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）： 对天然产品的作用方式的探索通常会阐明传统方法不容易研究生物学的基本领域。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在分子水平（通过鉴定其细胞内蛋白受体）如何起作用，可以导致在复杂的细胞内过程中鉴定关键蛋白质。从定义上讲，这些天然产物靶蛋白是“药物脆弱的”，因此可以用作新的药物靶标。 该提案重点介绍了两种天然产品的作用方式。抗炎真菌代谢产物异丙氧基蛋白和有效的抗肿瘤大花环二氨基二醇B，从冲绳鞭毛藻分离出来。异丙氧基酮阻断了NF-kappab的DNA结合活性，NF-kappab是介导促炎信号诱导的基因转录的关键转录因子。这是通过抑制i？b降解来介导的，i？b降解对NF-kappab的活性负面调节。尽管对异磷蛋白的抗炎作用的下游后果知之甚少，但这种天然产物的蛋白质靶标在Ikappab上游是未知的。为了确定异丙氧基的细胞内靶标的目标，我们修改了先前报道的合成途径，以生成生物素化的异位蛋白蛋白亲和力试剂。在这里，我们提供了一个48KDA蛋白的证据，该蛋白与该异丙代蛋白亲和力试剂共价和专门结合。这项研究的目的是理解异丙氧基的分子机制，通过纯化，鉴定和表征这种48KDA异位蛋白结合蛋白，抑制促炎信号转导。 抗肿瘤大花环二醇二醇B处于较早的阶段，处于作用阐明过程中。我们在这里提出了一种新型的循环合成断开策略，用于总合成这种有效的天然产物。这种合成的努力还将提供易于生物素化的亲和力试剂的易于生成，我们计划通过该试剂纯化，识别和克隆二苯胺B结合蛋白。这些两栖动物结合蛋白的生化和细胞生物学表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了相同的化学/生化/细胞生物学方法来识别和表征抗血管生成的天然产物的细胞内受体，富马吉林，抗炎剂，parthenolide和抗肿瘤化合物，抗肿瘤化合物，环氧素和eposymicincin。