Chemoenzymatic synthesis and pharmacological evaluation of designer plant meroterpenoids

项目摘要/摘要 开花植物和苔类等陆地植物产生一系列天然产品，具有各种不同的 生物功能。聚酮类化合物，包括部分来源于萜类化合物的结构 生物合成途径和部分来源于聚酮合成酶的生物合成途径吸引了 几十年来，科学家们从他们独特而多样的生物活动中脱颖而出。神经活性植物类化合物，如 大麻中的植物大麻素，代表着一系列特别令人兴奋的化合物，具有治疗作用 由于他们有能力跨越血脑屏障，并与GPCR靶点接触，因此有希望实现这一目标。然而，大部分 到目前为止，文献中存在的药理数据主要集中在大麻素，Δ9-四氢大麻酚 (Δ9-THC)和大麻二酚(CBD)，而来自其他生产商的次要成分和独特类似物仍然较少 研究得很好。缺乏这些化合物的药理数据的部分原因是较少积累的更稀有的 在本地生产者中种植分叶萜类化合物，在分布较少的植物(即特定的地钱属植物)中生产， 以及缺乏能够从一个共同的中间体产生几个类似物的聚合合成路线。 在真核宿主(即酵母)中异源生产植物分叶萜类化合物已经完成，但 由于植物戊烯环化催化活性低和表达不佳而导致途径瓶颈 酶(如THCA合成酶)。细菌衍生的环化酶产生相同的密钥 中间体是邻苯二酚甲醚，为生物催化剂的生成提供了一种有吸引力的替代方案 植物类化合物及其类似物的生产。在这里，我提出了新自行车运动的发展 细菌生物合成酶工程生物催化剂用于化学酶法生产稀有和 设计类似植物的硫代萜类产品及其药理学评价以评估治疗前景。 这项提案旨在解决目前存在的低积累的稀有硫代萜类化合物的供应问题。 本地生产商，使用工程生物催化剂生成新颖的、结构多样化的支架，并测试 此类化合物的药理作用(即治疗前景)。在目标1中，我将设计生物合成途径 最近从摩尔实验室中鉴定出的化学酶Clz9和Tcz9，可以通过化学酶作用产生分叶萜类化合物 具有交替的区域选择性和空间修饰。而神经活性的硫代萜类化合物主要是 由开花的陆地植物产生，其他物种，如地钱或海洋细菌，产生类似的外观 天然产品。在目标2中，我将从海洋细菌和地钱草源中鉴定新的戊烯基环化酶， 扩展生物催化剂工具包，用于生产植物类化合物，特别是具有独特结构的化合物 立体化学和更大的立体修饰。在目标3中，生产的化合物将受到预- 药代动力学实验以确定可能的代谢产物，以及这两种化合物及其代谢 将对产品进行评估，以确定其激活人脑GPCR靶标的能力。这项研究建议将 使用来自细菌宿主的生物催化剂生产稀有和设计的植物硫代萜类化合物，并鉴定哪种 生产的类似物具有值得注意的治疗潜力。

Project Summary/Abstract Land plants like flowering plants and liverworts produce an array of natural products with various biological functions. Polyketide meroterpenoids, comprising structures partially derived from terpenoid biosynthetic pathways and partially derived from polyketide synthase biosynthetic pathways, have attracted scientists for decades from their unique and diverse biological activity. Neuroactive plant meroterpenoids, like phytocannabinoids from Cannabis sativa, represent a particularly exciting suite of compounds with therapeutic promise due to their ability to cross the blood-brain barrier and engage GPCR targets. However, much of the pharmacological data present in the literature to-date has focused on the cannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), while minor constituents and unique analogs from other producers remain less well studied. Lack of pharmacological data for these compounds is partly due to low accumulation of more rare plant meroterpenoids in native producers, production in less widespread plants (i.e. specific liverwort species), and lack of convergent synthetic routes capable of producing several analogs from one common intermediate. Heterologous production of plant meroterpenoids has been accomplished in eukaryotic hosts (i.e. yeast) but suffers from a pathway bottleneck caused by low catalytic activity and poor expression of plant prenyl cyclization enzymes (e.g. THCA synthase). Bacterially derived cyclization enzymes that generate the same key intermediate, an ortho-quinone methide, provide an attractive alternative for biocatalyst generation toward production of plant-like meroterpenoids and their analogs. Here, I propose the development of new cyclization biocatalysts engineered from bacterial biosynthetic enzymes for chemoenzymatic production of rare and designer plant-like meroterpenoid products and their pharmacological evaluation to assess therapeutic promise. This proposal aims to address issues of supply present for rare meroterpenoids with low accumulation in native producers, generate novel, structurally diverse scaffolds using engineered biocatalysts, and test the pharmacology (i.e. therapeutic promise) of such compounds. In Aim 1, I will engineer biosynthetic pathway enzymes recently identified from the Moore lab, Clz9 and Tcz9, to chemoenzymatically produce meroterpenoids with alternative regioselectivity and steric modification. While neuroactive meroterpenoids are predominantly produced by flowering land plants, other species, like liverworts or marine bacteria, produce similar-looking natural products. In Aim 2, I will identify new prenyl cyclase enzymes from marine bacteria and liverwort sources, expanding the toolkit of biocatalysts for producing plant-like meroterpenoids, especially compounds with unique stereochemistry and larger steric modifications. In Aim 3, produced compounds will be subjected to pre- pharmacokinetics experiments to determine likely metabolic products, and both compounds and their metabolic products will be assessed for ability to activate GPCR targets from the human brain. This research proposal will produce rare and designer plant meroterpenoids using biocatalysts from bacterial hosts and identify which of the produced analogs possess noteworthy therapeutic potential.