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

描述(申请人提供)：同源重组(HR)系统负责修复DNA双链断裂(DSB)和链间交链(ICL)，这是最有害的DNA损伤。在酵母中，Rad52蛋白在HR中起着关键作用。相比之下，在哺乳动物中，Rad52基因敲除是可行的，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，RAD52的功能与BRCA2的功能重叠，并且RAD52基因的失活在人类BRCA2缺陷细胞中是致命的(冯等人，PNAS，2011)。BRCA2基因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RAD52的小分子抑制剂，以选择性地杀死BRCA2缺陷的乳腺癌细胞。这些抑制剂也将为分析RAD52提供一个有用的工具 在单元格中发挥作用。在体外，RAD52促进互补的单链DNA分子的退火。为了通过高通量筛选(HTS)确定RAD52 DNA退火酶活性的抑制物，我们建立了一种基于FRET的体外初步检测方法。该方法在MLPCN化合物文库(Z‘~0.85)的中试筛选中得到验证，该文库产生了5种推定的RAD52抑制剂(HITS)。已经开发了可靠的二级和三级检测方法来评估这些HITS的生物学意义。为了消除由于荧光干扰造成的假阳性，二次分析将使用DNA底物和一对不同于一次分析的荧光团。此外，使用放射性标记的DNA底物和凝胶电泳法的正交试验将被用来验证“真正的”命中。它的独特性 选定的抑制剂将使用结构上与RAD52无关的人类RAD51蛋白进行检测。已证实的RAD52抑制剂对BRCA2缺陷细胞(CAPAN-1)活性的影响将被测试。按优先顺序排列的抑制剂的结构活性关系(SAR)如下 开发的目的是提高它们的选择性和效力。在这项资助的继续，将使用几个第三级测试，包括RAD52 DNA结合，齐聚，和单链DNA退火法来研究所选化合物抑制RAD52的机制。优先化合物的治疗潜力将在免疫缺陷小鼠和移植的人类异种移植鼠身上进行检验。

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.