Fungal Acquisition, Isolation Chemistry, Structure Elucidation, and Bioassay

项目3由三角研究所(RTI)和MyCosynthetix公司合作完成。 [MSX]，两家公司都位于北卡罗来纳州的研究三角公园地区。后者提供了一种低于- 探索的来源材料(丝状真菌；具体目标1)，用于补充收集 项目2中描述的蓝藻的营养物质和培养。真菌将从MSX收集中选择 以一种确保最大化学多样性的方式，这将涉及地理、 生态和分类信息，以及关于产生次生物质能力的菌株的信息 代谢物。将使用前面显示的复合培养基来培养真菌，以支持第二代 新陈代谢。MSX将扩大有希望的线索的文化，提供几乎无限的所需供应 化合物。RTI将进行天然产物分离和结构鉴定研究(具体目标2)， 而这些将使用整个计划可用的全部生物资源进行监测， 包括在RTI恢复EZH2对组蛋白H3赖氨酸27的三甲基化活性的内部检测 (H3-K27)和/或对非致瘤性乳腺腺癌细胞的选择性致瘤活性 (具体目标3)以及项目1、核心A和核心C中描述的那些生物目标。 最近，EZH2的表达与肿瘤的转化和转移有直接的因果关系 人前列腺癌、乳腺癌和肾癌及其H3-K27三甲基化活性的抑制 在体外和人肿瘤移植瘤中，最终由细胞存活信号调节。抽提物的测试 恢复细胞EZH2 H3-K27三甲基化活性的化合物应揭示基于机制的 抗肿瘤药物先导。 项目3将与项目1互动，通过LC-MS协助样品的分离，并 以植物为基础的先导化合物的药理学评价。它将与项目2互动，以确定优先顺序 通过文献数据库(NAPRALERT)的样本和结构说明研究将通过 与微线圈核磁共振的合作。此外，样本将在生物检测中进行测试，以供 整个团队，如简介的图3所示。这样做的最终目标是获得最大的 从化学和真菌学的努力中获得的药理学价值，以造福整个计划。在这 我们还重新设计了我们的数据管理计划，整个计划将使用一个 用于协调、交换、存储和帮助确定数据优先级的关系型实时数据库(请参阅核心 d)。 总而言之，项目3带来了不同的来源材料，经过验证的化学，以及创新和互补 生物靶标，全部用于整个计划。从最初的开始，已经开始了一些细微的变化 其中大多数是对初步数据的补充(C节)。

Project 3 comprises a collaborative effort between Research Triangle Institute (RTI) and Mycosynthetix, Inc. [MSX), both located in the Research Triangle Park area of North Carolina. The latter provides an under- explored source material (filamentous fungi; specific aim 1) that serves to complement the collection of Dlants and culturing of cyanobacteria described in Project 2. Fungi will be selected from the MSX collection n a manner that insures maximum chemical diversity, and this will involve a combination of geographic, ecological, and taxonomic information, as well as information on a strains ability to produce secondary metabolites. Fungi will be cultured using complex media shown previously to support secondary metabolism. MSX will scale-up the cultures of promising leads, providing a nearly limitless supply of desired compounds. RTI will carry out natural product isolation and structure elucidation studies (specific aim 2), and these will be monitored using the full breadth of biological resources available to the entire program, including in-house assays at RTI for restoration of EZH2 trimethylation activity toward lysine 27 of histone H3 (H3-K27) and/or selective activity towards tumorigenic over non-tumorigenic breast adenocarcinoma cells (specific aim 3) as well as those biological targets described in Project 1, Core A, and Core C. EZH2 expression has been linked recently in a direct and causal fashion to transformation and metastasis of human prostate, breast, and renal cell carcinoma, with inhibition of its H3-K27 trimethylation activity regulated ultimately by cell survival signals in vitro and in human tumor xenografts. Testing of extracts for compounds that restore cellular EZH2 H3-K27 trimethylation activity should reveal mechanism-based antitumor drug leads. Project 3 will interact with Project 1 for assistance with dereplication of samples via LC-MS and for pharmacological evaluation of their plant-based leads. It will interact with Project 2 for prioritization of samples via literature databases (NAPRALERT), and structure elucidation studies will be facilitated via collaboration with the micro-coil NMR. Also, the samples will be tested in biological assays available to the entire team, as illustrated in Figure 3 of the Introduction. In doing so, the ultimate goal is to derive the most pharmacological value from the chemistry and mycology efforts for the benefit of the entire Program. In this A1 application, we have also re-designed our data management plans, and the entire Program will use a relational, real time database to coordinate, to exchange, to store, and to help prioritize the data (see Core D). In total, Project 3 brings diverse source materials, proven chemistry, and innovative and complementary biological targets, all for the use of the entire Program. Minor changes have been instituted since the initial application, and most of these constitute additions to the preliminary data (section C).