Discovery of protein-protein interaction inhibitors of Replication Protein A

复制蛋白 A 的蛋白质-蛋白质相互作用抑制剂的发现

• 批准号: 8456589
• 负责人: James Patrone
• 金额: $ 5.22万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456589
• 关键词: Affinity; Apoptosis; Binding; Binding Proteins; Binding Sites; Biological Assay; Breast Cancer Cell; Cancer cell line; Cell Cycle; Cell Cycle Checkpoint; Cell Death; Cells; Chemicals; Critical Pathways; DNA Damage; DNA Repair Pathway; Digit structure; Eukaryotic Cell; G2/M Checkpoint Pathway; Individual; Lead; Libraries; Link; Malignant Neoplasms; Molecular; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Play; Property; Protein Binding; Protein Inhibition; Proteins; Recruitment Activity; Role; Site; Small Interfering RNA; Structure; TREX1 gene; Techniques; Testing; base; cancer cell; cancer therapy; cytotoxicity; drug discovery; flexibility; inhibitor/antagonist; member; novel; protein protein interaction; public health relevance; repaired; replication factor A; research study; response; triple-negative invasive breast carcinoma

DESCRIPTION (provided by applicant): Due to the high rate of DNA damage within the cell cycle, DNA damage response and repair pathways are critical for the survival of eukaryotic cells. Beyond the survival of healthy cells, DNA repair pathways also play a role in the survival of cancer cells. The inhibition of these repair proteins represent a potential strategy for cancer therapy. The inhibition of these repair proteins represent a potential strategy for cancer therapy. Identifying and validating different targets within DNA damage response pathways is a strategy for attaining novel cancer targets from a validated paradigm. Replication protein A (RPA) was identified as a potential novel target for triple negative breast cancer (TNBC) from a siRNA screen of 20 TNBC cell lines performed by the Cortez group at Vanderbilt. RPA is a ssDNA binding protein responsible for recruiting and initiating multiple DNA damage response proteins such as Rad9, MRE11, and ATRIP and as such serves as both a G1/S and G2/M cell cycle checkpoint. Our hypothesis is that the inhibition of the protein-protein binding interface of RPA70N should allow for the blockage DNA damage response pathways and thus induce apoptosis in cancer cells. Due to the inherent difficulties with inhibiting protein-protein interactions, a fragment-based discovery approach will be undertaken in order to discover a selective inhibitor of the protein-protein binding interface of RPA70N. We will be using the technique of "SAR by NMR" to discover the lead molecule of appropriate potency. A 15,000 member fragment library has been screened using 1H/15N HSQC. We have identified 150 fragment hits from our library and currently have eleven co-crystal structures of molecules bound to RPA70N. Small libraries of the fragment hits will be generated and tested via 1H/15N HSQC to establish the SAR of the fragment molecules. Once the fragments have been optimized, we will link the two fragments together using a small subset of flexible linkers to generate a lead molecule. The lead molecule will be further optimized using structural information to fill any and all unoccupied binding nooks and crannies on RPA70N. Simultaneously, the pharmaceutical properties of the lead molecule will be taken into consideration and optimized. The lead molecule of sufficient potency and pharmaceutical properties will be used to validate our hypothesis that the inhibition of protein-protein interactions of RPA70N will lead to cancer cell death.

描述（申请人提供）：由于细胞周期中DNA损伤率很高，DNA损伤反应和修复途径对于真核细胞的生存至关重要。除了健康细胞的存活，DNA修复途径也在癌细胞的存活中发挥作用。这些修复蛋白的抑制代表了癌症治疗的潜在策略。这些修复蛋白的抑制代表了癌症治疗的潜在策略。 识别和验证DNA损伤反应途径内的不同靶标是从经验证的范例获得新的癌症靶标的策略。复制蛋白A（RPA）被鉴定为三阴性乳腺癌（TNBC）的潜在新靶点，来自范德比尔特的Cortez组对20个TNBC细胞系进行的siRNA筛选。RPA是一种ssDNA结合蛋白，负责募集和启动多种DNA损伤反应蛋白，如Rad 9、MRE 11和ATRIP，因此作为G1/S和G2/M细胞周期检查点。我们的假设是，RPA 70 N的蛋白质-蛋白质结合界面的抑制应允许阻断DNA损伤反应途径，从而诱导癌细胞凋亡。 由于抑制蛋白质-蛋白质相互作用的固有困难，将采用基于片段的发现方法来发现RPA 70 N蛋白质-蛋白质结合界面的选择性抑制剂。我们将使用“核磁共振SAR”技术来发现具有适当效力的先导分子。用1H/15 N HSQC筛选了15，000个成员的片段文库。我们已经从我们的文库中鉴定了150个片段命中，目前有11个与RPA 70 N结合的分子的共晶结构。将生成片段命中的小文库，并通过1H/15 N HSQC进行检测，以确定片段分子的SAR。一旦片段被优化，我们将使用一小部分柔性接头将两个片段连接在一起，以生成前导分子。先导分子将使用结构信息进一步优化，以填充RPA 70 N上任何和所有未占用的结合角落和缝隙。同时，将考虑并优化先导分子的药物性质。具有足够效力和药学性质的先导分子将用于验证我们的假设，即RPA 70 N的蛋白质-蛋白质相互作用的抑制将导致癌细胞死亡。