Identification of inhibitors of RAD54, an important DNA repair protein

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DNA修复的过程。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基因敲除显著增加小鼠对电离辐射和icl诱导剂的敏感性。虽然RAD54的生化活性已被很好地表征，但其具体的细胞功能仍有待阐明。本研究的目的是利用化学文库的高通量筛选（HTS）技术开发人类RAD54蛋白的特异性小分子抑制剂。特异性抑制剂为研究RAD54在人细胞中的功能提供了有价值的工具。由于DSB-和ICL诱导剂常用于抗癌治疗，特异性RAD54抑制剂也可能有助于提高治疗效果。我们之前的研究表明，RAD54利用ATP水解的能量促进Holliday结（关键的同源重组中间体）的分支迁移。假日结点的分支迁移是完成同源重组事件所必需的重要步骤。为了通过HTS鉴定RAD54分支迁移活性的抑制剂，我们开发了一种体外基于fret的初级试验。该试验在MLPCN化合物库（Z' >0.8）的中试筛选中得到验证，产生了10个暂定的RAD54抑制剂（命中）。已经开发出可靠的二级和三级分析来评估撞击的生物学意义。为了消除由于荧光干扰引起的假阳性，将使用与初级分析中荧光团不同的DNA底物进行二级分析。此外，使用放射性标记的Holliday结底物和凝胶电泳的正交试验将有助于优先考虑“真正的”命中。我们已经证明了两种测定法在消除假阳性方面的效率。所选抑制剂的特异性将使用与RAD54结构无关的人RAD51蛋白进行检测。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。优先抑制剂的构效关系（SAR）将被开发，以提高其选择性和效力。为了继续这项资助，所选化合物抑制RAD54的机制将通过几个三级分析进行研究，包括ATP水解、DNA结合、RAD54寡聚化和DNA易位。优先化合物的治疗潜力将通过移植人类异种移植物的免疫缺陷小鼠来检验。

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.