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: 10926246
• 负责人: Lin Du
• 金额: $ 130.48万
• 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/10926246
• 关键词: Advanced Development; Age Years; Biochemical; Biochemistry; Biological; Biological Assay; Biology; CCR; Cancer Biology; Cell Line; Cell physiology; Cellular biology; Characteristics; Chemicals; Chemistry; Chimeric Proteins; Collaborations; Collection; Competitive Binding; Complement; Computational Science; Computer Models; Crystallization; DNA; DNA Topoisomerases; Data; Data Analyses; Development; Digit structure; Disease; Enzyme-Linked Immunosorbent Assay; Evaluation; Exhibits; Extramural Activities; Family; Fibrolamellar Hepatocellular Carcinoma; Fingerprint; Fractionation; Gene Fusion; Genes; Genetic; Goals; HCT116 Cells; HIV; Human; Immune checkpoint inhibitor; Immunology; Immunotherapeutic agent; Individual; Industrialization; Intervention; Journals; Large Intestine Carcinoma; Lead; Legal patent; Letters; Libraries; Link; Malignant Neoplasms; Malignant neoplasm of liver; Manuscripts; Merkel cell carcinoma; Mesothelioma; Methodology; Microbe; Modification; Molecular; Molecular Biology; Molecular Probes; Molecular Target; Natural Products; Natural Products Chemistry; Natural Source; Neurosecretory Systems; New Agents; Oncogenic; Organic Synthesis; PRKCA gene; Pathway interactions; Patients; Penicillium; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Phosphotransferases; Photosensitizing Agents; Plakortis; Plants; Play; Porifera; Positioning Attribute; Process; Proliferating; Protein Chemistry; Protein-Serine-Threonine Kinases; Publications; Publishing; RNA; RNA Virus Infections; Reporting; Research; Resources; Role; Route; Scientist; Serine; Skin Cancer; Source; Specificity; Structure; Survival Rate; Synthesis Chemistry; Techniques; Technology; Topoisomerase; Translational Research; United States National Institutes of Health; Urochordata; X ray diffraction analysis; alternative treatment; analog; anti-cancer; assay development; bioactive natural products; cancer cell; cancer type; cytotoxic; cytotoxicity; design; diketopiperazine; experience; experimental study; fluorophore; high throughput screening; improved; inhibitor; insight; interdisciplinary approach; kinase inhibitor; marine; marine organism; monomer; nanomolar; novel; novel therapeutics; patient population; pembrolizumab; pharmacologic; preclinical development; programs; proto-oncogene protein c-cbl; response; scaffold; screening; screening panel; secondary metabolite; skills; structural biology; therapeutic development; tumor; tumorigenesis; ubiquitin ligase

The NPCS 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 2023, we have been continuing our research campaign to identify bioactive natural products that interact with a wide variety of molecular targets including the ubiquitin ligase Cbl-b, the Merkel cell carcinoma, the J-PKAc alpha kinase fusion protein, the topoisomerase-3B, the NCI60 cell line screening panel, and mesothelioma (MPM). Totally, the NPCS has completed 98 isolation projects leading to the identification of 267 natural products including 91 new compounds. Among all compounds, 151 of them including 47 new compounds exhibited bioactivities against the selected targets. (1) Cbl-b: The E3 ubiquitin-protein ligase Cbl-b represents an attractive target for immunotherapeutic intervention in cancer. In FY 2022, we reported the identification of two novel Cbl-b inhibitors, (+)-plakoramine A and (-)-plakoramine A, from a marine sponge Plakortis sp. We continued to reveal a previously undescribed, nonenzymatic route to form plakoramine A via photochemical conversion of its naturally occurring monomeric counterpart, plakinidine B, which stands for a new functional fluorophore with potential utility as both a photosensitizer and a photochemically triggered electrophilic agent. The plakoramine story has been successfully published on the ACS journal Organic Letters. (2) 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. Two new cytotoxic oxygenated diketopiperazines, brevianamides E1 and E2, were identified from a Penicillium brevicompactum. Both compounds showed selective cytotoxicity against the Merkel cell carcinoma cell line MCC13 cell line with IC50 values at 2.6 and 2.5 mM, respectively. (3) The J-PKAc alpha kinase fusion protein (PKADJ): Fibrolamellar Hepatocellular Carcinoma (FLHCC) is a rare liver cancer with a young patient population ( 35 years of age) and a 5-year survival rate of only 30-35%. The DNAJB1-PRKCA oncogenic gene fusion is specifically expressed in the tumor and exclusively detected in FLHCC patients. The DNAJB1-PRKCA gene fusion produces an enzymatically active chimeric protein J-PKAc alpha that is a key driver in the oncogenesis of FLHCC. A high-throughput, modified sandwich ELISA assay was developed by PCMBS to identify selective modulators of the J-PKAc alpha catalytic activity by screening the prefractionated natural product library recently created by the NCI Program for Natural Product Discovery (NPNPD). In FY 2023, the NPCS has been engaged in the bioassay-guided fractionations of 36 PKADJ-active extracts. The NPCS assisted PCMBS in the publication of the first PKADJ HTS manuscript on ACS Pharmacology & Translational Science by providing several bioactive natural products. Last year, we reported the identification of a novel PKADJ lead molecule aplithianine A (1) from a marine Aplidium sp. tunicate. Aplithianine A (1) showed potent inhibition against both J-PKAc alpha and wild-type PKAc alpha with an IC50 value 1 uM in the primary assay. Further mechanistic studies including co-crystallization and X-ray diffraction experiments revealed that 1 inhibited PKAc alpha catalytic activity by competitively binding to the ATP pocket. Human kinome profiling of 1 against a panel of 370 kinases revealed potent inhibition of select serine/threonine kinases in the CLK, DYRK, and PKG families with IC50 values ranging from 11-90 nM. An efficient, four-step total synthesis of 1 has been accomplished enabling further development of aplithianines as biologically relevant kinase inhibitors. Through the collaboration with the CCR medicinal chemistry accelerator, we have designed and synthesized more than 400 aplithianine analogs and successfully improved the biochemical potency to single digit nanomolar range. Based on data from the initial discovery and following optimization of alithianines, a manuscript and two provisional patent applications have been submitted and a following PCT application was also filed after one of the provisional applications. (4) Topoisomerase-3B: DNA Topoisomerase-3B (TOP3B) stands out among all mammalian topoisomerases due to its distinctive capability to resolve topological entanglements in both DNA and RNA. Deletion of the topoisomerase-3B (TOP3B) gene has been linked to various cancer types. This unique characteristic has made TOP3B a promising target for the development of new therapeutics for both cancer and RNA virus infections. The paired colorectal carcinoma HCT116 cell lines with (TOP3B-WT) and without TOP3B (TOP3B-KO) were developed for discovering TOP3B-specific proliferation modulators. In this high-throughput screening campaign, the NPCS has been engaged in the bioassay-guided fractionations of 38 TOP3B-active extracts leading to the identification of 76 active natural products including 20 new compounds. In FY2024, the NPCS 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.

