Structure and Mechanism of Chromatin-Bound PARP1

项目摘要 聚ADP-核糖聚合酶1（PARP 1）是DNA修复受损的肿瘤治疗的一个有前途的靶点。 肿瘤的一种PARP 1抑制剂（奥拉帕尼）在临床上用于治疗卵巢癌，约20个III期临床试验用于治疗卵巢癌。 PARP 1抑制剂的试验目前正在进行中。PARP 1是一种高度丰富的核蛋白， 结合受损的DNA将聚（ADP-核糖）的长链添加到其自身和许多其它核蛋白上。这 巴洛克式的翻译后修饰招募了DNA修复机制的关键成分。现有抑制剂 需要高有效剂量（100-400 mg/天），并且显示药物功效与 PARP 1活性的抑制，可能是因为它们也靶向大量结合到 未受损的染色质这种PARP 1群体具有许多核功能，这些功能超出了维持细胞核的功能。 基因组的完整性，如染色质压缩，转录和DNA甲基化的调节。 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 在染色质的背景下，与受损DNA和完整DNA的相互作用不同。我们的目标是了解选择性 抑制PARP 1的DNA结合活性构象是增加药物功效的潜在途径 和特异性。使用新的酶测定，我们将测试不同类型的激活的不同结果， DNA损伤。我们还建议研究PARP 1和活性低得多的 不太丰富的PARP 2。我们的研究将揭示PARP 1和PARP 2的构象多样性，并显示如何 这影响了复杂的反应机制，导致不同的下游信号事件。我们的机械 这些研究将为如何设计更好的筛选和检测方法提供关键见解， 选择性的、基于机制的PARP抑制剂。总之，我们提出的研究将有助于发展 用于癌症治疗的下一代PARP抑制剂。

PROJECT SUMMARY Poly(ADP-ribose) polymerase 1 (PARP1) is a promising target for cancer therapy in DNA repair-compromised tumors. One PARP1 inhibitor (Olaparib) is used in the clinic to treat ovarian cancer, and ~20 phase III clinical trials with inhibitors of PARP1 are currently ongoing. PARP1 is a highly abundant nuclear protein that upon binding damaged DNA adds long chains of poly(ADP-ribose) onto itself and many other nuclear proteins. This baroque post-translational modification recruits key components of the DNA repair machinery. Existing inhibitors of PARP1 require a high effective dose (100-400 mg/day) and show poor correlation of drug efficacy with inhibition of PARP1 activity, possibly because they also target inactive PARP1 that is abundantly bound to undamaged chromatin. This PARP1 population has many nuclear functions that extend beyond the maintenance of genome integrity, such as chromatin compaction, and the regulation of transcription and DNA methylation. 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. We aim to understand if selective inhibition of the DNA-bound active conformation of PARP1 is a potential approach for increasing drug efficacy and specificity. Using novel enzymatic assays, we will test the varied outcomes of activation by different types of DNA damage. We also propose to investigate the interplay between PARP1 and the much less active and less abundant PARP2. Our studies will reveal the conformational diversity of PARP1 and PARP2, and show how this affects the complex reaction mechanism, resulting in different downstream signaling events. Our mechanistic studies will provide key insights into how to design better screens and assays for the development of more selective, mechanism-based PARP inhibitors. Altogether our proposed research will aid the development of the next generation of PARP inhibitors for use in cancer therapy.