Natural Product Model of Action Studies

行动研究的天然产物模型

• 批准号: 7103657
• 负责人: CRAIG M CREWS
• 金额: $ 28.96万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7103657
• 关键词: Dinoflagellates; antineoplastics; binding proteins; biological products; chemical models; macrolide antibiotics; molecular cloning; peptide chemical synthesis; pharmacokinetics; pharmacology; protein purification; protein structure function

DESCRIPTION (provided by applicant): Exploration of a natural product's mode of action often sheds light on basic areas of biology not easily studied by traditional approaches. This bioorganic strategy to cell biology has led to significant advances in the study of immunoregulation using the natural products, FK506, cyclosporin, and rapamycin. In addition, the investigation of how potent biologically active natural products work at the molecular level (through the identification of their intracellular protein receptors), can lead to the identification of key proteins within a complex intracellular process. These natural product target proteins are, by definition, 'pharmaceutically vulnerable' and thus can serve as novel drug targets. This proposal focuses on the modes of action of two natural products; the anti-inflammatory fungal metabolite isopanepoxydone and the potent antitumor macrolide amphidinolide B isolated from an Okinawan dinoflagellate. Isopanepoxydone blocks the DNA binding activity of NF-kappaB, a key transcription factor that mediates pro-inflammatory signal-induced gene transcription. This is mediated via inhibition of I?B degradation, a protein that negatively regulates the activity of NF-kappaB. Although much is known about the downstream consequences of isopanepoxydone's anti-inflammatory action, the protein target of this natural product that lies upstream of IkappaB is unknown. Towards the goal of identifying the intracellular target of isopanepoxydone, we have modified our previously reported synthetic route to generate a biotinylated isopanepoxydone affinity reagent. Here, we present evidence for a 48kDa protein that covalently and specifically binds to this isopanepoxydone affinity reagent. The objective of this research is to understand the molecular mechanisms by which isopanepoxydone inhibits pro-inflammatory signal transduction through the purification, identification and characterization of this 48kDa isopanepoxydone binding protein. The anti-tumor macrolide amphidinolide B is at an earlier stage in the mode of action elucidation process. We propose here a novel retrosynthetic disconnection strategy for the total synthesis of this potent natural product. This synthetic effort will also afford the facile generation of a biotinylated affinity reagent, with which we plan to purify, identify and clone amphidinolide B binding proteins. Biochemical and cell biological characterization of these amphidinolide binding proteins will confirm their role in mediating the potent cytotoxicity of the natural product. We have successfully employed this same chemical/biochemical/cell biological approach to identify and characterize the intracellular receptors of the antiangiogenic natural product, fumagillin, the anti-inflammatory agent, parthenolide, and the antitumor compounds, epoxomicin and eponemycin.

描述（由申请人提供）： 对天然产物作用方式的探索通常可以揭示传统方法不易研究的生物学基本领域。这种细胞生物学的生物有机策略导致使用天然产物 FK506、环孢菌素和雷帕霉素的免疫调节研究取得了重大进展。此外，研究有效的生物活性天然产物如何在分子水平上发挥作用（通过识别其细胞内蛋白质受体），可以识别复杂的细胞内过程中的关键蛋白质。根据定义，这些天然产物靶蛋白“在药物上是脆弱的”，因此可以作为新的药物靶点。 该提案重点关注两种天然产物的作用方式；从冲绳甲藻中分离出的抗炎真菌代谢物 isopanepoxydone 和强效抗肿瘤大环内酯 amphidinolide B。 Isopanepoxydone 可阻断 NF-kappaB 的 DNA 结合活性，NF-kappaB 是介导促炎信号诱导基因转录的关键转录因子。这是通过抑制 IκB 降解来介导的，IκB 是一种负调节 NF-κB 活性的蛋白质。尽管人们对异环氧酮抗炎作用的下游后果了解很多，但这种位于 IkappaB 上游的天然产物的蛋白质靶标尚不清楚。为了确定异潘环氧酮的细胞内靶标，我们修改了之前报道的合成路线，以生成生物素化异潘环氧酮亲和试剂。在这里，我们提供了 48kDa 蛋白质与这种异潘环氧酮亲和试剂共价特异性结合的证据。本研究的目的是通过纯化、鉴定和表征这种 48kDa 异潘环氧酮结合蛋白，了解异潘环氧酮抑制促炎信号转导的分子机制。 抗肿瘤大环内酯类 Amphidinolide B 处于作用方式阐明过程的早期阶段。我们在这里提出了一种新颖的逆合成断开策略来全合成这种有效的天然产物。这项合成工作还将提供生物素化亲和试剂的简便生成，我们计划用它来纯化、鉴定和克隆两栖类内酯 B 结合蛋白。这些两栖类内酯结合蛋白的生化和细胞生物学特征将证实它们在介导天然产物的有效细胞毒性中的作用。我们已经成功地采用相同的化学/生化/细胞生物学方法来鉴定和表征抗血管生成天然产物夫马洁林、抗炎剂小白菊内酯以及抗肿瘤化合物环氧霉素和埃博霉素的细胞内受体。