Identification of inhibitors of RAD54, an important DNA repair protein

重要 DNA 修复蛋白 RAD54 抑制剂的鉴定

• 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
• 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

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。同源重组系统负责修复 DNA 双链断裂 (DSB) 和链间交联 (ICL)，这是最有害的 DNA 损伤。 RAD54是同源重组的关键蛋白之一。 RAD54 敲除显着增加小鼠对电离辐射和 ICL 诱导剂的敏感性。虽然 RAD54 的生化活性已得到很好的表征，但其特定的细胞功能仍有待阐明。该提案的目标是利用化学库的高通量筛选 (HTS) 开发人类 RAD54 蛋白的特异性小分子抑制剂。特定抑制剂为研究 RAD54 在人类细胞中的功能提供了一个有价值的工具。由于 DSB 和 ICL 诱导剂常用于抗癌治疗，因此特定的 RAD54 抑制剂也可能有助于提高治疗效果。 我们之前表明，RAD54 利用 ATP 水解的能量促进关键同源重组中间体霍利迪连接体的分支迁移。霍利迪连接体的分支迁移构成了完成同源重组事件所需的重要步骤。为了通过 HTS 鉴定 RAD54 分支迁移活性的抑制剂，我们开发了一种基于体外 FRET 的初级测定。该测定在 MLPCN 化合物库 (Z' >0.8) 的初步筛选中得到验证，产生了 10 种试验性 RAD54 抑制剂（命中）。已经开发出稳健的二级和三级测定法来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，将使用与初级测定中荧光团不同的 DNA 底物进行二级测定。此外，使用放射性标记霍利迪连接底物和凝胶电泳的正交测定将有助于优先考虑“真实”命中。我们已经证明了两种检测方法在消除假阳性方面的效率。将使用结构上与 RAD54 无关的人 RAD51 蛋白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。将开发优先抑制剂的结构活性关系（SAR），以提高其选择性和效力。在这项资助的延续中，将使用几种三级测定法来研究所选化合物抑制 RAD54 的机制，包括 ATP 水解、DNA 结合、RAD54 寡聚化和 DNA 易位。将使用移植了人类异种移植物的免疫缺陷小鼠来检查优先化合物的治疗潜力。