Covalent Modification of DNA and Protein by Bioactivated Antitumor Acylfulvenes

描述(申请人提供)：酰基富烯，伊鲁丁天然产物的衍生物，是一类很有前途的癌症治疗药物。它们以对肿瘤细胞的高治疗指数和选择性而闻名，临床试验正在进行中。酰基富烯是烷基化试剂，但DNA和蛋白质加合物形成的详细化学机制以及这与肿瘤细胞选择性的关系尚不清楚。该项目的长期目标是定义一种机制范例，说明酰基富烯的选择性毒性，这将导致开发组合药物和量身定做的治疗方法，旨在针对特定部位调整酰基富烯毒性。这项应用的目的是建立从酰基富烯生物激活到生物分子靶标修饰和细胞毒性的联系。其具体目的是(1)表征酰基富烯在体外的DNA烷基化模式，并区分直接修饰DNA的加合物和生物活化偶联反应的加合物；(2)表征酰基富烯抑制细胞氧化还原蛋白的机制；(3)确定生物激活还原酶对细胞中酰基富烯烷基化反应和细胞毒性的影响。目标1的研究方法包括合成化学激活的分子探针，这些探针将用于鉴定生物活性的酰基富烯进行DNA烷基化反应的产物。我们将使用化学表征方法来鉴定酰基富烯DNA加合物的结构。在目标2中，我们将使用酶抑制研究和基于质谱学的方法来构建蛋白质共价修饰的结构图谱。在目标3中，我们将使用还原酶过表达的细胞和稳定的肿瘤细胞系来测试在复杂的细胞环境中加合物形成的影响。酰基富烯是一类新的药物，通过与细胞中的生物分子共价连接并启动一系列导致细胞死亡的生物事件来杀死癌细胞。支配这一过程的最初化学转化以及它们在对酰基富烯敏感的细胞和不敏感的细胞中的差异尚不清楚。选择性靶向肿瘤细胞的能力是癌症治疗中的一个中心问题，因此了解这一机制对于设计更安全的癌症治疗策略是重要的。

DESCRIPTION (provided by applicant): Acylfulvenes, derivatives of illudin natural products, are a promising class of drugs for cancer treatment. They are notable for high therapeutic indices and selectivities for tumor cells, and clinical trials are underway. Acylfulvenes are alkylating agents, but the detailed chemical mechanisms of DNA and protein-adduct formation, and how this relates to tumor-cell selectivity is not known. The long-term goals of this project are to define a mechanistic paradigm accounting for the selective toxicities of acylfulvenes that will lead to the development of combined-agent and tailored therapies designed to site-specifically tune acylfulvene toxicity. The objectives of this application are to establish a link from acylfulvene bioactivation to biomolecular target modification and cytotoxicity. The specific aims are to (1) Characterize in vitro patterns of DNA alkylation by acylfulvenes and distinguish adducts that result from direct modification of DNA from those of bioactivation-coupled reactions; (2) Characterize the mechanism of inhibition of cellular redox proteins by acylfulvenes; (3) Determine the influence of a bioactivating reductase on acylfulvene alkylation reactions and toxicity in cells. The research approach for aim 1 involves the synthesis of chemically activated molecular probes that will be used to identify products of DNA alkylation by bioactivated acylfulvenes. We will use chemical characterization methods to identify the structures of acylfulvene DNA adducts. In aim 2, we will use enzyme-inhibition studies combined with a mass-spectrometry-based approach to structurally map protein covalent modification. In aim 3, we will use reductase-overexpressing cells and stable tumor cell lines to test the impact of adduct formation in the complex environment of the cell. Acylfulvenes are a new class of drugs that kill cancer cells by becoming covalently linked to biomolecules in the cell and initiating a sequence of biological events that result in cell death. The initial chemical transformations that govern this process and how they differ in cells that are sensitive to acylfulvenes versus those that are not are unknown. The ability to selectively target tumor cells is a central problem in cancer therapy and consequently understanding this mechanism is important for designing safer cancer therapy strategies.