Natural Products Discovery and Characterization Through Network Collaborations

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。在2022财年，我们一直在继续我们的研究活动，以确定与各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ccl -b，默克尔细胞癌，PKA激酶融合蛋白和拓扑异构酶3b。(1) PAX3-FOXO1：对habereri牙苔藻（Dentitheca habereri）的化学研究，鉴定出8个新的二酰基化的虫黄质生物碱，命名为dentithecamides A-H，以及3个先前报道的类似物，命名为zoamides B-D。这些化合物是首次报道的从水合物中提取的虫花黄质生物碱。牙酰胺A、B和zoamides B- d共用一个构象可移动的环庚二烯核心，它们抑制PAX3-FOXO1调控的转录活性，IC50值在1167 uM范围内。(2) Cbl-b: E3泛素蛋白连接酶Cbl-b是癌症免疫治疗干预的一个有吸引力的靶点。用生物测定法对海绵Plakortis sp.的活性组分进行分离，得到了二聚体生物碱plakoramine a .手性分离得到了对映体(+)-plakoramine a和(-)-plakoramine a，它们抑制了ccl -b的活性，IC50值分别为7.5和9.7 uM。(3)默克尔细胞癌：默克尔细胞癌（Merkel cell carcinoma， MCC）是一种罕见但具有高度侵袭性的神经内分泌皮肤癌。晚期MCC的治疗通常使用免疫检查点抑制剂，如avelumab或pembrolizumab。尽管这些药物的反应率相对较高，但只有不到一半的患者获得持久的益处；因此，迫切需要替代疗法。从玫瑰真菌中分离到一个新的11个氨基酸的线状肽roseabol A和已知化合物13-氧-反式9,10-环氧-11(E)-十八烯酸。13-氧-反式9,10-环氧-11(E)-十八烯酸对默克尔细胞癌有抑制作用，IC50值为16.5 uM。(4) PKA激酶融合蛋白（PKADJ）： DNAJB1-PRKACA （PKADJ）致癌基因融合已被确定为治疗罕见的纤维层状肝细胞癌的一个有吸引力的抗肿瘤靶点。通过筛选NCI天然产物发现计划（NPNPD）最近创建的预分离天然产物库，开发了一种高通量测定方法来鉴定PKADJ催化活性的选择性调节剂。2022年，NPCS已经参与了35种pkjj活性提取物的生物测定引导分离。从这些项目中共鉴定出9个活性化合物。采用生物测定法对海洋被囊动物Aplidium sp.的提取物进行分离，发现了两种新的生物碱：Aplithianine a和b。Aplithianine a对PKADJ和宽型PKA均有抑制作用，IC50值为1.1 uM。(5) Topoisomerase-3b: Topoisomerase-3b （TOP3B）是一种在转录过程中通过催化DNA单链瞬间断裂和完整链通过断裂来改变DNA拓扑结构的酶。有研究表明，包括SARS-CoV-2在内的阳性RNA病毒在宿主体内的高效复制需要TOP3B，因此将其作为一个有吸引力的潜在治疗靶点。在这项新的高通量筛选活动中，NPCS已经参与了18种top3b活性提取物的生物测定引导分离。我们正在从这些项目中分离和鉴定活性天然产物。(6) Discorhabdins：我们进行了discorhabdinl的构效关系研究，这是一种天然产物，显示出有希望的前列腺癌异种移植初步结果。FY22分离出一系列新的discorhabdin天然产物，以discorhabdin L为骨架生成半合成衍生物，探究先导化合物的构效关系。我们将通过优化基于分析指纹的去复制和项目选择管道，继续提高基于热液相色谱的生物测定导向分离平台的效率。我们还将结合新的计算建模和有机合成技术/方法，以建立我们在结构修改，目标识别和活性优化方面的专业知识。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。天然产物化学部分在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。

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 2022, 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 Merkel cell carcinoma, the PKA kinase fusion protein, and the topoisomerase-3b. (1) PAX3-FOXO1: Chemical investigation of the marine hydroid Dentitheca habereri led to the identification of eight new diacylated zoanthoxanthin alkaloids, named dentithecamides A-H , along with three previously reported analogues, zoamides B-D. These compounds are the first zoanthoxanthin alkaloids to be reported from a hydroid. Dentithecamides A and B along with zoamides B-D, which all share a conformationally mobile cycloheptadiene core, inhibited PAX3-FOXO1 regulated transcriptional activity with IC50 values in the range of 1167 uM. (2) Cbl-b: The E3 ubiquitin-protein ligase Cbl-b represents an attractive target for immunotherapeutic intervention in cancer. Bioassay-guided fractionation of the active fractions of a marine sponge Plakortis sp. afforded an unprecedented dimeric alkaloid plakoramine A. Chiral separation of the racemic plakoramine A led to the purified enantiomers (+)-plakoramine A and (-)-plakoramine A which inhibited the Cbl-b activities with IC50 values at 7.5 and 9.7 uM, respectively. (3) Merkel cell carcinoma: Merkel cell carcinoma (MCC) is a rare but highly aggressive neuroendocrine skin cancer. The treatment of advanced MCC often utilizes immune checkpoint inhibitors such as avelumab or pembrolizumab. Despite relatively high response rates to these agents, less than half of patients achieve durable benefit; thus, alternative treatments are urgently needed. A new 11 amino acid linear peptide named roseabol A and the known compound 13-oxo-trans-9,10-epoxy-11(E)-octadecenoic acid were isolated from the fungus Clonostachys rosea. 13-oxo-trans-9,10-epoxy-11(E)-octadecenoic acid showed inhibitory activity against Merkel cell carcinoma with an IC50 value of 16.5 uM. (4) The PKA kinase fusion protein (PKADJ): The DNAJB1-PRKACA (PKADJ) oncogenic gene fusion has been identified as an attractive antitumor target against the rare fibrolamellar hepatocellular carcinoma. A high-throughput assay was developed to identify selective modulators of the PKADJ catalytic activity by screening the prefractionated natural product library recently created by the NCI Program for Natural Product Discovery (NPNPD). In 2022, the NPCS has been engaged in the bioassay-guided fractionations of 35 PKADJ-active extracts. Totally 9 active compounds have been identified from these projects so far. Bioassay-guided fractionation of the extract of a marine tunicate, Aplidium sp., led to the discovery of two novel alkaloids, aplithianines A and B. Aplithianine A showed potent inhibition against both PKADJ and wide-type PKA with an IC50 value of 1.1 uM. (5) Topoisomerase-3b: Topoisomerase-3b (TOP3B) is an enzyme that alters DNA topology during transcription by catalyzing transient single strand DNA breaks and passage of the intact strand through this break. TOP3B was proposed as an attractive potential therapeutic target following studies showing that it is required for the efficient replication of positive-sense RNA viruses including SARS-CoV-2 in hosts. In this new high-throughput screening campaign, the NPCS has been engaged in the bioassay-guided fractionations of 18 TOP3B-active extracts. We are in the process of isolation and identification of active natural products from these projects. (6) Discorhabdins: We have undertaken a structure-activity relationship study of discorhabdin L, a natural product that showed promising preliminary prostate cancer xenograft results. In FY22, a series of new discorhabdin natural products were isolated and discorhabdin L was used as a scaffold for the generation of semi-synthetic derivatives probing the structure activity relationship of the lead compound. 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. We will also incorporate new computational modeling and organic synthesis techniques/methodologies to establish our expertise in structure modification, target identification, and activity optimization. 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. 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.