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。在2021财年，我们一直在继续我们的研究活动，以确定与多种分子靶点相互作用的生物活性天然产物，包括致癌转录因子PAX3-FOXO1、泛素连接酶Cbl-b、细胞成熟调节因子Tbata、NADPH氧化酶亚型DUOX和PKA激酶融合蛋白。Neopetrothiazide，分离自一株Neopetrosia sp.海绵，代表了一种新的异喹啉醌，它对PAX3-FOXO1驱动的荧光素酶的表达具有很强的抑制作用，IC50值为1.4微摩尔。通过对最新优化的脉冲序列进行复杂的核磁共振实验的综合分析，确定了由14个连续的非质子化中心(C、N和S)组成的史无前例的新四氢噻嗪熔融结构支架。从海绵Pusosuberites sp.的提取物中分离得到3个新的芳基生物碱，命名为Suberitamines A-C。在北卡罗来纳州海岸收集的。Suberitamide A和B对Cbl-b有抑制作用，Cbl-b是一种E3泛素连接酶，是免疫细胞功能的重要调节因子，其IC50值约为11微摩尔。应用新的核磁共振方法，包括PIP HSQMBC IPAP实验，测量了关键的长程异核(C，H)偶合常数，辅助了琥珀酰胺A的结构解释。生物测定引导下的中华乳杆菌提取物的分离。软珊瑚鉴定了一系列由萜类化合物衍生的亚精胺和亚精胺，命名为Sinularamide A-G。所有分离的化合物都具有Cbl-b抑制活性，其IC50值约为6.5-33微摩尔。从HTS的预分解库中快速分离和鉴定生物活性天然产物的新方法已经被开发并应用于最近针对PKA激酶融合蛋白的分离项目中。在较短的时间内，从6种植物和海洋提取物中快速分离出12种PKA融合抑制剂，其中包括一种新型的嘌呤-噻嗪-咪唑偶联生物碱。我们将通过优化基于分析指纹的去复制和项目选择流水线，继续提高我们基于HTS的生物测定导向分离平台的效率。该项目的长期重点是开发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. In FY 2021, we have been continuing our research campaign to identify bioactive natural products that interact with a wide variety of molecular targets including the oncogenic transcription factor PAX3-FOXO1, the ubiquitin ligase Cbl-b, the hymocyte maturation regulator Tbata, the NADPH oxidase isoforms DUOX, and PKA kinase fusion protein. Neopetrothiazide, isolated from a Neopetrosia sp. sponge, represents a novel isoquinoline quinone that showed potent inhibition against PAX3-FOXO1-driven luciferase expression with an IC50 value of 1.4 micromolar . The unprecedented thiazide-fused structural scaffold of neopetrothiazide comprised of 14 contiguous nonprotonated centers (C, N, and S) was assigned by comprehensive analysis of sophisticated NMR experiments with newly optimized pulse sequences. Three new aryl alkaloids named suberitamides A-C were isolated from an extract of the marine sponge Pseudosuberites sp. collected along the coast of North Carolina. Suberitamides A and B inhibited Cbl-b, an E3 ubiquitin ligase that is an important modulator of immune cell function, with IC50 values of approximately 11 micromolar . Structure elucidation of suberitamide A was aided by the application of new NMR methodologies including the PIP HSQMBC IPAP experiment for measuring key long-range heteronuclear (C, H) coupling constants. Bioassay-guided separation of the extract of a Sinularia sp. soft coral led to the identification of a series of terpenoid-derived spermidine and spermine amides that were named sinularamides A-G. All of the isolated compounds showed Cbl-b inhibitory activities with IC50 values that ranged from approximately 6.5 to 33 micromolar . New methodologies for rapid isolation and identification of biologically active natural products from prefractionated libraries in HTS have been developed and applied in recent isolation projects against the PKA kinase fusion protein. Twelve PKA-fusion inhibitors including a novel purine-thiazine-imidazole-conjugated alkaloid have been rapidly isolated from six plant and marine extracts within a short period. We will continue to improve the efficiency of our HTS-based bioassay-guided fractionation platform by optimizing the analytical-fingerprint-based dereplication and project selection pipelines. The long-term 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.