Development of RAD52 Inhibitors to Induce Lethality of BRCA2-Deficient Cells

描述（由申请人提供）：同源重组（HR）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。在酵母中，Rad 52蛋白在HR中起关键作用。相反，在哺乳动物中，Rad 52敲除是可行的，并且没有显示出明显的DNA修复和重组表型。然而，最近发现，在哺乳动物中，RAD 52的功能与BRCA 2的功能重叠，并且RAD 52基因的失活在人BRCA 2缺陷型细胞中是致命的（Feng et al.，PNAS，2011）。BRCA 2基因的突变是导致数百万人死亡的家族性乳腺癌的原因。我们将利用这一非凡的发现，开发小分子的RAD 52抑制剂，以选择性地杀死BRCA 2缺陷的乳腺癌细胞。这些抑制剂也将为分析RAD 52提供有用的工具 在细胞中发挥作用。 在体外，RAD 52促进互补ssDNA分子的退火。为了通过高通量筛选（HTS）鉴定RAD 52 DNA退火活性的抑制剂，我们开发了基于FRET的体外初步测定。在MLPCN化合物文库的中试筛选（Z' ~0.85）中验证了该测定法，其产生了五种推定的RAD 52抑制剂（命中）。已经开发了稳健的二级和三级测定来评价这些命中的生物学意义。为了消除由于荧光干扰导致的假阳性，将使用二次试验，该二次试验采用具有一对不同于一次试验的荧光团的DNA底物。此外，使用放射性标记的DNA底物和凝胶电泳的正交试验将用于验证“真实”命中。的特异性 将使用结构上与RAD 52无关的人RAD 51蛋白来检查所选的抑制剂。将测试经确认的RAD 52抑制剂对BRCA 2缺陷型细胞（Capan-1）的活力的影响。优先抑制剂的结构活性关系（SAR）将是 以提高其选择性和效力。在继续这一授权，RAD 52抑制所选化合物的机制将使用几个三级测定，包括RAD 52 DNA结合，寡聚化，和ssDNA退火进行研究。将使用具有移植的人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。

DESCRIPTION (provided by applicant): The system of homologous recombination (HR) is responsible for the repair of DNA double-stranded breaks (DSB) and inter-strand cross-links (ICL), the most harmful DNA lesions. In yeast, Rad52 protein plays a key role in HR. In contrast, in mammals Rad52 knockouts are viable and show no distinct DNA repair and recombination phenotype. However, recently it was discovered that in mammals the function of RAD52 overlaps with that of BRCA2 and that inactivation of the RAD52 gene is lethal in human BRCA2-deficient cells (Feng et al., PNAS, 2011). Mutations in the BRCA2 gene are responsible for familial breast cancer that claims millions of lives. We will take advantage of this remarkable discovery by developing small-molecule inhibitors of RAD52 in order to selectively kill BRCA2-deficient breast cancer cells. The inhibitors will also present a useful tool for analysis of RAD52 function in the cell. In vitro, RAD52 promotes annealing of complementary ssDNA molecules. In order to identify inhibitors of the RAD52 DNA annealing activity by high throughput screening (HTS) we developed an in vitro FRET- based primary assay. The assay was validated in a pilot screen of the MLPCN compound library (Z' ~0.85) that yielded five putative RAD52 inhibitors (hits). Robust secondary and tertiary assays have been developed to evaluate the biological significance of these hits. To eliminate false positives due to fluorescence interference, the secondary assay will be used that employs DNA substrates with a pair of fluorophores that are different than in the primary assay. Additionally, an orthogonal assay using radioactively-labeled DNA substrates and gel-electrophoresis will be used to validate "true" hits. The specificity of the selected inhibitors will be examined using human RAD51 protein that is structurally unrelated to RAD52. The effect of confirmed RAD52 inhibitors on viability of BRCA2-deficient cells (Capan-1) will be tested. 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 RAD52 inhibition by the selected compounds will be investigated using several tertiary assays including RAD52 DNA binding, oligomerization, and ssDNA annealing. The therapeutic potential of the prioritized compounds will be examined using immuno-deficient mice with transplanted human xenografts.