Natural Products Discovery and Characterization Through Network Collaborations

NPCS利用高通量筛选技术来帮助识别能够特异性地与选定的生化目标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2023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，包括泛素连接酶Cbl-b、默克尔细胞癌、j - ppkac α激酶融合蛋白、拓扑异构酶3b、NCI60细胞系筛选小组和间皮瘤（MPM）。NPCS共完成98个分离项目，鉴定出267个天然产物，其中新化合物91个。结果表明，其中151个化合物（包括47个新化合物）对所选靶点具有生物活性。(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点。在2022财年，我们报告了从海绵Plakortis sp中鉴定出两种新的ccl - B抑制剂，(+)-plakoramine A和(-)-plakoramine A。我们继续揭示了一种以前未描述的非酶途径，通过光化学转化其天然存在的单体对偶物plakinidine B形成plakoramine A， plakoramine B代表一种新的功能荧光团，具有光敏剂和光化学触发的亲电剂的潜在用途。关于plakoramine的报道已经成功地发表在美国化学学会的期刊《有机通讯》上。(2)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。从一株短囊青霉菌中分离到两种新的细胞毒性氧合二酮哌嗪，即短甲酰胺E1和E2。两种化合物对默克尔细胞癌细胞MCC13均表现出选择性细胞毒性，IC50值分别为2.6和2.5 mM。(3) J-PKAc α激酶融合蛋白（PKADJ）：纤维层状肝细胞癌（FLHCC）是一种罕见的肝癌，患者群体年轻（35岁），5年生存率仅为30-35%。DNAJB1-PRKCA致癌基因融合在肿瘤中特异性表达，并且仅在FLHCC患者中检测到。DNAJB1-PRKCA基因融合产生酶活性嵌合蛋白j - ppkac α，这是FLHCC肿瘤发生的关键驱动因素。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文库，PCMBS开发了一种高通量、改进的夹心ELISA方法，以鉴定j - ppkac α催化活性的选择性调节剂。在2023财年，NPCS已经参与了36个pkjj活性提取物的生物测定引导分离。NPCS通过提供几种具有生物活性的天然产品，协助PCMBS在ACS药理学与转化科学上发表了第一篇PKADJ HTS手稿。去年，我们报道了一种新的PKADJ引线分子阿plithi氨酸a(1)从海洋阿plidium sp. tunicate中鉴定出来。阿普氨酸A(1)对j - ppkac α和野生型ppkac α均有抑制作用，IC50值为1 uM。进一步的机制研究包括共结晶和x射线衍射实验表明，1通过与ATP袋竞争性结合来抑制ppkac α的催化活性。1对370种激酶的人类激酶组分析显示，它对CLK、DYRK和PKG家族中选定的丝氨酸/苏氨酸激酶具有有效的抑制作用，IC50值在11-90 nM之间。一个高效的，四步全合成1已经完成，使阿普硫氨酸作为生物学相关激酶抑制剂的进一步发展。通过与CCR药物化学加速器的合作，我们设计并合成了400多种阿普氨酸类似物，并成功地将生化效价提高到个位数纳摩尔范围。根据alithianines的初步发现和随后的优化数据，已提交了一份手稿和两份临时专利申请，并在一份临时申请之后提交了一份PCT申请。(4)拓扑异构酶3b: DNA拓扑异构酶3b （TOP3B）因其解决DNA和RNA拓扑纠缠的独特能力而在所有哺乳动物拓扑异构酶中脱颖而出。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希望的靶点。利用带（TOP3B- wt）和不带（TOP3B- ko）的配对结直肠癌HCT116细胞系，发现TOP3B特异性增殖调节剂。在这项高通量筛选活动中，NPCS已经参与了38种top3b活性提取物的生物测定引导分离，从而鉴定出76种活性天然产物，其中包括20种新化合物。在2024财年，NPCS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hts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。

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.