Structure and Mechanism of Chromatin-Bound PARP1

项目摘要 核酶PARP 1（PARPi）的抑制剂在临床上使用，并作为治疗剂进行了高级试验， DNA损伤修复缺陷的癌症。PARP 1是一种高度丰富的蛋白质，它塑造染色质结构， 但一旦检测到受损的DNA，它就会变得有酶活性，并形成聚（ADP）-核糖（PAR）链， 自身。其他核蛋白如组蛋白的PAR化由最近发现的组蛋白介导。 PAR化因子1（HPF 1）。PAR化使紧密的染色质松弛，并作为招募DNA的信号 修复机制，但访问的DNA损伤修复机制也依赖于行动的ATP依赖 染色质重塑因子和组蛋白伴侣。PARP抑制剂在癌症患者中的疗效是 归因于合成致死性：同时关闭PARP介导的DNA修复患者 DNA修复途径缺陷（即BRCA 1/2阴性）或诱导的DNA损伤（即化疗或化疗） 放射疗法），导致压倒性的DNA损伤和癌细胞死亡。尽管现有的成功 PARP 1抑制剂，高有效剂量，药物疗效与PARP 1活性抑制的相关性差， 癌细胞和对PARPi的抗性表明还有改进的空间。 我们的目标是使用严格的定量，结构和机制的方法来研究PARP 1 在染色质的情况下，以及在存在或不存在 其辅助蛋白HPF 1。我们的目标是发现下一代PARPi是否应该优化绑定 更紧密地与PARP 1-HPF 1复合物结合，或者相反地阻止这种复合物的形成，以及这些是否 对于不同的临床应用（即癌症与心血管或神经疾病），特性应该不同。 我们还试图研究选择性抑制PARP 1的DNA结合活性构象是否是一个重要因素。 潜在的方法，以减少药物的量，一个人将需要管理，通过照亮 PARP 1通过结构生物学结合到完整染色质与受损染色质的构象多样性。最后我们 将确定PAR化对染色质重塑者提供更好的DNA进入能力的影响 修理机器。我们的机制研究将为如何设计更好的筛选或测定提供关键见解， 开发更具选择性和基于机制的PARP抑制剂。总的来说，我们提出的研究 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.