Discovery of UDP-Glycosyltransferases and BAHD-Acyltransferases Involved in the Biosynthesis of the Antidiabetic Plant Metabolite Montbretin A.

Plant specialized metabolism serves as a rich resource of biologically active molecules for drug discovery. The acylated flavonol glycoside montbretin A (MbA) and its precursor myricetin 3-O-(6'-O-caffeoyl)-glucosyl rhamnoside (mini-MbA) are potent inhibitors of human pancreatic α-amylase and are being developed as drug candidates to treat type-2 diabetes. MbA occurs in corms of the ornamental plant montbretia (Crocosmia x crocosmiiflora), but a system for large-scale MbA production is currently unavailable. Biosynthesis of MbA from the flavonol myricetin and MbA accumulation occur during early stages of corm development. We established myricetin 3-O-rhamnoside (MR), myricetin 3-O-glucosyl rhamnoside (MRG), and mini-MbA as the first three intermediates of MbA biosynthesis. Contrasting the transcriptomes of young and old corms revealed differentially expressed UDP-sugar-dependent glycosyltransferases (UGTs) and BAHD-acyltransferases (BAHD-ATs). UGT77B2 and UGT709G2 catalyze the consecutive glycosylation of myricetin to produce MR and of MR to give MRG, respectively. In addition, two BAHD-ATs, CcAT1 and CcAT2, catalyze the acylation of MRG to complete the formation of mini-MbA. Transcript profiles of UGT77B2, UGT709G2, CcAT1, and CcAT2 during corm development matched the metabolite profile of MbA accumulation. Expression of these enzymes in wild tobacco (Nicotiana benthamiana) resulted in the formation of a surrogate mini-MbA, validating the potential for metabolic engineering of mini-MbA in a heterologous plant system.

A Total Synthesis of Bifidenone.

The first total synthesis of bifidenone, a novel natural tubulin polymerization inhibitor, has been achieved in 12 steps starting from commercially available 1,4-dioxaspiro[4.5]decan-8-one. The synthesis includes a newly developed method to generate the dihydrobenzodioxolone core by palladium-catalyzed aerobic dehydrogenation. The three stereocenters were installed with an AD-mix-ß dihydroxylation step followed by a late-stage palladium-catalyzed decarboxylation-allylation procedure. The absolute stereochemistry of 3 was determined via 13a by single-crystal X-ray analysis.

Isolation and Identification of the Novel Tubulin Polymerization Inhibitor Bifidenone.

The pursuit of structurally novel compounds has led to the isolation of a series of neolignans (2-6), for which the structures have been determined from microgram quantities using microcryoprobe NMR technology. Compounds 2-6 provided some unexpectedly clear structure-activity relationship data, with compound 2 demonstrating significantly more potency in the in vitro cytotoxicity assay than the other analogues. Further screening found that compound 2 induces apoptosis with activation of caspase 3/7. The NCI Compare algorithm suggested that compound 2 acts through the inhibition of tubulin/microtubule dynamics. Compound 2 was confirmed to be a tubulin polymerization inhibitor that binds directly to tubulin.

Exploring diterpene metabolism in non-model species: transcriptome-enabled discovery and functional characterization of labda-7,13E-dienyl diphosphate synthase from Grindelia robusta.

Grindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surfaces, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G. robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the in vitro and in vivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G. robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts.

Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells.

Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol.

Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare.

Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual ßα-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids.

Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination.

The structural scaffolds of many complex natural products are produced by multifunctional type I polyketide synthase (PKS) enzymes that operate as biosynthetic assembly lines. The modular nature of these mega-enzymes presents an opportunity to construct custom biocatalysts built in a lego-like fashion by inserting, deleting, or exchanging native or foreign domains to produce targeted variants of natural polyketides. However, previously engineered PKS enzymes are often impaired resulting in limited production compared to native systems. Here, we show a versatile method for generating and identifying functional chimeric PKS enzymes for synthesizing custom macrolactones and macrolides. PKS genes from the pikromycin and erythromycin pathways were hybridized in Saccharomyces cerevisiae to generate hybrid libraries. We used a 96-well plate format for plasmid purification, transformations, sequencing, protein expression, in vitro reactions and analysis of metabolite formation. Active chimeric enzymes were identified with new functionality. Streptomyces venezuelae strains that expressed these PKS chimeras were capable of producing engineered macrolactones. Furthermore, a macrolactone generated from selected PKS chimeras was fully functionalized into a novel macrolide analogue. This method permits the engineering of PKS pathways as modular building blocks for the production of new antibiotic-like molecules.

Dereplication of natural products using minimal NMR data inputs.

