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

在化学系生命过程化学(CLP)计划的支持下，约翰霍普金斯大学的Marc Greenberg教授和他的研究团队正在研究选择性形成DNA-蛋白质交联链(DPC)的分子的发展。DPC是一种细胞毒性形式的DNA损伤，可以有效地阻止复制和转录。尽管DPC具有生物学意义，但它们往往被其他形式的损害所掩盖，例如DNA-DNA链间交联链(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涉及基因表达的过程的后果。这些分子在细胞的复杂环境中可能特别有用，在这种环境中，混合物的形成使阐明单个化学产品的后果变得特别困难。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。在为这个项目做出贡献的同时获得培训的学生，将包括那些在科学界历史上代表性不足的群体。在这一奖项下，格林伯格实验室将专注于双亲电体的合成努力，这些亲电体利用氮芥的高反应性来快速烷化DNA。然而，其中一个高活性的氯乙基将被一种更具选择性的电泳剂取代，该电泳剂旨在优先与赖氨酸的侧链氨基反应。这样，将不利于形成单烷基化产物，而单烷基化产物是氮芥酸反应性的主要来源。此外，还将确定形成DPC的电泳剂相对于ICL的选择性。选择性DPC的形成将在核小体核心颗粒以及细胞中进行测试。与ICL相比，优先的DPC形成将使用不同的细胞系来探索，这些细胞系在修复一个病变家族方面存在缺陷。例如，假设缺少SPRTN的细胞将对分子高度敏感。SPRTN是一种启动DPC修复的蛋白酶。在缺乏SPRTN的细胞中选择性地形成DPC将使探索替代的DPC修复系统成为可能。对这些双亲电体的研究可能为发现通过在靶向细胞中形成DPC而发挥作用的治疗剂提供基础。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.