Covalent Modification of DNA and Protein by Bioactivated Antitumor Acylfulvenes

描述（由申请人提供）：Acylfulvenes 是伞菌素天然产物的衍生物，是一类有前景的癌症治疗药物。它们以高治疗指数和对肿瘤细胞的选择性而闻名，临床试验正在进行中。酰基富烯是烷化剂，但 DNA 和蛋白质加合物形成的详细化学机制以及其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个解释酰基富烯选择性毒性的机制范式，这将导致开发旨在特异性调节酰基富烯毒性的组合药物和定制疗法。本申请的目的是建立从酰富烯生物活化到生物分子靶标修饰和细胞毒性的联系。具体目标是 (1) 表征酰基富烯 DNA 烷基化的体外模式，并将 DNA 直接修饰产生的加合物与生物活化偶联反应的加合物区分开来； (2) 表征酰基富烯抑制细胞氧化还原蛋白的机制； (3)确定生物活性还原酶对酰基富烯烷基化反应和细胞毒性的影响。目标 1 的研究方法涉及化学活化分子探针的合成，该探针将用于识别生物活化酰基富烯的 DNA 烷基化产物。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与基于质谱的方法相结合来绘制蛋白质共价修饰的结构图。在目标 3 中，我们将使用还原酶过表达细胞和稳定的肿瘤细胞系来测试加合物形成在细胞复杂环境中的影响。酰富烯是一类新型药物，通过与细胞中的生物分子共价连接并引发一系列导致细胞死亡的生物事件来杀死癌细胞。控制这一过程的初始化学转化以及它们在对酰基富烯敏感的细胞与对酰基富烯不敏感的细胞中有何不同尚不清楚。选择性靶向肿瘤细胞的能力是癌症治疗的核心问题，因此了解这一机制对于设计更安全的癌症治疗策略非常重要。

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.