Structure and Mechanism of Chromatin-Bound PARP1

项目总结 核酶PARP1抑制剂(PARPI)正作为治疗药物在临床和高级试验中使用 有DNA损伤的癌症修复缺陷。PARP1是一种高度丰富的蛋白质，可以形成染色质结构， 但一旦检测到损伤，DNA就会变得具有酶活性，并构建聚(ADP)-核糖链(PAR) 单打独斗。其他核蛋白，如组蛋白的旁化是由新近发现的组蛋白介导的 磷酸化因子1(HPF1)。PAR化松弛紧密的染色质，作为招募DNA的信号 修复机制，但DNA损伤修复机制的获得也依赖于ATP依赖的动作 染色质重塑因子和组蛋白伴侣蛋白。PARP抑制剂对癌症患者的疗效是 归因于合成致死性：同时关闭PARP介导的DNA修复 DNA修复途径缺陷(即BRCA1/2阴性)或诱导DNA损伤(即化疗或 放射治疗)，导致压倒性的DNA损伤和癌细胞死亡。尽管现有的 PARP1抑制剂，高效剂量，药效与抑制PARP1活性相关性差 癌细胞和对PARPI的耐药性表明还有改进的空间。 我们的目标是使用严格的定量、结构和机械方法来研究PARP1如何 在染色质的上下文中，以及在存在或不存在的情况下，与受损的与完整的DNA的相互作用不同 其辅助蛋白HPF1。我们的目标是发现下一代PARPI是否应该针对绑定进行优化 更紧密地与PARP1-HPF1复合体结合，或者阻止这种复合体的形成，以及这些 不同的临床应用(如癌症与心血管或神经疾病)的特性应该有所不同。 我们还试图调查选择性地抑制DNA结合的PARP1的活性构象是否是一种 减少需要服用的药物数量的潜在方法，通过说明 PARP1的构象多样性通过结构生物学与完整染色质和受损染色质结合。最后，我们 将决定PAR化对染色质重构体提供更好地获取DNA的能力的影响 修理机器。我们的机理研究将为如何设计更好的筛查或检测方法提供关键见解 更具选择性和基于机理的PARP抑制剂的开发。总之，我们提议的研究 将为下一代用于癌症治疗的PARP抑制剂的开发提供信息。

PROJECT SUMMARY Inhibitors of the nuclear enzyme PARP1 (PARPi) are in clinical use and advanced trials as therapeutics for cancers with DNA damage repair defects. PARP1 is a highly abundant protein that shapes chromatin structure, but upon detecting damaged DNA becomes enzymatically active and builds chains of poly(ADP)-ribose (PAR) on itself. PARylation of other nuclear proteins such as histones is mediated by the recently discovered Histone PARylation Factor 1 (HPF1). PARylation relaxes compact chromatin and serves as a signal to recruit the DNA repair machinery, but access of the DNA damage repair machinery also relies on the action of ATP-dependent chromatin remodeling factors and histone chaperones. The efficacy of PARP inhibitors in cancer patients is attributed to synthetic lethality: Simultaneously shutting down PARP-mediated DNA repair in patients with deficiencies in DNA repair pathways (i.e. BRCA1/2 negative) or with induced DNA damage (i.e. chemo- or radiotherapy), leads to overwhelming DNA damage and cancer cell death. Despite the success of existing inhibitors of PARP1, high effective dosages, poor correlation of drug efficacy with inhibition of PARP1 activity in cancer cells, and resistance to PARPi suggest room for improvement. Our goal is to use rigorous quantitative, structural, and mechanistic approaches to investigate how PARP1 interacts differently with damaged vs. intact DNA in the context of chromatin, and in the presence or absence of its accessory protein HPF1. We aim to discover whether next-generation PARPi should be optimized for binding more tightly to the PARP1-HPF1 complex, or instead prevent this complex from forming, and whether these properties should differ for different clinical applications (i.e. cancer vs. cardiovascular or neurological diseases). We also seek to investigate whether selective inhibition of the DNA-bound active conformation of PARP1 is a potential approach to lowering the amount of drug one would need to administer, by illuminating the conformational diversity of PARP1 bound to intact vs. damaged chromatin through structural biology. Finally, we will determine the effect of PARylation on the ability of chromatin remodelers to provide better access to the DNA repair machinery. Our mechanistic studies will yield key insights into how to design better screens or assays for the development of more selective and mechanism-based PARP inhibitors. Altogether our proposed research will inform the development of the next generation of PARP inhibitors for use in cancer therapy.