The XPA scaffold protein in Nucleotide Excision Repair

总结 核苷酸切除修复（NER）通过去除有害的DNA损伤来保护人类细胞，但修复DNA损伤的作用是通过修复DNA损伤来实现的。 NER对DNA的损伤可降低顺铂等抗肿瘤药物的疗效。NER基因是 癌症中经常发生错义突变，NER基因表达减少或功能丧失突变 ERCC 1和ERCC 2分别与顺铂治疗后患者结局改善相关 治疗该提案调查了肿瘤突变引起的NER能力降低的假设， 与对铂（Pt）试剂的更高敏感性相关。它的重点是NER支架蛋白XPA， 是正确组装和组织NER机械所必需的。XPA是NER的“阿喀琉斯之踵” 因为它与DNA底物和几乎所有核心NER蛋白相互作用。我们最近的出版物（i）显示 NER被抑制与其伴侣支架RPA相互作用的XPA突变所抑制，以及（ii）鉴定XPA 肿瘤基因组中破坏NER的突变，包括我们目前的工作表明的一些高度 可能会破坏XPA与RPA的相互作用。因此，XPA代表了研究 这一假说认为，降低的NER能力与对DNA损伤剂的敏感性相关。目标1将测试 假设XPA中错义突变可导致NER缺陷，从而降低修复能力并致敏 肿瘤细胞与Pt剂的比例。将使用高通量PCR筛选XPA突变以降低NER能力。 报告基因测定，以选择在表达XPA的细胞中进一步表征NER缺陷的那些 变种人然后，我们将确定其功能障碍的机制，并测试其对铂剂的敏感性。目的 2将使用基于片段的发现方法来开发破坏XPA-RPA的小分子抑制剂 相互作用，使得能够进一步测试NER容量和对Pt试剂的敏感性之间的相关性。一个高度 将通过NMR和结合位置和方向筛选小分子片段的精选文库 将由X射线晶体学来定义。经过包括结构分析、设计、 和评价，将验证连接片段化合物用于物理抑制XPA-RPA相互作用， 抑制NER，并在癌细胞系中引起对Pt试剂的敏感性。这些目标不仅将 测试NER缺乏与对Pt试剂敏感性之间的相关性，但也产生工具化合物 为进一步验证抑制NER的治疗价值奠定了基础。他们还将提供宝贵的见解 更接近于在临床中使用Pt敏感性预测因子，并探索NER抑制如何影响 对其他DNA损伤剂的敏感性最终，我们试图了解肿瘤基因组景观如何 使癌细胞易于对药物敏感，从而能够识别对药物敏感的患者肿瘤。 单独使用DNA损伤剂或需要与NER抑制剂联合治疗。

SUMMARY Nucleotide excision repair (NER) protects human cells by removing harmful DNA damage, but repair of damaged DNA by NER can reduce the efficacy of some antitumor drugs such as cisplatin. NER genes are frequently missense mutated in cancers and decreased expression or loss of function mutation of NER genes ERCC1 and ERCC2, respectively, has been shown to correlate with improved patient outcomes after cisplatin treatment. This proposal investigates the hypothesis that reduced NER capacity arising from tumor mutations correlates with greater sensitivity to platinum (Pt) agents. It focuses on the NER scaffolding protein XPA, which is required for proper assembly and organization of the NER machinery. XPA is an “Achilles Heel” of NER because it interacts with the DNA substrate and nearly all core NER proteins. Our recent publications (i) show NER is suppressed by XPA mutations that inhibit interaction with its partner scaffold RPA, and (ii) identify XPA mutations from tumor genomes that disrupt NER, including some that our current work suggests are highly likely to disrupt the interaction of XPA with RPA. Thus, XPA represents the ideal factor to investigate the hypothesis that reduced NER capacity correlates with sensitivity to DNA damaging agents. Aim 1 will test the hypothesis that missense mutations in XPA can lead to NER defects that reduce repair capacity and sensitize tumor cells to Pt agents. XPA mutations will be screened for reduced NER capacity using a high throughput reporter assay to select those for which NER deficiency will be further characterized in cells expressing XPA mutants. We will then determine the mechanism of their dysfunction and test their sensitivity to Pt agents. Aim 2 will use a fragment-based discovery approach to develop small molecule inhibitors that disrupt the XPA-RPA interaction to enable further tests of the correlation between NER capacity and sensitivity to Pt agents. A highly curated library of small molecular fragments will be screened by NMR and the binding location and orientation of ‘hits’ will be defined by X-ray crystallography. After cycles of optimization involving structural analysis, design, and evaluation, linked fragment compounds will be validated for physically inhibiting XPA-RPA interaction, suppressing NER, and eliciting sensitivity to Pt agents in cancer cell lines. Together, these aims will not only test the correlation between NER deficiency and sensitivity to Pt agents, but also generate tool compounds that lay the foundation for testing the therapeutic value of inhibiting NER. They will also provide valuable insights to move closer to the use of Pt sensitivity predictors in the clinic and explore how NER inhibition affects sensitivity to other DNA damaging agents. Ultimately, we seek to understand how the tumor genomic landscape predisposes cancer cells to drug sensitivity to enable identification of patient tumors that will be sensitive to DNA damaging agents alone or require combinatorial treatment with NER inhibitors.