NPCS 利用高通量筛选技术来帮助识别能够与选定的生化靶标或过程特异性相互作用或调节其功能的化合物和提取物。采用生物测定引导的天然产物提取物化学分馏来分离和纯化各个生物活性化合物。这些化合物的鉴定和结构表征为开发可以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。除了广泛的核磁共振和质谱分析之外，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进行严格评估。 2023 财年，我们一直在继续开展研究活动，以确定与多种分子靶标相互作用的生物活性天然产物，包括泛素连接酶 Cbl-b、默克尔细胞癌、J-PKAc α 激酶融合蛋白、拓扑异构酶-图3B，NCI60细胞系筛选组和间皮瘤(MPM)。 NPCS 总共完成了 98 个分离项目，鉴定了 267 种天然产物，其中包括 91 种新化合物。在所有化合物中，其中 151 种（包括 47 种新化合物）对所选靶标表现出生物活性。 (1) 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标。 2022 财年，我们报告了从海洋海绵 Plakortis sp 中鉴定出两种新型 Cbl-b 抑制剂：(+)-plakoramine A 和 (-)-plakoramine A。我们继续揭示了一种先前未描述的非酶促途径，通过其天然存在的单体对应物 plakinidine B 的光化学转化来形成 plakoramine A，plakinidine B 代表一种新的功能性荧光团，具有作为光敏剂和光化学触发亲电子剂的潜在用途。 Plakoramine 的故事已成功发表在 ACS 期刊 Organic Letters 上。 (2)默克尔细胞癌：默克尔细胞癌(MCC)是一种罕见但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，例如 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获益；因此，迫切需要替代治疗。从短致青霉中鉴定出两种新的细胞毒性含氧二酮哌嗪类化合物：短酰胺 E1 和 E2。两种化合物均对 Merkel 细胞癌细胞系 MCC13 细胞系表现出选择性细胞毒性，IC50 值分别为 2.6 和 2.5 mM。 （3）J-PKAcα激酶融合蛋白（PKADJ）：纤维板层肝细胞癌（FLHCC）是一种罕见的肝癌，患者人群年轻（35岁），5年生存率仅为30-35% 。 DNAJB1-PRKCA 致癌基因融合体在肿瘤中特异性表达，并且仅在 FLHCC 患者中检测到。 DNAJB1-PRKCA 基因融合产生一种具有酶活性的嵌合蛋白 J-PKAc α，它是 FLHCC 肿瘤发生的关键驱动因素。 PCMBS 开发了一种高通量、改良的夹心 ELISA 测定法，通过筛选 NCI 天然产物发现计划 (NPNPD) 最近创建的预分级天然产物库来鉴定 J-PKAc α 催化活性的选择性调节剂。 2023 财年，NPCS 已参与 36 种 PKADJ 活性提取物的生物测定指导分馏。 NPCS 通过提供多种生物活性天然产物，协助 PCMBS 发表了关于 ACS 药理学和转化科学的第一份 PKADJ HTS 手稿。去年，我们报道了从海洋 Aplidium sp. 中鉴定出一种新型 PKADJ 先导分子 aplithianine A (1)。被囊类。 Aplithianine A (1) 对 J-PKAc α 和野生型 PKAc α 均表现出有效抑制作用，在初步测定中 IC50 值为 1 uM。包括共结晶和 X 射线衍射实验在内的进一步机制研究表明，1 通过与 ATP 口袋竞争性结合来抑制 PKAc α 催化活性。 1 针对一组 370 种激酶的人类激酶组分析揭示了对 CLK、DYRK 和 PKG 家族中选定丝氨酸/苏氨酸激酶的有效抑制，IC50 值范围为 11-90 nM。 1 的高效四步全合成已经完成，从而能够进一步开发 aplithianines 作为生物学相关的激酶抑制剂。通过与CCR药物化学加速器的合作，我们设计合成了400多个aplithianine类似物，并成功将生化效价提高到个位数纳摩尔范围。基于最初发现和随后优化 alithianines 的数据，已提交一份手稿和两份临时专利申请，并在其中一份临时申请之后提交了后续 PCT 申请。 (4) 拓扑异构酶-3B：DNA 拓扑异构酶-3B (TOP3B) 在所有哺乳动物拓扑异构酶中脱颖而出，因为它具有解决 DNA 和 RNA 中拓扑缠结的独特能力。拓扑异构酶 3B (TOP3B) 基因的缺失与多种癌症类型有关。这种独特的特性使 TOP3B 成为开发癌症和 RNA 病毒感染新疗法的有前途的目标。开发了含有（TOP3B-WT）和不含TOP3B（TOP3B-KO）的配对结直肠癌HCT116细胞系，用于发现TOP3B特异性增殖调节剂。在这次高通量筛选活动中，NPCS 对 38 种 TOP3B 活性提取物进行了生物测定指导的分馏，从而鉴定了 76 种活性天然产物，其中包括 20 种新化合物。 2024 财年，NPCS 将通过优化基于分析指纹的去重复和项目选择流程，继续提高基于 HTS 的生物测定引导分馏平台的效率。我们还将整合新的计算模型和有机合成技术/方法，以建立我们在结构修饰、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 内广泛的化学多样性来实现潜在的抗癌和抗艾滋病毒应用。它依赖于与 MTP 测定开发和筛选部分、化学多样性开发部分以及蛋白质化学和分子生物学部分的紧密集成，以进行提取物筛选、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。我们组建了一个由具有有机合成、化学生物学、分子药理学、计算科学和光谱分析专业知识的校内和校外合作伙伴组成的广泛联盟，以帮助表征和推进我们的天然产品发现。天然产物化学科在 NCI 内具有独特的地位，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，可以提供对新靶标、途径或细胞过程功能的深入了解。它们在剖析和理解癌症发生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。

