Natural Product Model of Action Studies

描述(由申请人提供)： 探索一种天然产物的作用模式，往往有助于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了使用天然产物FK506、环孢素和雷帕霉素进行免疫调节的研究取得了重大进展。此外，对具有强大生物活性的天然产物如何在分子水平上发挥作用的研究(通过鉴定它们的胞内蛋白受体)可以导致识别复杂的胞内过程中的关键蛋白质。根据定义，这些天然产物靶标蛋白在药物上是脆弱的，因此可以作为新的药物靶标。 这项建议侧重于两种天然产物的作用模式：抗炎真菌代谢物异环氧酮和从冲绳甲藻中分离出来的有效抗肿瘤大环内酯类化合物两性内酯B。异环氧酮阻断核因子-kappaB的DNA结合活性，核因子-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I？B降解来实现的，I？B降解是一种负向调节核因子-kappaB活性的蛋白质。虽然人们对异环氧酮的抗炎作用的下游影响已知很多，但这种位于IkappaB上游的天然产物的蛋白质靶标尚不清楚。为了确定异环氧酮的胞内靶标，我们改进了以前报道的合成路线，生成了一种生物素化的异环氧酮亲和试剂。在这里，我们提出了一个48 kDa的蛋白质与这种异环氧酮亲和试剂共价并特异结合的证据。本研究的目的是通过对48 kDa异环氧酮结合蛋白的纯化、鉴定和鉴定，了解异环氧酮抑制促炎信号转导的分子机制。 抗肿瘤大环内酯类化合物两性内酯B的作用机制还处于早期阐明阶段。我们在这里提出了一种新的反合成断链策略，用于这一有效天然产物的全合成。这项合成工作还将提供一种生物素化亲和试剂的简便生成，我们计划用它来纯化、鉴定和克隆两性内酯B结合蛋白。这些两面针内酯结合蛋白的生化和细胞生物学特性将证实它们在调节天然产物的强大细胞毒性方面的作用。我们已经成功地使用了同样的化学/生化/细胞生物学方法来鉴定和表征抗血管生成天然产物伏马西林、抗炎剂巴特内酯以及抗肿瘤化合物环氧米星和依诺霉素的细胞内受体。

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.