A strategy for the dereplication of a complete or a partial structure using (1)H NMR, (1)H-(13)C HSQC and (1)H-(1)H COSY spectral data, a molecular formula composition range and structural fragments against a massive database of about 22 million compounds is considered. As the increasing availability of public online databases containing natural products continues to grow the potential of utilizing these resources for dereplication purposes increases. This work examines approaches for NMR dereplication of natural products and includes a comparison with approaches for molecular formula and mass-based dereplication. The strategy is an application of computer-assisted structure elucidation using ACD/Structure Elucidator and data obtained from the ChemSpider database hosted by the Royal Society of Chemistry.

Digging Deep for New Compounds from the Compass Plant, Silphium laciniatum.

The compass plant, Silphium laciniatum, is an iconic perennial plant of the North American tallgrass prairie. The plants of the tallgrass prairie historically have been subjected to a number of biological and environmental stresses. Among the adaptations developed by S. laciniatum is a large deep taproot. An investigation of the secondary metabolites found in the root of a S. laciniatum specimen has led to the identification of 15 new terpenoids (3-8, 10-17, and 22), which were screened for cytotoxic activity in the NCI-H460 human large-cell lung carcinoma cell line.

Diterpenes from the endangered goldenrod Solidago shortii.

Species extinction is tantamount to loss of chemical diversity, and so it is important to seize all opportunities to study species on the brink of extinction. Such studies are often hampered by the limited material available, but that obstacle is surmountable through collaboration with botanical gardens and advances in instrumentation. The goldenrod Solidago shortii is one example of an endangered species native to the United States. From S. shortii, one known diterpene (1), two new diterpenes (2 and 3), and three new hydrolysis products (4-6) are described. This work was made possible through collaboration with the Missouri Botanical Garden and with the use of highly sensitive microcryoprobe NMR technology for structure elucidation and VCD spectroscopy for the determination of absolute configuration.

Acetylated dammarane-type bisdesmosides from Combretum inflatum.

The first study of the chemical constituents of Combretum inflatum has resulted in the isolation of seven new acetylated dammarane-type bisdesmosides (1-7). Their structures were determined from microgram quantities on hand using Bruker BioSpin TCI 1.7 mm MicroCryoProbe technology, ESIMS, and comparison to data found in the literature. Compounds 1-7 were screened for inhibition of an Escherichia coli strain UTI89 biofilm, MRSA inhibition, and cytotoxicity in NCI-H460 human lung cancer cells. Compounds 3-7 reduced the growth of MRSA at 16 µg/mL by 71-45%, and compound 7 had an IC50 value of 3.9 µM in NCI-H460.

Cytotoxic and antibacterial beilschmiedic acids from a Gabonese species of Beilschmiedia.

High-throughput natural products chemistry methods have facilitated the isolation of eight new (1-8) and two known (9 and 10) beilschmiedic acid derivatives from the leaves of a Gabonese species of Beilschmiedia. Compounds 3-10 were isolated in microgram quantities, and the NMR data for structure elucidation and dereplication were acquired utilizing a Bruker BioSpin TCI 1.7 mm MicroCryoProbe. All of the compounds were screened for cytotoxic and antibacterial activity against NCI-H460 human lung cancer cells and a clinical isolate of methicillin-resistant Staphylococcus aureus, respectively. This is the first report of cytotoxic activity for the endiandric/beilschmiedic acid class of compounds.

Isolation of apoptosis-inducing stilbenoids from four members of the Orchidaceae family.

High-throughput natural product research produced a suite of anticancer hits among several species of the Orchidaceae family (Oncidium microchilum, O. isthmi, and Myrmecophila humboldtii). A commercial Oncidium sp. was also examined as a convenient source of additional material. Isolation and structure elucidation led to the identification of fifteen stilbenoids including a new phenanthraquinone and two new dihydrostilbenes. NMR data for structure elucidation and dereplication were acquired utilizing a Bruker BioSpin TCI 1.7-mm MicroCryoProbe or a 5-µL CapNMR capillary microcoil. Several compounds inhibited proliferation of NCI-H460 and M14 cancer cell lines. All compounds were also examined for their ability to induce apoptosis. Apoptosis induction was determined by measuring caspase 3/7 activation and LDH release in a NCI-H460 cell line. Based on these results, a portion of the extract from a commercially available Oncidium sp. was chemically modified in an attempt to obtain additional phenanthraquinones.

Novel pentadecenyl tetrazole enhances susceptibility of methicillin-resistant Staphylococcus aureus biofilms to gentamicin.

One method that bacteria employ to reduce their susceptibility to antibiotics is the formation of biofilms. We developed a robust 6-well plate biofilm assay to evaluate early-stage discovery compounds against methicillin-resistant Staphylococcus aureus (MRSA). Tissue culture-treated 6-well plates were selected for this assay because they facilitate the adherence of MRSA and enable accurate determination of the number of CFU in each well. The MRSA biofilms formed in this assay exhibit increased tolerances to clinically used antibiotics. Using this biofilm assay, we identified a novel potentiator of gentamicin against MRSA biofilms. The combination of gentamicin and pentadecenyl tetrazole is superior to clinically used MRSA antibiotics against these MRSA biofilms. This novel combination also exhibits synergistic effects on MRSA planktonic cells. This plant-derived compound reveals promise for its effectiveness and warrants further lead optimization as an antibiotic and aminoglycoside potentiator.

