Tuning PARP-1 retention and release on DNA breaks

DNA结构的断裂对基因组的完整性造成持续的压力，并且它们构成了 染色体重排和基因突变的重大风险，可能会挑战一个人的福祉。 促进癌症的发展。有几种细胞机制可以监控 基因组的状态，并迅速启动修复机制，以应对DNA损伤，使健康的 基因组会遗传给下一代聚（ADP-核糖）聚合酶-1，或PARP-1，是一个主要的反应， DNA结构的断裂。PARP-1具有独特的催化活性，可以合成ADP-1的聚合物。 核糖作为靶蛋白的翻译后修饰，主要是PARP-1本身（自修饰）。 在与DNA断裂结合后，PARP-1活性被“打开”以调节DNA损伤修复途径， 从而促进细胞存活。相反，过度的DNA损伤导致PARP-1活性水平升高 导致细胞死亡因此，PARP-1活性的调节是决定一个细胞命运的关键因素。 cell.重要的是，PARP-1（PARPi）的抑制剂最近已经成为治疗糖尿病的有前景的治疗剂。 治疗癌症和炎症。尽管对PARPi的兴趣越来越大， PARP-1活性在DNA修复、转录调节和凋亡信号传导中的作用， 对PARP-1催化活性和调节机制的了解有限。本研究的目的 该项目旨在填补我们对PARP-1如何被激活的知识的主要空白， 蛋白质（HPF 1），随后在检测健康细胞中DNA损伤的过程中沉默，以及它如何 在目前针对癌症和炎症中的PARP-1的努力中，小分子可以最好地抑制。 氢-氘交换结合质谱（HXMS）和晶体学是主要的 我们将应用这些结构工具来了解PARPi对PARP-1动力学的影响，以及PARP-1如何在 DNA损伤反应最初被激活，随后通过自动修饰而沉默。的 结构和蛋白质动力学实验将与PARP-1催化的生化分析相结合 和DNA结合，以及基于我们的结构和生物化学工作的PARP-1功能的细胞分析。在 此外，药物化学将用于将变构PARP-1“捕获”到PARPi中 这些化合物用于增加癌症临床中靶向这种酶的功效。而且安 一种新兴的基于PARPi的成像方法，使用乳腺癌患者来源的已建立的肿瘤测定， 异种移植将决定我们产生的PARPi化合物参与/杀死癌细胞的程度 并影响携带肿瘤异种移植物的小鼠的存活率。PARP-1活性和调节的拟议研究 将推进PARP-1生物功能的现有模型，并产生新的小分子工具， 推进对PARP-1生物学的理解，并可能代表癌症临床的新药。

Breaks in the structure of DNA are a persistent stress on the integrity of the genome, and they pose a substantial risk of chromosomal rearrangement and genetic mutation that can challenge the well-being of an organism and promote the development of cancer. There are several cellular mechanisms that monitor the state of the genome and rapidly initiate repair mechanisms in response to DNA damage so that a healthy genome is passed on to the next generation. Poly(ADP-ribose) Polymerase-1, or PARP-1, is a primary responder to breaks in the structure of DNA. PARP-1 has a unique catalytic activity that synthesizes polymers of ADP- ribose as a posttranslational modification on target proteins, primarily on PARP-1 itself (automodification). Upon binding to DNA breaks, PARP-1 activity is “turned on” to modulate DNA damage repair pathways and thereby promote cell survival. In contrast, excessive DNA damage leads to an elevated level of PARP-1 activity that results in cell death. Regulation of PARP-1 activity is therefore a critical factor in determining the fate of a cell. Importantly, inhibitors of PARP-1 (PARPi) have recently emerged as promising therapeutic agents for the treatment of cancer and inflammation. Despite a growing interest in PARPi and the discovery of expanded roles for PARP-1 activity in DNA repair, transcriptional regulation, and apoptotic signaling, there are still limited insights into the mechanism of PARP-1 catalytic activity and regulation. The objective of this research program is to fill major gaps in our knowledge of how PARP-1 is activated, modulated by a critical accessory protein (HPF1), and subsequently silenced in the process of detecting DNA damage in healthy cells and how it can be best inhibited by small molecules in current efforts to target PARP-1 in cancer and inflammation. Hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and crystallography are the major structural tools that we will apply to understand the impact of PARPi on PARP-1 dynamics and how PARP-1 in the DNA damage response is initially activated and then subsequently silenced through automodification. The structural and protein dynamics experiments will be combined with biochemical analysis of PARP-1 catalysis and DNA binding, and cell-based analysis of PARP-1 function based on our structural and biochemical work. In addition, medicinal chemistry will be employed to engineer allosteric PARP-1 “trapping” into PARPi compounds in order to increase the efficacy of targeting this enzyme in the cancer clinic. Moreover, an emerging PARPi-based imaging approach using established tumor assays with breast cancer patient-derived xenografts will determine the degree to which the PARPi compounds that we generate engage/kill cancer cells and impact survival of mice carrying tumor xenografts. The proposed studies of PARP-1 activity and regulation will advance current models of PARP-1 biological functions and generate new small molecule tools that will advance the understanding of PARP-1 biology and potentially represent new medicines for the cancer clinic.