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

验证复制蛋白 A 的蛋白结合域作为癌症靶点

• 批准号: 8631295
• 负责人: STEPHEN W. FESIK
• 金额: $ 32.53万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8631295
• 关键词: Affect; Affinity; Antineoplastic Agents; Attenuated; Binding; Binding Proteins; Binding Sites; Biological; Biology; Breast; Breast Cancer Cell; Breast Cancer Treatment; Cancer Patient; Cancer cell line; Cell Line; Cell membrane; Cell physiology; Cells; Chemicals; Cisplatin; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA Replication Damage; DNA Structure; DNA damage checkpoint; Data; Defect; Future; Genetic; Germ Cells; Goals; Grant; Growth; Lead; Malignant - descriptor; Malignant Neoplasms; Mediating; Methods; Molecular; N-terminal; Oncogene Activation; Oncogenes; Pathway interactions; Peptides; Pharmaceutical Preparations; Phenotype; Process; Protein Binding; Protein Binding Domain; Proteins; Recruitment Activity; Risk; Role; SS DNA BP; Signal Transduction; Site; Small Interfering RNA; Stress; Structure; TREX1 gene; Technology; Testing; Therapeutic; Therapeutic Agents; Validation; base; biological adaptation to stress; cancer cell; cancer therapy; cell growth; cellular targeting; cytotoxic; cytotoxicity; design; drug discovery; high throughput screening; inhibitor/antagonist; innovation; lead series; malignant phenotype; new therapeutic target; novel; prevent; protein protein interaction; public health relevance; repaired; replication factor A; response; scaffold; small molecule; therapeutic target; tool; triple-negative invasive breast carcinoma; tumor

Project Summary 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. Interferring 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 test 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 signifance 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. !

项目摘要 从范德比尔特进行的siRNA筛选中，复制蛋白A（RPA）被鉴定为新靶点 三阴性乳腺癌（TNBC）在筛选中，RPA的敲低导致细胞生长减弱， 测试所有19种TNBC细胞系。RPA是异源三聚体单链（ss）DNA结合蛋白，由以下组成： 70、32和14 kDa亚基，对真核DNA复制、损伤反应和修复至关重要。 RPA作为DNA加工蛋白组装和启动DNA损伤的支架发挥作用 响应路径。同时，RPA作为ssDNA的保护层，通过防止 在复制焦点处的异常DNA结构。 在DNA加工过程中，RPA 70亚基的A、B和C结构域负责结合ssDNA。 RPA 70亚基的N-末端结构域与多种蛋白质相互作用，如p53、RAD 9、ATRIP、 和Mre 11，介导DNA损伤反应途径。通过阻塞干扰RPA 70 N的作用 70 N亚基募集DNA加工蛋白的能力应该对几种关键的DNA有影响 损伤反应途径，如p53介导的那些和ATR途径。此外，这种抑制 应该产生比siRNA筛选更有选择性的效果，因为RPA的ssDNA结合功能应该 不受影响。 使用传统方法抑制蛋白质-蛋白质相互作用是困难的。以便选择性 通过抑制RPA 70 N介导的蛋白质-蛋白质相互作用，我们提出发现一种有效的钉合螺旋， 肽探针和小分子探针。我们已经鉴定了候选钉合螺旋肽探针， 与RPA 70 N紧密结合并穿透细胞。此外，我们还发现了一种化合物， 使用基于NMR的片段筛选对RPA 70 N上的相邻位点进行了分析，并且还鉴定了跨越 使用高通量筛选两个结合位点。基于结构的优化产生了多个 以亚微摩尔亲和力结合RPA 70 N的先导化合物系列。在本提案中，我们的目标是 确认肽作为细胞探针的适用性，并获得有效的小分子探针， 适用于在广泛的细胞系中进行测试。我们将使用这些探针来检验选择性的假设， 抑制RPA 70 N介导的蛋白质-蛋白质相互作用是癌症治疗的有效方法。我们将 进一步确定这些抑制剂活性的分子决定因素，并确定其功能意义 RPA 70 N抑制。如果成功，我们将确定一种有希望的针对癌症的策略。在 此外，我们将产生有用和有效的探针分子，不仅可以用来使 对RPA生物学的进一步了解，也将进一步优化成治疗乳腺癌的药物 癌症和其他类型的肿瘤。 !