Chemoenzymatic synthesis and pharmacological evaluation of designer plant meroterpenoids

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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.