Mass Spectrometry Studies on Radical Reactions of DNA

描述（由申请人提供）：迄今为止发现的最有效的抗癌药物家族，烯二炔的作用被认为是基于活性的？？？-双基，从每条双链DNA中提取一个氢原子。不幸的是，这些药物的高毒性阻碍了它们的临床应用。与它们的作用有关的各种问题，包括它们在DNA中的结合位点、双自由基形成的机制、它对不同DNA位点的可及性以及由此产生的DNA自由基的命运，正在被科学家们解决，以促进更好的抗肿瘤药物的合理设计。然而，该过程的关键部分，双基中间体与DNA和其他细胞成分的反应，仍然几乎完全未被探索。尽管控制这些反应的因素的信息对于合理的药物设计至关重要，但由于研究这种高活性物种的实验困难，人们对药物形成的双基几乎一无所知。本研究的目的是提供有关这些双自由基在细胞损伤中的作用的信息，并为药物开发提供控制其反应性和选择性的方法。将采用一种独特的质谱法（在NIH的支持下开发）研究气相中的生物自由基反应。该方法涉及将带电基团连接到感兴趣的二元基上，以便在FT-ICR质谱仪中进行操作，其中中性生物分子通过激光诱导的声波解吸引入。在过去，这种方法可以描述单自由基和双自由基与小DNA组分、氨基酸和小肽的反应的新结构/反应性关系。建议的研究将包括1)进一步研究双自由基的反应性和选择性，以及多自由基的包含，2)检查更复杂的生物聚合物，以推进反应性范式，为DNA和蛋白质提供预测能力，3)开发溶液中生成双自由基的方法，并测试它们的DNA切割能力，最后，4)质谱技术将在商业仪器中实施，使其他科学家能够使用它，并将追求自由基反应的肽测序潜力。此外，通过提高对DNA氧化损伤的基础知识，本研究促进了对衰老和包括癌症在内的各种疾病的一般认识。

DESCRIPTION (provided by applicant): The action of the most potent family of anticancer agents discovered thus far, the enediynes, is believed to be based on reactive ???-biradicals that abstract a hydrogen atom from each strand of double-stranded DNA. Unfortunately, the high toxicity of these agents hinders their clinical use. Various issues relating to their action, including their binding sites in DNA, the mechanism of biradical formation, its accessibility to various DNA sites, and the fate of the resulting DNA radicals, are being addressed by scientists to advance the rational design of better antitumor drugs. However, the key part of the process, the reaction of the biradical intermediate with DNA and other cellular components, remains almost entirely unexplored. Although information on the factors that control these reactions is critically needed for rational drug design, nearly nothing is known about the biradicals formed from drugs because of severe experimental difficulties in studying such highly reactive species. The goal of this research is to provide information on the role of these biradicals in cellular damage, and to advance approaches of controlling their reactivity and selectivity for drug development. A unique mass spectrometric method (developed with NIH support) for the study of biological radical reactions in the gas phase will be employed. The method involves attaching a charged group to a biradical of interest for manipulation in an FT-ICR mass spectrometer into which neutral biomolecules are introduced by laser-induced acoustic desorption. In the past, this method allowed the delineation of novel structure/reactivity relationships for reactions of mono- and biradicals with small DNA components, amino acids and small peptides. The proposed research will include 1) further studies on "tuning" of the reactivity and selectivity of biradicals, and inclusion of polyradicals, 2) examination of more complex biopolymers in order to advance a reactivity paradigm that provides predictive power for DNA and proteins, 3) development of methodology for the generation of biradicals in solution and testing of their DNA cleaving ability, and finally, 4) the mass spectrometry technology will be implemented in commercial instruments to make it accessible to other scientists, and the potential for peptide sequencing of radical reactions will be pursued. Furthermore, by improving the fundamental knowledge on oxidative DNA damage, this research advances the general understanding of aging and various diseases, including cancer. This research will drastically improve the fundamental knowledge on oxidative DNA and protein damage. Systematic examination of drug intermediate/DNA and protein interactions will enable the design of better synthetic antitumor drugs for the treatment of, for example, lung, mouth and ovarian cancers. Further, this research advances the general understanding of aging and various diseases, including cancer.