Covalent Modification of DNA and Protein by Bioactivated Antitumor Acylfulvenes

描述（申请人提供）：酰基fulvenes是天然发光素的衍生物，是一类很有前途的癌症治疗药物。它们具有较高的治疗指数和对肿瘤细胞的选择性，临床试验正在进行中。酰基fulvenes是烷基化剂，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个解释酰基fulvenes选择性毒性的机制范例，这将导致开发联合药物和量身定制的疗法，设计用于特异性调节酰基fulvenes毒性。本申请的目的是建立从酰基氟烯生物活化到生物分子靶标修饰和细胞毒性的联系。具体目的是：(1)表征酰基黄烯对DNA烷基化的体外模式，并区分DNA直接修饰产生的加合物与生物活化偶联反应产生的加合物；(2)表征酰基fulvenes抑制细胞氧化还原蛋白的机制；(3)确定一种生物活化还原酶对酰基氟烯烷基化反应和细胞毒性的影响。目标1的研究方法包括化学活化分子探针的合成，这些探针将用于鉴定生物活化的酰基fulvenes的DNA烷基化产物。我们将使用化学表征方法来鉴定酰基fulvene DNA加合物的结构。在目标2中，我们将使用酶抑制研究结合基于质谱的方法来绘制蛋白质共价修饰的结构图。在目标3中，我们将使用过表达还原酶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fulvenes是一类新型药物，通过与细胞内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。控制这一过程的初始化学转化，以及它们在对酰基fulvenes敏感的细胞和对酰基fulvenes不敏感的细胞中有何不同，都是未知的。选择性靶向肿瘤细胞的能力是癌症治疗中的一个核心问题，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.