Natural Products Discovery and Characterization Through Network Collaborations

我们利用高通量筛选技术，以帮助确定化合物和提取物，可以特异性地与选定的生化靶标或过程相互作用或调节其功能。生物测定引导的天然产物提取物的化学分级分离用于分离和纯化单个生物活性化合物。这些化合物的鉴定和结构表征为开发可与所需分子靶标相互作用的潜在药物先导物或生物探针提供了新的结构类别或分子支架。除了广泛的NMR和质谱分析，我们的工作还包括严格评估新化合物的效力，分子靶点特异性和作用模式。一种名为eudistines的新型海洋生物碱被鉴定出来，这些代谢物被证明可以破坏转录辅激活因子p300和HIF-1 α之间的相互作用。这些化合物具有前所未有的稠合四环核心骨架，由两个与咪唑环稠合的嘧啶环组成，咪唑环还含有嵌入的胍和脒官能团。新的分子结构的eudistidine C最初分配从光谱数据（主要是NMR和MS）的综合分析。由于在分子的核心中缺乏质子化的碳和氮原子，结构的解析提出了一些非常重大的挑战。应用复杂的核磁共振实验和痛苦的数据分析，最终允许分配的新结构。与化学生物学实验室的工作人员一起完成了一项综合努力，以验证结构并为生物学评价提供额外的材料。通过一步合成证实了eudistidine C的新型杂环结构，该合成涉及eudistidine A与甲氧基苯基-氨基咪唑试剂反应以产生在所有方面与天然产物相同的合成产物。eudistidine C支架现在可以以直接和可扩展的方式合成，可以提供潜在的治疗先导化合物或分子探针来研究p300/HIF-1 α相互作用以及这些蛋白质在肿瘤对低氧条件的反应中所起的作用。提交了一份EIR，以涵盖Eudistidines的发现和合成，并编写了一份描述其分离、结构解析和合成的高影响力出版物。从葡萄牙科英布拉大学的Jorge萨尔瓦多教授获得一系列半合成植物三萜衍生物，并在所有MTL和DTP测定中进行测试。这些化合物之一显示出对黑素瘤和结肠癌细胞系的选择性细胞毒性活性，并且NCI 60细胞数据的Cell Miner生物信息学分析显示出与临床使用的B-Raf抑制剂的强相关性。所有敏感细胞系都具有V600 E突变的B-Raf（组成型活性B-Raf）。临床药物抑制B-Raf的激酶活性。我们研究的化合物不抑制B-Raf或C-Raf的激酶活性，但它确实导致B-Raf和C-Raf的细胞水平显著降低。正在编写一份环境影响报告和一份说明这些调查结果的手稿。其他值得注意的发现包括星状肽A和B，它们是来自海绵Stelletta sp.的新型HIV抑制性环状缩酚肽，和stelliosphaerol A和B，来自厄瓜多尔真菌内生菌的倍半甘油-多元醇缀合物。后一个项目是与耶鲁大学的斯科特·斯特罗贝尔教授合作完成的。

We utilized high throughput screening technologies to help identify compounds and extracts that can specifically interact with or modulate the function of selected biochemical targets or processes. Bioassay-guided chemical fractionation of natural products extracts is employed to isolate and purify the individual bioactive compounds. Identification and structural characterization of these compounds provides new structural classes or molecular scaffolds for the development of potential drug leads or biological probes that can interact with the desired molecular target. In addition to extensive NMR and mass spectroscopic analyses, our efforts include rigorous evaluation of a new compound's potency, molecular target specificity, and mode of action. A new class of marine alkaloids named the eudistidines was identified and these metabolites were shown to disrupt interactions between the transcriptional co-activator p300 and HIF-1 alpha. These compounds have an unprecedented fused tetracyclic core skeleton, comprised of two pyrimidine rings fused with an imidazole ring that also contains embedded guanidine and amidine functionalities. The novel molecular architecture of eudistidine C was initially assigned from a comprehensive analysis of spectroscopic data (primarily NMR and MS). Elucidation of the structure presented some very significant challenges due to a lack of protonated carbon and nitrogen atoms in the core of the molecule. Application of sophisticated NMR experiments and pains-taking data analysis finally allowed assignment of the novel structures. A synthetic effort to verify the structure and provide additional material for biological evaluates was accomplished in conjunction with staff in the Chemical Biology Laboratory. The novel heterocyclic architecture of eudistidine C was confirmed by a one-step synthesis that involved reaction of eudistidine A with a methoxyphenyl-aminoimidazole reagent to generate a synthetic product that was identical in all respects with the natural product. The eudistidine C scaffold, which can now be synthesized in a straightforward and scalable manner, may provide potential therapeutic lead compounds or molecular probes to study p300/HIF-1 alpha interactions and the role these proteins play in tumor response to low oxygen conditions. An EIR was filed to cover the discovery and synthesis of the eudistidines and a high impact publication that describes their isolation, structural elucidation, and synthesis has been prepared. A series of semisynthetic plant triterpene derivatives were obtained from Professor Jorge Salvador at the University of Coimbra, Portugal and tested in all of the MTL and DTP assays. One of these compounds showed selective cytotoxic activity against melanoma and colon cancer cell lines and Cell Miner bioinformatic analysis of the NCI 60-cell data showed a strong correlation with clinically used B-Raf inhibitors. All of the sensitive cell lines have V600E mutated B-Raf (constitutive active B-Raf). The clinical agents inhibit the kinase activity of B-Raf. The compound we investigated did not inhibit the kinase activity of B-Raf or C-Raf, but it did result in significant reductions in cellular levels of B-Raf and C-Raf. An EIR and a manuscript describing these findings is in preparation. Other notable discoveries included stellettapeptins A and B, novel HIV-inhibitory cyclic depsipeptides from the sponge Stelletta sp., and stelliosphaerol A and B, sesquiterpene-polyol conjugates from an Ecuadorian fungal endophyte. The latter project was done in conjunction with Professor Scott Strobel at Yale University.