项目成果

Four New Pregnane-10,2-carbolactones from an Epipolasis sp. Marine Sponge.

来自 Epipolasis sp 的四种新 Pregnane-10,2-carbolactones。

• 发表时间: 2021
• 影响因子: 1.7
• 作者: Kang, Unwoo;Wang, Dongdong;Bokesch, Heidi R;Gustafson, Kirk R
• 通讯作者: Gustafson, Kirk R

Structure Revision of Poecillastrin C and the Absolute Configuration of the β-Hydroxyaspartic Acid Residue.

Poecillastrin C 的结构修正和β-羟基天冬氨酸残基的绝对构型。

• 发表时间: 2017-10-06
• 影响因子: 5.2
• 作者: Irie, Raku;Takada, Kentaro;Ise, Yuji;Ohtsuka, Susumu;Okada, Shigeru;Gustafson, Kirk R;Matsunaga, Shigeki
• 通讯作者: Matsunaga, Shigeki

Cembranoids from a Chinese Collection of the Soft Coral Lobophytum crassum.

来自中国软珊瑚 Lobophytum crasum 收藏的西松烷类化合物。

• 发表时间: 2016-06-03
• 影响因子: 5.4
• 作者: Zhao, Min;Cheng, Shimiao;Yuan, Weiping;Xi, Yiyuan;Li, Xiubao;Dong, Jianyong;Huang, Kexin;Gustafson, Kirk R;Yan, Pengcheng
• 通讯作者: Yan, Pengcheng

Cytotoxic Triterpenes from Salacia crassifolia and Metabolite Profiling of Celastraceae Species.

来自五层龙的细胞毒性三萜和卫矛科物种的代谢物分析。

• 发表时间: 2018-06-20
• 作者: Espindola, Laila S;Dusi, Renata G;Demarque, Daniel P;Braz;Yan, Pengcheng;Bokesch, Heidi R;Gustafson, Kirk R;Beutler, John A
• 通讯作者: Beutler, John A

Biotransformed Metabolites of the Hop Prenylflavanone Isoxanthohumol.

啤酒花异黄酮异黄腐酚的生物转化代谢物。

• 发表时间: 2019-01-22
• 作者: Kim, Hyun Jung;Yim, Soon;Han, Fubo;Kang, Bok Yun;Choi, Hyun Jin;Jung, Da;Williams, Darren R;Gustafson, Kirk R;Kennelly, Edward J;Lee, Ik
• 通讯作者: Lee, Ik