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。RAD 54是同源重组的关键蛋白之一。RAD 54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RAD 54的生物化学活性已被很好地表征，但其特定的细胞功能仍有待阐明。本发明的目的是利用化学文库的高通量筛选（HTS）来开发人RAD 54蛋白的特异性小分子抑制剂。特异性抑制剂为研究RAD 54在人类细胞中的功能提供了有价值的工具。由于DSB-和ICL-诱导剂通常用于抗癌治疗，特异性RAD 54抑制剂也可能有助于提高治疗效果。 我们以前表明，RAD 54促进分支迁移霍利迪连接，关键同源重组中间体，使用ATP水解的能量。霍利迪连接的分支迁移构成完成同源重组事件所需的重要步骤。为了通过HTS鉴定RAD 54分支迁移活性的抑制剂，我们开发了基于FRET的体外初步测定。在MLPCN化合物文库（Z' >0.8）的中试筛选中验证了该测定法，其产生了10种试验性的RAD 54抑制剂（命中）。已经开发了稳健的二级和三级测定来评价命中的生物学意义。为了消除由于荧光干扰导致的假阳性，将使用二次试验，该二次试验使用的DNA底物的荧光团与一次试验中的荧光团不同。此外，使用放射性标记的霍利迪连接基板和凝胶电泳的正交试验将有助于优先考虑“真正的”命中。我们已经证明了这两种检测在消除假阳性方面的效率。所选抑制剂的特异性将使用结构上与RAD 54无关的人RAD 51蛋白进行检查。将使用基于细胞的系统来确定经确认的RAD 54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。在继续这一授权，RAD 54抑制选定的化合物的机制将使用几个三级检测，包括ATP水解，DNA结合，RAD 54寡聚化，和DNA易位进行研究。将使用移植有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。

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.