Identification of inhibitors of RAD54, an important DNA repair protein

描述(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DNA双链断裂(DSB)和链间交链(ICL)的修复，这是危害最大的DNA损伤。RAD54是同源重组的关键蛋白之一。RAD54基因敲除显著增加了小鼠对电离辐射和ICL诱导剂的敏感性。虽然RAD54的生化活性已被很好地描述，但其特定的细胞功能仍有待阐明。这项建议的目标是利用高通量化学文库筛选(HTS)开发特定的人类RAD54蛋白小分子抑制剂。特定的抑制剂为研究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诱导剂通常用于抗癌治疗，特异性RAD54抑制剂也可能有助于提高治疗效果。我们之前已经证明，RAD54利用ATP水解的能量促进关键的同源重组中间产物Holliday连接的分支迁移。Holliday连接的分支迁移是完成同源重组事件所需的重要步骤。为了通过HTS确定RAD54分支迁移活性的抑制剂，我们建立了一种基于FRET的体外初步检测方法。该分析在MLPCN化合物文库(Z‘&gt；0.8)的中试筛选中得到验证，该文库产生了10个试探性RAD54抑制剂(HITS)。为了评估HITS的生物学意义，已经开发了可靠的二级和三级检测方法。为了消除由于荧光干扰造成的假阳性，将使用与一次检测中不同的荧光基团的DNA底物进行二次检测。此外，使用放射性标记的Holliday连接底物和凝胶电泳法的正交分析将有助于确定“真正的”命中的优先顺序。我们已经证明了这两种检测方法在消除假阳性方面的有效性。将使用结构上与RAD54无关的人RAD51蛋白来检验所选抑制剂的特异性。基于细胞的系统将被用来确定确认的RAD54抑制剂对DNA修复、同源重组和细胞活力的影响。将开发优先排列的抑制剂的结构活性关系(SAR)，以提高它们的选择性和效力。在这项资助的继续，将使用几个三级分析方法来研究所选化合物抑制RAD54的机制，包括ATP水解、DNA结合、RAD54寡聚和DNA易位。优先考虑的化合物的治疗潜力将通过移植人类异种移植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.