Natural Products Discovery and Characterization Through Network Collaborations

通过网络合作发现和表征天然产品

• 批准号: 10262372
• 负责人: Kirk Gustafson
• 金额: $ 149.12万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262372
• 关键词: Advanced Development; Alkaloids; Amidines; Architecture; Biochemical; Biochemistry; Biocompatible Materials; Bioinformatics; Biological; Biological Assay; Biology; CCR; Cancer Biology; Carbon; Cell Line; Cell physiology; Cells; Cellular biology; Chemicals; Chemistry; Clinical; Collaborations; Collection; Complement; Computational Science; Data; Data Analyses; Development; Disease; EP300 gene; Evaluation; Exhibits; Extramural Activities; Fractionation; Genetic; Goals; Guanidines; HIF1A gene; HIV; Imidazole; Immunology; Individual; Industrialization; Laboratories; Malignant Neoplasms; Microbe; Molecular; Molecular Biology; Molecular Probes; Molecular Target; Mutate; Names; Natural Products; Natural Products Chemistry; New Agents; Nitrogen; Organic Synthesis; Oxygen; Pain; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phosphotransferases; Plants; Play; Portugal; Positioning Attribute; Process; Protein Chemistry; Proteins; Publications; Pyrimidine; Reaction; Reagent; Research Personnel; Resources; Role; Scientist; Series; Skeleton; Source; Specificity; Structure; Synthesis Chemistry; Technology; Testing; Transcription Coactivator; Triterpenes; United States National Institutes of Health; Universities; anti-cancer; assay development; base; cancer cell; colon cancer cell line; cytotoxic; experience; experimental study; high throughput screening; inhibitor/antagonist; insight; interdisciplinary approach; marine organism; melanoma; novel; preclinical development; professor; response; scaffold; screening; skills; spectroscopic data; structural biology; therapeutic lead compound; tumor

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.

我们利用高通量筛选技术来帮助鉴定能够特异性地与选定的生化靶点或过程相互作用或调节其功能的化合物和提取物。天然产物萃取物的生物测定引导化学分馏被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潜在的药物先导物或生物探针提供了新的结构类别或分子支架，可以与期望的分子靶标相互作用。除了广泛的核磁共振和质谱分析，我们的工作还包括对新化合物的效力、分子靶标特异性和作用方式的严格评估。研究人员发现了一类新的海洋生物碱，它们被称为eudistidines，这些代谢物被证明可以破坏转录共激活因子p300和HIF-1 α之间的相互作用。这些化合物具有前所未有的融合四环核心骨架，由两个嘧啶环和一个咪唑环融合组成，咪唑环还含有嵌入的胍和脒功能。eudistidine C的新分子结构最初是通过对光谱数据（主要是核磁共振和质谱）的综合分析确定的。由于分子核心缺乏质子化的碳原子和氮原子，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艰苦的数据分析最终允许分配新的结构。与化学生物学实验室的工作人员一起完成了一项综合工作，以核实结构并为生物学评估提供额外的材料。用甲氧基苯基氨基咪唑试剂一步合成的方法证实了异丁二啶C新的杂环结构，合成产物与天然产物完全相同。eudistidine C支架，现在可以以一种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-1 α相互作用以及这些蛋白在低氧条件下肿瘤反应中的作用。已提交EIR以报道eudistidines的发现和合成，并准备了一篇高影响力的出版物，描述了它们的分离，结构解析和合成。从葡萄牙科英布拉大学Jorge Salvador教授处获得了一系列半合成植物三萜衍生物，并在所有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系的选择性细胞毒活性，对NCI 60细胞数据的cell Miner生物信息学分析显示，它与临床使用的B-Raf抑制剂有很强的相关性。所有的敏感细胞系都有V600E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。我们研究的化合物没有抑制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。这个项目的重点是利用NPR中广泛的化学多样性，用于潜在的抗癌和抗艾滋病毒应用。它依赖于与MTP分析开发和筛选部门、化学多样性开发部门以及蛋白质化学和分子生物学部门的密切整合，以进行提取物筛选、数据分析、生物分析支持和分离化合物的功能分析。我们的CCR合作伙伴研究癌症生物学、遗传学和免疫学方面，为靶点选择和随后的化合物评估提供专业知识。我们在有机合成、化学生物学、分子药理学、计算科学和光谱分析方面组建了一个广泛的校内和校外合作伙伴联盟，以帮助表征和推进我们的天然产品发现。天然产物化学部门拥有25年研究NPR提取物化学的经验，开发和应用靶向筛选分析的成熟能力，以及在癌症和HIV生物学方面具有广泛专业知识的CCR研究人员，在NCI中具有独特的定位，将基于分子靶标的发现与天然产物化学相结合。天然产物是结构复杂性和生物活性的来源，可以为新靶点、途径或细胞过程的功能提供见解。它们在剖析和理解癌症发展和进展的复杂性方面发挥着重要作用，因此持续的天然产物发现工作可以补充CCR和NCI的目标。