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。于2021财政年度，我们继续进行研究活动，以识别可与多种分子靶标相互作用的生物活性天然产物，包括致癌转录因子PAX 3-FOXO 1、泛素连接酶Cbl-b、细胞成熟调节因子Tbata、NADPH氧化酶亚型DUOX及PKA激酶融合蛋白。从Neopetrosia sp.海绵中分离的新石油噻嗪代表了一种新型异喹啉醌，其对PAX 3-FOXO 1驱动的荧光素酶表达显示出强效抑制作用，IC 50值为1.4微摩尔。通过对具有新优化脉冲序列的复杂NMR实验的综合分析，分配了由14个连续非质子化中心（C、N和S）组成的新替噻嗪的前所未有的噻嗪融合结构支架。从沿着北卡罗来纳州海岸采集的海绵Pseudosuberites sp.的提取物中分离得到三个新的芳基生物碱，命名为辛二酰胺A-C。辛二酰胺A和B抑制Cbl-B（一种E3泛素连接酶，是免疫细胞功能的重要调节剂），IC 50值约为11微摩尔。通过应用新的NMR方法，包括PIP HSQMBC IPAP实验测量关键的长程杂原子（C，H）耦合常数，辅助辛二酰胺A的结构解析。通过生物测定引导分离软珊瑚提取物，鉴定出一系列萜类衍生的亚精胺和精胺酰胺，命名为sinularamides A-G。所有分离的化合物均显示出Cbl-b抑制活性，IC 50值范围为约6.5至33微摩尔。从HTS的预分级文库中快速分离和鉴定具有生物活性的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。从6种植物和海洋提取物中快速分离得到12种PKA融合抑制剂，其中包括一种新的嘌呤-噻嗪-咪唑共轭生物碱。我们将通过优化基于分析指纹的去复制和项目选择流程，继续提高基于HTS的生物测定引导分馏平台的效率。该项目的长期重点是利用NPR中广泛的化学多样性，用于潜在的抗癌和抗HIV应用。它依赖于与MTP检测开发和筛选部分，化学多样性开发部分和蛋白质化学和分子生物学部分的紧密整合，用于提取物筛选，数据分析，生物测定支持和分离化合物的功能分析。我们的CCR合作者研究癌症生物学，遗传学和免疫学方面，为靶点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广泛的校内和校外合作伙伴联盟，他们拥有有机合成，化学生物学，分子药理学，计算科学和光谱分析方面的专业知识，以帮助表征和推进我们的天然产物发现。凭借25年研究NPR提取物化学的经验，经过验证的开发和应用靶向筛选测定的能力，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，天然产物化学科在NCI内具有独特的定位，将联合收割机分子靶向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，可以提供对新靶点，途径或细胞过程功能的见解。它们在解剖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。

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.