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

项目摘要 植物一直是药用天然产物的重要来源，约25%的FDA批准的药物 灵感来自植物化学物质。然而，基于植物的新药的发现和开发， 天然产物是具有挑战性的，由于三个主要瓶颈：（1）从复杂的化合物中重新发现已知化合物 植物提取物在生物活性指导的发现方法，（2）难以规模化生产的化学 通过有机合成或源植物提取的复杂铅结构，以及（3）铅的有限多样化 由于分子结构的复杂性。植物组学技术与植物合成生物学研究进展 通过基因引导的发现，为天然产物药物发现的这些一般瓶颈提供解决方案 approach.本文中，新植物代谢物的优先级排序可以通过基于质谱（MS）的方法来实现。 植物提取物的代谢组学和从植物基因组预测生物合成基因。异源 生物合成基因的表达允许靶植物天然产物的来源无关性生产， 微生物和真核宿主生物。生物合成途径的遗传修饰使得 目标天然产物类似物。植物环肽是一类具有开发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。我们实验室 最近鉴定了一种新的植物特异性肽环化酶，称为BURP结构域，它是一种自催化的环化酶。 铜依赖酶家族参与核糖体编码和后分泌的 修饰肽（RIPPs）通过酪氨酸和色氨酸进行侧链大环化。我们的目标是 克服上述瓶颈的植物为基础的药物发现，通过开发工作流程， 从植物中系统地发现环肽，表征BURP结构域肽环化酶， 环植物肽的体内或体外生产，并建立生物催化平台， 通过BURP结构域环化酶的环状植物肽用于治疗评价。在此，系统识别 新的侧链-大环RiPP类别将通过连接候选化合物的串联MS光谱来完成。 肽分析物与植物基因组和转录组中的BURP结构域基因。我们将研究其结构 和已知的自催化类的BURP结构域肽环化酶的功能，表征BURP结构域 肽环化酶，其作用于单独的前体肽底物并鉴定参与N-和 BURP结构域衍生的RIPP的C末端保护。最后，从抗病毒环肽生物碱库， 本研究将从枣树中提取抗癌活性肽，并从青冠草中提取抗癌活性肽 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价。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 药用植物天然产物的发现和开发。

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.