Discovery, Biosynthesis and Engineering of Side-Chain-Macrocyclic Plant Peptides

侧链大环植物肽的发现、生物合成和工程化

• 批准号: 10653247
• 负责人: Roland David Kersten
• 金额: $ 33.97万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10653247
• 关键词: Alkaloids; Anabolism; Biological Assay; Biological Availability; C-terminal; Cells; Celosia; Chemicals; Chinese; Complex; Copper; Cyclic Peptides; Development; Engineering; Enzymes; FDA approved; Family; Genes; Genetic; In Vitro; Lead; Libraries; Mass Spectrum Analysis; Medicinal Plants; Medicine; Mining; Modification; Molecular; Natural Product Drug; Natural Products; Oral; Organic Synthesis; Organism; Pathway interactions; Peptide Library; Peptides; Periodicity; Pharmaceutical Preparations; Pharmacologic Substance; Plant Extracts; Plant Genes; Plant Genome; Plant Sources; Plants; Production; Research; Route; Source; Specificity; Structure; Technology; Tertiary Protein Structure; Testing; Trees; Tryptophan; Tyrosine; Viral; Viral Cancer; Ziziphus; analog; anti-cancer; cancer therapy; design; drug development; drug discovery; genetic information; genome resource; guided inquiry; in vivo; manufacturing facility; metabolomics; microbial; peptide natural products; plant genetics; plant growth/development; plant metabolites; synthetic biology; tandem mass spectrometry; therapeutic evaluation; transcriptome

PROJECT SUMMARY Plants have been an important source of medicinal natural products, as ~25% of FDA-approved drugs are inspired by plant chemicals. However, discovery and development of new pharmaceuticals based on plant natural products is challenging due to three main bottlenecks: (1) rediscovery of known compounds from complex plant extracts during bioactivity-guided discovery approaches, (2) difficult scaled production of chemically complex lead structures by organic synthesis or source plant extraction, and (3) limited diversification of lead structures due to molecular complexity. Recent advances in plant omics-technologies and plant synthetic biology offer solutions to these general bottlenecks of natural product drug discovery by a gene-guided discovery approach. Herein, prioritization of new plant metabolites can be enabled by mass spectrometry (MS)-based metabolomics of plant extracts and prediction of biosynthetic genes from plant genomes. Heterologous expression of biosynthetic genes allows source-independent production of target plant natural products in microbial and eukaryotic host organisms. Genetic modification of biosynthetic pathways enables the formation of target natural product analogs. Cyclic plant peptides are a class of plant natural products with potential applications in antiviral and anticancer therapy due to oral bioavailability, stability and diversifiability. Our lab recently characterized a new plant-specific peptide cyclase called the BURP domain, which is an autocatalytic copper-dependent enzyme family involved in the biosynthesis of ribsomally encoded and posttranslationally modified peptides (RiPPs) with side-chain-macrocyclizations via tyrosines and tryptophans. Our objective is to overcome the aforementioned bottlenecks of plant-based drug discovery by developing a workflow for the systematic discovery of cyclic peptides from plants, characterizing BURP domain peptide cyclases for scaled in vivo or in vitro production of cyclic plant peptides and establishing a biocatalytic platform for the diversification of cyclic plant peptides via BURP domain cyclases for therapeutic evaluation. Herein, systematic identification of new side-chain-macrocyclic RiPP classes will be accomplished by connecting tandem MS spectra of candidate peptide analytes to BURP domain genes in plant genomes and transcriptomes. We will investigate the structure and function of known autocatalytic classes of BURP domain peptide cyclases, characterize BURP domain peptide cyclases, which act on separate precursor peptide substrates and identify enzymes involved in N- and C-terminal protection of BURP-domain-derived RiPPs. Finally, libraries of antiviral cyclopeptide alkaloids from Chinese date tree (Ziziphus jujuba) and anticancer celogentin peptides from Celosia argentea will be generated by design and optimization of biocatalytic routes and evaluated in in vivo cell-based assays. As plant genomic resources are rapidly growing, the proposed research will set the stage for using plant genetic information for the discovery and development of medicinal plant natural products.

项目概要 植物一直是药用天然产物的重要来源，约 25% 的 FDA 批准药物是 灵感来自于植物化学物质。然而，基于植物的新药物的发现和开发 天然产物由于三个主要瓶颈而具有挑战性：（1）从复杂的化合物中重新发现已知化合物 生物活性引导发现方法中的植物提取物，(2) 难以大规模生产化学物质 通过有机合成或来源植物提取来实现复杂的铅结构，以及（3）铅的有限多样化 由于分子结构的复杂性。植物组学技术和植物合成生物学的最新进展 通过基因引导的发现为天然产物药物发现的这些普遍瓶颈提供解决方案 方法。在此，可以通过基于质谱 (MS) 的方法对新植物代谢物进行优先排序 植物提取物的代谢组学和植物基因组生物合成基因的预测。异源的 生物合成基因的表达允许目标植物天然产物的来源独立生产 微生物和真核宿主生物。生物合成途径的基因修饰能够形成 目标天然产物类似物。环植物肽是一类具有潜力的植物天然产物 由于口服生物利用度、稳定性和多样性，其在抗病毒和抗癌治疗中的应用。我们的实验室 最近表征了一种新的植物特异性肽环化酶，称为 BURP 结构域，它是一种自催化 铜依赖性酶家族参与核糖体编码和翻译后生物合成 通过酪氨酸和色氨酸进行侧链大环化的修饰肽（RiPP）。我们的目标是 通过开发一个工作流程来克服上述基于植物的药物发现的瓶颈 从植物中系统地发现环肽，表征 BURP 结构域肽环化酶 体内或体外生产环状植物肽并建立多样化的生物催化平台 通过 BURP 结构域环化酶对环状植物肽进行治疗评估。在此，系统地鉴定 新的侧链大环 RiPP 类别将通过连接候选的串联 MS 谱来完成 植物基因组和转录组中 BURP 结构域基因的肽分析物。我们将研究结构 以及 BURP 结构域肽环化酶的已知自催化类别的功能，表征 BURP 结构域 肽环化酶，作用于单独的前体肽底物并识别参与 N- 和 BURP 结构域衍生的 RiPP 的 C 端保护。最后，抗病毒环肽生物碱文库 将产生枣树（Ziziphus jujuba）和来自鸡冠花的抗癌celogentin肽 通过设计和优化生物催化途径并在体内基于细胞的测定中进行评估。作为植物基因组 资源正在迅速增长，拟议的研究将为利用植物遗传信息奠定基础 药用植物天然产物的发现和开发。