter-strand cross-links (ICL), the most harmful DNA lesions. RAD54 is one of the key proteins of homologous recombination. RAD54 knock-outs significantly increase mice sensitivity to ionizing radiation and ICL-inducing agents. While the biochemical activities of RAD54 are well characterized, its specific cellular functions remain to be elucidated. The goal of this proposal is to develop specific small- molecule inhibitors of human RAD54 protein using high throughput screening (HTS) of chemical libraries. Specific inhibitors present a valuable tool to study RAD54 functions in human cells. Since DSB- and ICL- inducing agents are commonly used in anticancer therapy, specific RAD54 inhibitors may also help to increase the therapy efficacy. We previously showed that RAD54 promotes branch migration of Holliday junctions, key homologous recombination intermediates, using the energy of ATP hydrolysis. Branch migration of Holliday junctions constitutes an important step that is required for completion of homologous recombination events. In order to identify inhibitors of the RAD54 branch migration activity by HTS we developed an in vitro FRET-based primary assay. The assay was validated in the pilot screen of the MLPCN compound library (Z' >0.8) that yielded ten tentative RAD54 inhibitors (hits). Robust secondary and tertiary assays have been developed to evaluate the biological significance of hits. To eliminate false positives due to fluorescence interference, a secondary assay using DNA substrates with a different fluorophore than that in the primary assay will be used. Additionally, an orthogonal assay using radioactively-labeled Holliday junction substrates and gel-electrophoresis will help to prioritize "true" hits. We have demonstrated the efficiency of both assays in elimination of false positives. The specificity of th selected inhibitors will be examined using human RAD51 protein that is structurally unrelated to RAD54. Cell-based systems will be used to determine the effect of confirmed RAD54 inhibitors on DNA repair, homologous recombination, and cell viability. 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 RAD54 inhibition by the selected compounds will be investigated using several tertiary assays including ATP hydrolysis, DNA binding, RAD54 oligomerization, and DNA translocation. The therapeutic potential of the prioritized compounds will be examined using immune-deficient mice with transplanted human xenografts.

描述（由申请人提供）：我们的长期目标是了解人类细胞中同源重组系统促进的DNA修复过程。同源重组系统负责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。 RAD54是同源重组的关键蛋白质之一。 RAD54敲除显着提高小鼠对电离辐射和ICL诱导剂的敏感性。虽然RAD54的生化活性表征得很好，但其特异性细胞功能仍有待阐明。该提案的目的是使用化学文库的高吞吐量筛选（HTS）开发特定的小分子抑制剂。特异性抑制剂提出了研究人类细胞中RAD54功能的宝贵工具。由于DSB和ICL-诱导剂通常用于抗癌治疗中，因此特定的RAD54抑制剂也可能有助于提高治疗疗效。我们先前表明，使用ATP水解的能量，RAD54促进了霍利迪连接，关键同源重组中间体的分支迁移。霍利迪连接的分支迁移构成了完成同源重组事件所必需的重要步骤。为了鉴定HTS的RAD54分支迁移活性的抑制剂，我们开发了基于体外FRET的主要测定法。该测定在MLPCN化合物库（Z'> 0.8）的试验屏幕上进行了验证，该库产生了十个暂定的Rad54抑制剂（hits）。已经开发出强大的二级和第三次测定，以评估命中的生物学意义。为了消除由于荧光干扰而导致的假阳性，将使用使用与主要测定法不同的DNA底物的次级测定。此外，使用放射标记的Holliday结底物和凝胶电泳的正交测定将有助于优先考虑“ True”命中率。我们已经证明了这两种测定法在消除误报方面的效率。将使用与RAD54无关的人Rad51蛋白来检查所选抑制剂的特异性。基于细胞的系统将用于确定已确认的RAD54抑制剂对DNA修复，同源重组和细胞活力的影响。优先抑制剂的结构活动关系（SAR）将开发出来，以提高其选择性和效力。在持续此授予的过程中，将使用几种第三次分析，包括ATP水解，DNA结合，RAD54低聚和DNA易位，研究所选化合物抑制RAD54的机制。将使用具有移植的人异种移植物的免疫缺陷小鼠检查优先化合物的治疗潜力。