Small molecule inhibitors as a new approach to study human RAD51 recombinase

 描述(申请人提供)：同源重组(HR)途径在修复最有害的DNA损伤、DNA双链断裂(DSB)和链间交链(ICL)中发挥关键作用。HR的故障会导致基因组不稳定、癌症和遗传性疾病。RAD51是HR的一个关键蛋白，它促进作为HR的基本步骤的DNA链交换。然而，RAD51在人类细胞中的DNA链交换机制和特定功能仍有待阐明。传统的遗传方法很难应用，因为RAD51对细胞活力是必不可少的。在这里，我们建议开发小分子抑制剂来研究RAD51的活性和功能。这些抑制物也可能被用作开发联合疗法的原型，以使癌细胞对放射和化疗敏感。由于数以百万计的癌症患者通常接受放射治疗和化疗，其疗效的提高将对公共医疗保健产生重大影响。能够干扰蛋白质特定功能的小分子化合物(Mw&lt；500 Da)成为现代生物学的重要工具。小分子抑制剂的作用速度很快，而且往往是可逆的。它们可以在生物体或细胞发育的任何时间点引入，并以剂量依赖的方式应用，这在细胞存活所必需的蛋白质研究中尤其重要，如RAD51。此前，通过高通量筛选，我们鉴定了几种RAD51的小分子抑制剂，并首次证明了它们在细胞中的生物活性。在这里，我们将使用这些抑制剂来分析RAD51 DNA链交换的机制以及RAD51在人类细胞中的功能。利用细胞和动物模型，我们将探索RAD51抑制剂单独或与其他DNA修复途径的抑制剂联合使用增加化疗药物对癌细胞的杀伤力的能力。

 DESCRIPTION (provided by applicant): The homologous recombination (HR) pathway plays a critical role in the repair of the most harmful DNA lesions, DNA double-strand breaks (DSB) and inter-strand cross-links (ICL). Malfunction of HR causes genome instability, cancer, and genetic diseases. RAD51, a key HR protein, promotes DNA strand exchange, a basic step of HR. However, the mechanism of DNA strand exchange and specific functions of RAD51 in human cells remain to be elucidated. Traditional genetic approaches are difficult to apply because RAD51 is essential for cell viability. Here, we propose to develop small molecule inhibitors to study RAD51 activities and functions. These inhibitors may also be used as prototypes for development of combination therapies to sensitize cancer cells for radiation and chemotherapy. Because millions of cancer patients commonly undergo radiation therapy and chemotherapy, improvement of their efficacy will have a significant impact on the public healthcare. Small molecule compounds (MW < 500 Da) that can perturb specific functions of proteins became an important tool in modern biology. Small molecule inhibitors act rapidly and often reversibly. They can be introduced at any point of organism or cell development and applied in a dose-dependent manner, which is especially important in studies of proteins essential for cell viability, like RAD51. Previously, by high throughput screening we identified several small molecule inhibitors of RAD51 and demonstrated for the first time their biological activity in the cell. Here, we will use these inhibitors to analyze the mechanism of RAD51 DNA strand exchange and the functions of RAD51 in human cells. Using cellular and animal models we will explore the ability of the RAD51 inhibitors alone or in combination with inhibitors of othe DNA repair pathways to increase killing of cancer cells by chemotherapeutic agents.