Chemoenzymatic synthesis and pharmacological evaluation of designer plant meroterpenoids

项目总结/摘要 陆地植物如开花植物和地钱产生一系列天然产物， 生物功能。包含部分衍生自萜类化合物的结构的聚酮部分萜类化合物 生物合成途径和部分来源于聚酮合酶生物合成途径，吸引了 几十年来，科学家们从它们独特而多样的生物活动中获益匪浅。具有神经活性的植物部分萜类化合物，如 植物大麻素，代表了一组特别令人兴奋的化合物， 由于它们能够穿过血脑屏障并与GPCR靶点结合，因此具有良好的前景。然而，大部分 迄今为止，文献中的药理学数据集中在大麻素，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 好好研究。缺乏这些化合物的药理学数据，部分原因是由于更稀有的 在本地生产者中的植物部分萜类，在分布不太广的植物中的生产（即特定的苔类植物物种）， 以及缺乏能够从一种共同中间体生产几种类似物的会聚合成路线。 在真核宿主（即酵母）中已经完成了植物类萜的异源生产，但 由于植物异戊二烯环化的低催化活性和低表达导致的途径瓶颈 酶（例如THCA合酶）。细菌衍生的环化酶产生相同的键 中间体，邻醌甲基化物，为生物催化剂生成提供了一种有吸引力的选择， 产生植物样的部分萜类化合物及其类似物。在这里，我建议开发新的环化 由细菌生物合成酶工程化的生物催化剂，用于化学酶促生产稀有和 设计师植物样meroterpenoid产品及其药理学评价，以评估治疗前景。 该提案旨在解决目前存在的低积累的稀有部分萜类化合物的供应问题， 天然生产者，使用工程生物催化剂产生新颖的，结构多样的支架，并测试 这些化合物的药理学（即治疗前景）。在目标1中，我将设计生物合成途径 最近从摩尔实验室鉴定出的酶Clz 9和Tcz 9，用于化学酶促产生类萜 具有可选择的区域选择性和空间修饰。虽然神经活性部分萜类主要是 由开花的陆地植物产生，其他物种，如地钱或海洋细菌， 天然产品。在目标2中，我将从海洋细菌和苔类植物中鉴定新的异戊二烯环化酶， 扩大生物催化剂的工具包，用于生产植物样的部分萜类化合物，特别是具有独特生物活性的化合物， 立体化学和更大的空间修饰。在目标3中，将对所产生的化合物进行预处理。 药代动力学实验，以确定可能的代谢产物，以及化合物及其代谢产物 将评估产品激活人脑GPCR靶点的能力。这项研究计划将 使用来自细菌宿主的生物催化剂生产稀有和设计的植物meroterpenoids，并确定哪些是 所产生的类似物具有显著的治疗潜力。

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.