DNA-Protein Cross-link Formation by Chimeric Bis-electrophiles

在化学系生命过程化学（CLP）项目的支持下，约翰霍普金斯大学的Marc Greenberg教授和他的研究小组正在研究用于选择性形成DNA-蛋白质交联（DPC）的分子的发展。DPC是DNA损伤的细胞毒性形式，其有效地阻断复制和转录。尽管它们具有生物学意义，但DPC经常被其他形式的损伤所掩盖，例如DNA-DNA链间交联（ICL）。这些分子的发展可能有助于研究DPC在试管、细胞甚至生物体中的形成。选择性DPC的形成将使科学家能够研究这种细胞毒性损伤是如何修复的，并确定它们的形成对基因表达过程的影响。这些分子在细胞的复杂环境中可能特别有用，其中混合物的形成使得特别难以阐明单个化学产物的后果。该项目将为学生提供几个研究领域的广泛培训，包括合成有机化学，生物化学和细胞生物学。在为该项目做出贡献的同时获得培训的学生将包括那些来自历史上在科学领域代表性不足的群体的学生。根据该奖项，格林伯格实验室将专注于双亲电体的合成工作，这些亲电体利用氮分子的高反应性快速烷基化DNA。然而，其中一个高度反应性的氯乙基基团将被替换为更有选择性的亲电试剂，该亲电试剂被设计为优先与赖氨酸的侧链氨基基团反应。以这种方式，将不利于形成占氮芥反应性的大部分的单烷基化产物。此外，将确定亲电试剂形成DPC相对于ICL的选择性。将在核小体核心颗粒以及细胞中测试选择性DPC形成。与ICL相比，优先DPC形成将使用一种损伤家族修复缺陷的各种细胞系进行探测。例如，假设缺乏SPRTN（一种启动DPC修复的蛋白酶）的细胞将对这些分子过敏。在缺乏SPRTN的细胞中选择性DPC形成将使得能够探索替代DPC修复系统。这些双亲电体的研究可能为发现通过在靶细胞位点形成DPC起作用的治疗剂提供基础。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Marc Greenberg of Johns Hopkins University and his research team are studying the development of molecules for the selective formation of DNA-protein crosslinks (DPCs). DPCs are a cytotoxic form of DNA damage that potently block replication and transcription. Despite their biological significance, DPCs are often overshadowed by other forms of damage, such as DNA-DNA interstrand crosslinks (ICLs). The development of these molecules could be useful for studying DPC formation in the test tube, in cells and possibly even organisms. Selective DPC formation would enable scientists to study how such cytotoxic lesions are repaired and determine the consequences of their formation on processes involved in genetic expression. These molecules could be especially useful in the complex environment of cells, where the formation of mixtures makes it particularly difficult to elucidate the consequences of individual chemical products. This project will provide students with broad training in several research areas, including synthetic organic chemistry, biochemistry, and cell biology. The students that obtain training while contributing to this project, will include those from groups that have historically been underrepresented in the sciences.Under this award, the Greenberg laboratory will focus synthetic efforts on bis-electrophiles that take advantage of the high reactivity of nitrogen mustards to rapidly alkylate DNA. However, one of the highly reactive chloroethyl groups will be replaced with a more selective electrophile that is designed to preferentially react with the side-chain amino group of lysines. In this way, the formation of monoalkylation products that account for the majority of nitrogen mustard reactivity will be disfavored. Furthermore, the selectivity of the electrophile to form DPCs over ICLs will be determined. Selective DPC formation will be tested in nucleosome core particles, as well as in cells. Preferential DPC formation compared to ICLs will be probed using various cell lines that are deficient in repair of one lesion family. For instance, it is hypothesized that cells lacking SPRTN, a protease that initiates DPC repair, will be hypersensitive to the molecules. Selective DPC formation in cells that lack SPRTN would enable the exploration of alternative DPC repair systems. Studies with these bis-electrophiles may provide the foundation for the discovery of therapeutic agents that act by forming DPCs in targeted cellular loci.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.