Natural Product Model of Action Studies

描述（由申请人提供）： 对天然产物作用方式的探索，往往能揭示传统方法难以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FK 506，环孢菌素和雷帕霉素进行免疫调节研究的重大进展。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（通过鉴定其细胞内蛋白受体），可以鉴定复杂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是“药学上脆弱的”，因此可以作为新的药物靶标。 该建议侧重于两种天然产物的作用模式;抗炎真菌代谢物异丙环氧酮和从冲绳甲藻中分离的有效抗肿瘤大环内酯amphidinaldehyde B。异戊环氧酮阻断NF-κ B的DNA结合活性，NF-κ B是介导促炎信号诱导的基因转录的关键转录因子。这是通过抑制介导的I？B降解，一种负调节NF-κ B活性的蛋白质。尽管对异丙环氧酮抗炎作用的下游结果了解很多，但这种天然产物位于IkappaB上游的蛋白质靶标是未知的。为了确定异丙环氧酮的细胞内靶点，我们修改了我们以前报道的合成路线，以产生生物素化的异丙环氧酮亲和试剂。在这里，我们提出了一个48 kDa的蛋白质，共价和特异性结合到这个异丙环氧酮亲和试剂的证据。本研究的目的是通过纯化、鉴定和表征该48 kDa异戊环氧酮结合蛋白来了解异戊环氧酮抑制促炎信号转导的分子机制。 抗肿瘤大环内酯类药物两性霉素B B的作用机制尚处于早期研究阶段。在这里，我们提出了一种新的逆合成断开策略，这种有效的天然产物的全合成。这一合成的努力也将提供一个生物素化的亲和试剂，我们计划纯化，鉴定和克隆双端B结合蛋白的简易生成。这些两亲性结合蛋白的生物化学和细胞生物学特性将证实它们在介导天然产物的强效细胞毒性中的作用。我们已经成功地采用这种相同的化学/生物化学/细胞生物学方法来识别和表征抗血管生成的天然产物，烟曲霉素，抗炎剂，parthenophosphate，和抗肿瘤化合物，epoxomicin和eponemycin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.