Covalent Modification of DNA and Protein by Bioactivated Antitumor Acylfulvenes

项目总结： 酰基富烯是伊利定天然产物的衍生物，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和蛋白质加合物的详细化学机理 形成，以及这如何与肿瘤细胞的选择性有关尚不清楚。这个项目的长期目标是 为了定义一种机械论范式，解释酰基富烯的选择性毒性，这将导致 开发针对特定部位调整酰基富烯毒性的联合制剂和量身定做的疗法。 该应用目的是建立从酰基富烯生物活化到生物分子靶标的联系 修饰和细胞毒性。其具体目的是(1)通过以下方法来表征DNA烷基化的体外模式 酰基富烯，并区分直接修饰DNA和生物活化的加合物- 偶联反应；(2)表征酰基富烯抑制细胞氧化还原蛋白的机制；(3) 测定一种生物活化还原酶对酰基富烯烷基化反应和细胞毒性的影响。 目标1的研究方法包括合成化学激活的分子探针，这将是 用于鉴定生物活性的酰基富烯与DNA烷基化的产物。我们将使用化学物质 鉴定酰基富烯DNA加合物结构的表征方法。在目标2中，我们将使用 酶抑制研究与基于质谱学的蛋白质结构图谱相结合 共价键修饰。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 加合物形成对细胞复杂环境的影响。 相关性： 酰基富烯是一类新的药物，通过与生物分子共价连接在一起来杀死癌细胞. 并启动一系列导致细胞死亡的生物事件。最初的化学物质 支配这一过程的转化以及它们在对酰基富烯敏感的细胞中有何不同 那些不是的是未知的。选择性靶向肿瘤细胞的能力是癌症的中心问题。 因此，了解这一机制对于设计更安全的癌症治疗方法非常重要。 战略。

Project Summary: Acylfulvenes, derivatives of illudin natural products, are a promising class of drugs for cancer treatment. They are notable for high therapeutic indices and selectivitiesfor 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 selectivetoxicities of acylfulvenes that will lead to the development of combined-agent and tailored therapies designed to site-specifically tune acylfulvene toxicity. The objectivesof 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 bioactivatedacylfulvenes. 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-overexpressingcells and stable tumor cell lines to test the impact of adduct formation in the complex environment of the cell. Relevance: 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 selectivelytarget tumor cells is a central problem in cancer therapy and consequently understanding this mechanism is important for designing safer cancer therapy strategies.