Natural Products Discovery and Characterization Through Network Collaborations

我们利用高通量筛选技术来帮助识别能够与选定的生化目标或过程特定地相互作用或调节其功能的化合物和提取物。采用生物测定导向的天然产物提取物的化学分级来分离和纯化单独的生物活性化合物。这些化合物的鉴定和结构表征为开发能够与期望的分子靶标相互作用的潜在药物先导或生物探针提供了新的结构类别或分子支架。除了广泛的核磁共振和质谱分析，我们的努力还包括对新化合物的效力、分子靶标特异性和作用模式进行严格评估。一类新的海洋生物碱被鉴定出来，命名为eudistidines，这些代谢物被证明破坏了转录共激活因子p300和HIF-1α之间的相互作用。这些化合物具有史无前例的稠合四环核心骨架，由两个嘧啶环和一个咪唑环融合而成，咪唑环还包含嵌入的胍和脒官能团。通过对光谱数据(主要是核磁共振和质谱学)的综合分析，首次确定了新的尤迪斯替丁C的分子结构。由于分子核心缺乏质子化的碳和氮原子，对结构的阐明提出了一些非常重大的挑战。应用复杂的核磁共振实验和费力的数据分析，最终确定了新的结构。与化学生物学实验室的工作人员一起完成了验证结构并为生物学评估提供额外材料的综合努力。通过一步法合成得到与天然产物完全相同的合成产物，合成产物与甲氧基苯氨基咪唑试剂反应，得到与天然产物完全相同的合成产物。Eudistidine C支架现在可以直接和可扩展的方式合成，可能提供潜在的治疗先导化合物或分子探针来研究p300/HIF-1α相互作用以及这些蛋白在低氧条件下肿瘤反应中所起的作用。提交了一份EIR文件，报道了尤迪斯蒂丁的发现和合成，并准备了一份高影响力的出版物，描述了它们的分离、结构阐明和合成。一系列半合成植物三萜衍生物是从葡萄牙科英布拉大学的豪尔赫·萨尔瓦多教授那里获得的，并在所有的MTL和DTP分析中进行了测试。其中一种化合物显示出对黑色素瘤和结肠癌细胞株的选择性细胞毒活性，Cell Miner对NCI 60细胞数据的生物信息学分析表明，与临床使用的B-Raf抑制剂有很强的相关性。所有敏感细胞系均存在V600E突变的B-Raf(构成活性B-Raf)。临床药物可抑制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，但它确实导致细胞内B-Raf和C-Raf水平的显著降低。该项目的重点是开发NPR内广泛的化学多样性，用于潜在的抗癌和抗艾滋病毒应用。它依靠与MTP分析开发和筛选科、化学多样性开发科以及蛋白质化学和分子生物学科的紧密结合，对分离出的化合物进行提取物筛选、数据分析、生物测定支持和功能分析。我们的CCR合作者研究癌症生物学、遗传学和免疫学的各个方面，为靶标选择和后续化合物评估提供专业知识。我们已经组建了一个由拥有有机合成、化学生物学、分子药理学、计算科学和光谱分析专业知识的墙内和墙外合作伙伴组成的广泛联盟，以帮助确定和推进我们的天然产品发现。天然产品化学科拥有25年研究NPR提取物化学的经验，具有开发和应用靶向筛选分析的成熟能力，并能够接触到在癌症和艾滋病毒生物学方面具有广泛专业知识的CCR研究人员，天然产品化学科在NCI中具有独特的地位，将基于分子靶标的发现与天然产品化学相结合。天然产物是结构复杂性和生物活性的来源，可以提供对新靶点、途径或细胞过程的功能的洞察。它们在解剖和理解癌症发展和进展的错综复杂的过程中发挥着重要作用，因此持续的天然产品发现努力可以补充CCR和NCI的目标。

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. The focus of this project is to exploit the vast spectrum of chemical diversity within the NPR for potential anticancer and anti-HIV applications. It relies on close integration with the MTP Assay Development and Screening Section, Chemical Diversity Development Section, and the Protein Chemistry and Molecular Biology Section for extract screening, data analysis, bioassay support, and functional analysis of isolated compounds. Our CCR collaborators who study aspects of cancer biology, genetics, and immunology provide expertise for target selection and subsequent compound evaluation. We have assembled a broad consortium of intramural and extramural partners with expertise in organic synthesis, chemical biology, molecular pharmacology, computational sciences, and spectroscopic analysis to help characterize and advance our natural product discoveries. With 25 years of experience studying the chemistry of extracts from the NPR, a proven capability to develop and apply targeted screening assays, and access to CCR investigators with broad expertise in cancer and HIV biology, the Natural Products Chemistry Section is uniquely positioned within the NCI to combine molecular target-based discovery with natural products chemistry. Natural products are a source of structural complexity and biological activity that can provide insight on the function of new targets, pathways, or cellular processes. They play an important role in dissecting and understanding the intricacies of cancer development and progression, so continued natural products discovery efforts can complement the goals of the CCR and NCI.