Validating the protein binding domain of replication protein A as a cancer target

描述(申请人提供)：在Vanderbilt进行的siRNA筛查中，复制蛋白A(RPA)被鉴定为三阴性乳腺癌(TNBC)的新靶点。在筛选中，RPA被敲除导致所有19个被测试的TNBC细胞系的细胞生长减弱。RPA是一种由70、32和14 kDa亚基组成的异三聚体单链DNA结合蛋白，对真核细胞的DNA复制、损伤反应和修复是必不可少的。RPA作为DNA加工蛋白组装和启动DNA损伤反应通路的支架发挥作用。同时，RPA作为单链DNA的保护层，防止在复制中心形成异常的DNA结构。在DNA加工过程中，RPA70亚单位的A、B和C结构域负责与ssDNA结合。RPA70亚基的N-末端结构域与多种蛋白质相互作用，如P53、RAD9、ARIP和Mre11，这些蛋白质介导DNA损伤反应通路。通过阻断70N亚基招募DNA加工蛋白的能力来干扰RPA70N的作用，应该会对几个关键的DNA损伤反应途径产生影响，如由p53和ATR途径介导的那些。此外，这种抑制应该比siRNA筛选产生更具选择性的效果，因为RPA的ssDNA结合功能不应该受到影响。用传统的方法很难抑制蛋白质之间的相互作用。为了选择性地抑制RPA70N介导的蛋白质-蛋白质相互作用，我们建议同时发现一个有效的钉合螺旋多肽探针和一个小分子探针。我们已经确定了一种与RPA70N紧密结合并穿透细胞的候选螺旋多肽探针。此外，我们使用基于核磁共振的片段筛选鉴定了与RPA70N上两个相邻位点结合的化合物，并使用高通量筛选鉴定了跨越两个结合位点的分子。基于结构的优化已经产生了多个先导系列化合物，它们以亚微摩尔亲和力与RPA70N结合。在这项提案中，我们的目标是确认多肽作为细胞探针的适用性，并获得适合在广泛的细胞系中进行测试的有效的小分子探针。我们将使用这些探针来验证选择性抑制RPA70N介导的蛋白质-蛋白质相互作用是癌症治疗的有效方法的假设。我们将进一步定义这些抑制剂活性的分子决定因素，并确定RPA70N抑制的功能意义。如果成功， 我们将确定一种有前景的针对癌症的策略。此外，我们还将生产出有用和有效的探针分子，这些分子不仅可以用来进一步了解RPA生物学，而且还可以进一步优化为治疗乳腺癌和其他肿瘤类型的药物。

DESCRIPTION (provided by applicant): From a siRNA screen performed at Vanderbilt, replication protein A (RPA) was identified as a novel target for triple negative breast cancer (TNBC). In the screen, knockdown of RPA resulted in attenuated cell growth of all 19 TNBC cell lines tested. RPA is a heterotrimeric single-stranded (ss) DNA-binding protein composed of 70, 32, and 14 kDa subunits and is essential for eukaryotic DNA replication, damage response, and repair. RPA functions as a scaffold upon which DNA processing proteins assemble and initiate DNA damage response pathways. Concurrently, RPA serves as a protective layer for ssDNA by preventing formation of aberrant DNA structures at replication foci. During DNA processing, the A, B, and C domains of the RPA70 subunit are responsible for binding ssDNA. The N-terminal domain of the RPA70 subunit interacts with a variety of proteins, such as p53, RAD9, ATRIP, and Mre11, that mediate DNA damage response pathways. Interfering with the action of RPA70N by blocking the ability of the 70N subunit to recruit DNA processing proteins should have an effect on several critical DNA damage response pathways, such as those mediated by p53 and the ATR pathway. Further, this inhibition should produce a more selective effect than the siRNA screen, as the ssDNA binding functions of RPA should not be affected. The inhibition of protein-protein interactions is difficult using traditional methods. In order to selectively inhibit RPA70N-mediated protein-protein interactions, we propose to discover both a potent stapled helix peptide probe and a small molecule probe. We have identified a candidate stapled helix peptide probe that binds tightly to RPA70N and penetrates cells. In addition, we have identified compounds that bind to two adjacent sites on RPA70N using an NMR-based fragment screen, and have also identified molecules that span the two binding sites using a high throughput screen. Structure-based optimizations have produced multiple lead series of compounds that bind to RPA70N with sub-micromolar affinities. In this proposal, our goal is to confirm the suitability of the peptide as a cellular probe and to obtain potent small molecule probes that are suitable for testing in a wide panel of cell lines. We will use these probes to tes the hypothesis that selective inhibition of RPA70N-mediated protein-protein interactions is a valid approach for cancer therapy. We will further define the molecular determinants of activity for these inhibitors and determine the functional significance of RPA70N inhibition. If successful, we will have identified a promising strategy for targeting cancer. In addition, we will have produced useful and potent probe molecules that could not only be used to enable further understanding of RPA biology, but also be further optimized into a drug for the treatment of breast cancer and other tumor types.