Antibiotic indole sesquiterpene alkaloid from Greenwayodendron suaveolens with a new natural product framework.

High-throughput natural products chemistry methods have led to the isolation of three new (1-3) and two known indole sesquiterpene alkaloids (4, 5) from Greenwayodendron suaveolens. Their structures were determined using CapNMR and MS. Pentacyclindole (1) was determined to possess a new natural product framework. Pentacyclindole (1) and polyalthenol (4) showed activity against clinical isolates of Staphylococcus aureus with polyalthenol (4) demonstrating a MIC(90) of 4 microg/mL.

Antibiofilm phenylethanoid glycosides from Penstemon centranthifolius.

Bioassay-guided fractionation of the antibacterial ethyl acetate-ethanol (50 : 50) extract obtained from the aerial parts of Penstemon centranthifolius led to the isolation of six phenylethanoid glycosides (1-6) and eleven iridoid glycosides (7-17). Their structures were determined on the basis of spectroscopic analysis and comparison with the literature. Among them, two phenylethanoid glycosides, 4'''-O-acetylverbascoside (1) and verbascoside (2), were found to show significant inhibition of the formation of bacterial biofilms by Escherichia coli UTI89. Compound 1 showed 77% biofilm inhibition at 2.5 microg/mL, and compound 2 showed 60% inhibition at 5 microg/mL.

Dammarane-type triterpene glycosides from Oncoba manii active against methicillin-resistant Staphylococcus aureus.

Drug-resistant bacteria are becoming more prevalent both in the community and in hospitals. In a search for new antibiotic leads, we used a high-throughput natural products chemistry approach to isolate one new (1) and two known (2, 3) dammarane-type triterpenes with mass-limited material from the African plant Oncoba manii. The new compound was determined by spectroscopic methods to be 1beta,2alpha,3beta,20(R)-tetrahydroxydammar-24-ene 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranoside. Compounds 1 and 2 inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA).

Bifidenone: Structure-Activity Relationship and Advanced Preclinical Candidate.

Bifidenone is a novel natural tubulin polymerization inhibitor that exhibits antiproliferative activity against a range of human cancer cell lines, making it an attractive candidate for development. A synthetic route was previously developed to alleviate supply constraints arising from its isolation in microgram quantities from a Gabonese tree. Using that previously published route, we present here 42 analogues that were synthesized to examine the structure-activity relationship of bifidenone derivatives. In addition to in vitro cytotoxicity data, data from murine xenograft and pharmacokinetic studies were used to evaluate the analogues. Compounds 45b and 46b were found to demonstrate promising efficacy in murine xenograft experiments, and 46b had significantly more potent in vitro antiproliferative activity against taxane-resistant cell lines compared to that of paclitaxel.

Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450 CYP725A4.

Natural product metabolic engineering potentially offers sustainable and affordable access to numerous valuable molecules. However, challenges in characterizing and assembling complex biosynthetic pathways have prevented more rapid progress in this field. The anticancer agent Taxol represents an excellent case study. Assembly of a biosynthetic pathway for Taxol has long been stalled at its first functionalization, putatively an oxygenation performed by the cytochrome P450 CYP725A4, due to confounding characterizations. Here, through combined in vivo (Escherichia coli), in vitro (lipid nanodisc), and metabolite stability assays, we verify the presence and likely cause of this enzyme's inherent promiscuity. Thereby, we remove the possibility that promiscuity simply existed as an artifact of previous metabolic engineering approaches. Further, spontaneous rearrangement and the stabilizing effect of a hydrophobic overlay suggest a potential role for nonenzymatic chemistry in Taxol's biosynthesis. Taken together, this work confirms taxadiene-5α-ol as a primary enzymatic product of CYP725A4 and provides direction for future Taxol metabolic and protein engineering efforts.

Cyclolignans from Scyphocephalium ochocoa via high-throughput natural product chemistry methods.

Two 2,7'-cyclolignans, ocholignans A and B, were obtained as mass-limited samples from Scyphocephalium ochocoa via high-throughput natural products chemistry methods. The rapid structure elucidation of each compound was primarily facilitated by NMR data acquisition using a capillary-scale NMR probe, CapNMR probe. Ocholignan A was found to possess significant in vitro antibacterial activity against Gram-positive bacteria methicillin-resistant Staphylococcus aureus ATCC 33591 and S. aureus 78-13607A with a MIC of 16 microg/mL, respectively.