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: 10702593
• 负责人: Lin Du
• 金额: $ 124.47万
• 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/10702593
• 关键词: 2019-nCoV; Advanced Development; Alkaloids; Amino Acids; Biochemical; Biochemistry; Biological; Biological Assay; Biology; CCR; Cancer Biology; Cell physiology; Cellular biology; Chemicals; Chemistry; Chimeric Proteins; Collaborations; Collection; Complement; Computational Science; Computer Models; Cyclic AMP-Dependent Protein Kinases; DNA; DNA Single Strand Break; Data Analyses; Development; Disease; Enzymes; Evaluation; Exhibits; Extramural Activities; FOXO1A gene; Fibrolamellar Hepatocellular Carcinoma; Fingerprint; Fractionation; Gene Fusion; Generations; Genetic; Genetic Transcription; Goals; HIV; Immune checkpoint inhibitor; Immunology; Immunotherapeutic agent; Individual; Industrialization; Intervention; Investigation; Lead; Libraries; Malignant Neoplasms; Malignant neoplasm of prostate; Merkel cell carcinoma; Methodology; Microbe; Modification; Molecular; Molecular Biology; Molecular Conformation; Molecular Probes; Molecular Target; Names; Natural Products; Natural Products Chemistry; Neurosecretory Systems; New Agents; Nuclear Pore Complex; Octadecenoic Acids; Oncogenic; Organic Synthesis; PAX3 gene; PRKACA gene; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Plakortis; Plants; Play; Porifera; Positioning Attribute; Process; Protein Chemistry; RNA Viruses; Reporting; Research; Resources; Role; Scientist; Series; Skin Cancer; Source; Specificity; Structure; Structure-Activity Relationship; Synthesis Chemistry; Techniques; Technology; Topoisomerase; United States National Institutes of Health; Urochordata; Xenograft procedure; alternative treatment; analog; anti-cancer; assay development; base; bioactive natural products; cancer cell; dimer; enantiomer; experience; fungus; high throughput screening; improved; insight; interdisciplinary approach; marine organism; novel; pembrolizumab; preclinical development; programs; protein kinase A kinase; proto-oncogene protein c-cbl; response; scaffold; screening; skills; structural biology; therapeutic target; transcription factor; ubiquitin ligase

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.

我们利用高通量筛选技术来帮助识别可以与选定的生化靶点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。 2022 财年，我们一直在继续开展研究活动，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、默克尔细胞癌、PKA 激酶融合蛋白质和拓扑异构酶-3b。 (1) PAX3-FOXO1：对海洋水螅 Dentitheca habereri 的化学研究导致鉴定出八种新的二酰化 zoanthoxanthin 生物碱，命名为 dentithecamides A-H ，以及之前报道的三种类似物 zoamides B-D。这些化合物是第一个从水螅中报道的花黄素生物碱。 Dentithecamides A 和 B 以及 zoamides B-D 均具有构象移动的环庚二烯核心，可抑制 PAX3-FOXO1 调节的转录活性，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标。生物测定引导下的海洋海绵 Plakortis sp 活性组分的分离。提供了前所未有的二聚生物碱 plakoramine A。外消旋 plakoramine A 的手性分离产生了纯化的对映体 (+)-plakoramine A 和 (-)-plakoramine A，它们抑制 Cbl-b 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（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。从真菌 Clonostachysrosea 中分离出一种名为 Roseabol A 的新 11 氨基酸线性肽和已知化合物 13-oxo-trans-9,10-epoxy-11(E)-octadeceneic Acid。 13-oxo-trans-9,10-epoxy-11(E)-octadeceneic Acid 对 Merkel 细胞癌具有抑制活性，IC50 值为 16.5 uM。 (4) PKA激酶融合蛋白(PKADJ)：DNAJB1-PRKACA (PKADJ)致癌基因融合体已被确定为针对罕见的纤维板层肝细胞癌的有吸引力的抗肿瘤靶点。通过筛选 NCI 天然产物发现计划 (NPNPD) 最近创建的预分级天然产物库，开发了一种高通量测定法来鉴定 PKADJ 催化活性的选择性调节剂。 2022 年，NPCS 已开始对 35 种 PKADJ 活性提取物进行生物测定指导的分级分离。迄今为止，已从这些项目中鉴定出总共 9 种活性化合物。对海洋被囊动物 Aplidium sp. 的提取物进行生物测定指导的分馏，发现了两种新型生物碱：aplithianine A 和 B。 Aplithianine A 对 PKADJ 和宽型 PKA 均表现出有效的抑制作用，IC50 值为 1.1 uM 。 (5) 拓扑异构酶-3b：拓扑异构酶-3b (TOP3B) 是一种在转录过程中通过催化瞬时单链 DNA 断裂以及完整链通过该断裂而改变 DNA 拓扑结构的酶。研究表明，TOP3B 是包括 SARS-CoV-2 在内的正义 RNA 病毒在宿主体内有效复制所必需的，因此被提议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 参与了 18 种 TOP3B 活性提取物的生物测定指导分级分离。我们正在从这些项目中分离和鉴定活性天然产物。 (6)Discorhabdins：我们对discorhabdin L进行了结构-活性关系研究，这是一种天然产物，初步显示出有希望的前列腺癌异种移植结果。 2022财年，一系列新的discorhabdin天然产物被分离出来，并使用discorhabdin L作为支架来生成半合成衍生物，探索先导化合物的结构活性关系。我们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。