Metabolism of carcinogenic pyrrolizidine alkaloids and pyrrolizidine alkaloid N -oxides by rat primary hepatocytes generate the same characteristic DHP-DNA adducts
Not provided.
Environmental Sciences & Ecology↗
SEARCH · Engineering Papers
Results for “alkaloids”
Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Alkaloids are associated with increased microbial diversity and metabolic function in poison frogs
Shifts in host-associated microbiomes can impact both host and microbes. It is of interest to understand how perturbations, like the introduction of exogenous chemicals, impact microbiomes. In poison frogs (family Dendrobatidae), the skin microbiome is exposed to alkaloids that the frogs sequester for defense. These alkaloids are antimicrobial; however, their effect on the frogs’ skin microbiome is unknown. To test this, we characterized microbial communities from field-collected dendrobatid frogs. Then, we conducted a laboratory experiment to monitor the effect of the alkaloid decahydroquinoline (DHQ) on the microbiome of two frog species with contrasting alkaloid loads in nature. In both datasets, we found that alkaloid-exposed microbiomes were more phylogenetically diverse, with an increase in diversity among rare taxa. Further, to better understand the isolate-specific response to alkaloids, we cultured microbial isolates from poison frog skin and found that many isolates exhibited enhanced growth or were not impacted by the addition of DHQ. To further explore the microbial response to alkaloids, we sequenced the metagenomes from high- and low-alkaloid frogs and observed a greater diversity of genes associated with nitrogen and carbon metabolism in high-alkaloid frogs. From these data, we hypothesized that some strains may metabolize the alkaloids. We used stable isotope tracing coupled to nanoSIMS (nanoscale secondary ion mass spectrometry), which supported the idea that some of these isolates are able to metabolize DHQ. Together, these data suggest that poison frog alkaloids open new niches for skin-associated microbes with specific adaptations, such as alkaloid metabolism, that enable survival in this environment.
59 BASIC BIOLOGICAL SCIENCES↗
Evaluating kratom alkaloids using PHASE
Kratom is a botanical substance that is marketed and promoted in the US for pharmaceutical opioid indications despite having no US Food and Drug Administration approved uses. Kratom contains over forty alkaloids including two partial agonists at the mu opioid receptor, mitragynine and 7-hydroxymitragynine, that have been subjected to the FDA’s scientific and medical evaluation. However, pharmacological and toxicological data for the remaining alkaloids are limited. Therefore, we applied the Public Health Assessment via Structural Evaluation (PHASE) protocol to generate in silico binding profiles for 25 kratom alkaloids to facilitate the risk evaluation of kratom. PHASE demonstrates that kratom alkaloids share structural features with controlled opioids, indicates that several alkaloids bind to the opioid, adrenergic, and serotonin receptors, and suggests that mitragynine and 7-hydroxymitragynine are the strongest binders at the mu opioid receptor. Subsequently, the in silico binding profiles of a subset of the alkaloids were experimentally verified at the opioid, adrenergic, and serotonin receptors using radioligand binding assays. The verified binding profiles demonstrate the ability of PHASE to identify potential safety signals and provide a tool for prioritizing experimental evaluation of high-risk compounds.
60 APPLIED LIFE SCIENCES↗
Characterization of the Entry Steps in Diterpenoid Alkaloid Biosynthesis
The diterpenoid alkaloids are a group of specialized metabolites where the terpenoid and alkaloid classes intersect, and which are found primarily within the Aconitum (Wolf’s-Bane) and Delphinium (Larkspur) genera. While there is considerable interest in these compounds for their wide range of bioactivities, their structural complexity poses a significant challenge for chemical synthesis. Biosynthesis offers an appealing alternative for production, however, little progress has been made towards elucidation of their biosynthetic pathways. Here, we employ a comparative transcriptomics approach to identify six enzymatic steps in the biosynthesis of atisinium, conserved across both Delphinium grandiflorum and Aconitum plicatum . Key to this pathway is a reductase which selectively incorporates ethanolamine over ethylamine into the diterpenoid scaffold. While the majority of diterpenoid alkaloids contain an ethylamine moiety, we demonstrate through isotope labeling in Aconitum callus cultures and a computational metabolomics approach that ethanolamine is, unintuitively, the preferred source of nitrogen for these metabolites. Identification of these enzymes and production of a key intermediate in a heterologous host paves the way for biosynthetic production of this group of metabolites with promise for medicinal applications.
biosynthesis↗
A yeast platform for high-level synthesis of tetrahydroisoquinoline alkaloids
The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate ( S )-reticuline to 4.6 g L –1 , a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.
59 BASIC BIOLOGICAL SCIENCES↗
Ergot alkaloid consumption alters serotonin receptor-induced vasoactivity in ovine umbilical vasculature
Consumption of ergot alkaloids during the second half of gestation has been shown to decrease umbilical artery vasoactivity resulting in decreased birth weights. Negative vascular effects of ergot alkaloids are mediated predominantly through serotonergic and adrenergic receptors in other tissues. Vasoactivity of serotonin (5-HT) receptors 5-HT 2A and 5-HT 1B/1D in umbilical artery and vein from ewes receiving endophyte-infected seed (E + 1.77 mg ergovaline/hd/d) or a control total mixed ration (CON; 0 mg ergovaline/hd/d) tall fescue seed at d-110 and d-133 of gestation was evaluated. Gravid reproduction tracts were collected from ewes. Two-mm sections of umbilical artery and vein were exposed to increasing concentrations of a 5-HT 1B/1D agonist and 5-HT 2A agonist. The 5-HT 1B/1D agonist did not stimulate a contractile response in artery or vein or either gestation time point. 5-HT 2A agonist caused large responses in artery with greatest occurring at d-110 and decreasing in magnitude as days of gestation increased ( p < 0.05). On d-110 and 133 of gestation, arteries from CON ewes had greater contractile response than arteries collected from E+ ewes ( p < 0.05). Veins responded to increasing concentrations of the 5-HT 2A agonist. Maximal d-110 vein response was greater than d-133 when exposed to 5-HT 2A agonist ( p < 0.05). Unlike the artery, veins from E+ ewes had greater d-133 contractile response than CON ( p < 0.05). Vascular contractions of umbilical artery and vein are induced by 5-HT 2A receptor activity and not 5-HT 1B/1D . Umbilical artery 5-HT 2A receptor activity was more sensitive to seed treatment and could be responsible for ergot alkaloid-induced intra-uterine growth restriction.
Toxicology↗
Diterpenoid Alkaloid Biosynthesis in the Delphinium and Aconitum Genera
Diterpenoid alkaloids are known for their range of potential medicinal applications and are found primarily in the Delphinium and Aconitum genera. We sequenced RNA isolated from different tissue types of Delphinium grandiflorum, Aconitum plicatum, and Aconitum lycoctonum to identify six entry steps in their biosynthesis.
biosynthesis↗
Bioinspired Diversification Approach Toward the Total Synthesis of Lycodine-Type Alkaloids
Nitrogen heterocycles (azacycles) are common structural motifs in numerous pharmaceuticals, agrochemicals, and natural products. Many powerful methods have been developed and continue to be advanced for the selective installation and modification of nitrogen heterocycles through C–H functionalization and C–C cleavage approaches, revealing new strategies for the synthesis of targets containing these structural entities. Here, we report the first total syntheses of the lycodine-type Lycopodium alkaloids casuarinine H, lycoplatyrine B, lycoplatyrine A, and lycopladine F as well as the total synthesis of 8,15-dihydrohuperzine A through bioinspired late-stage diversification of a readily accessible common precursor, N-desmethyl-β-obscurine. Key steps in the syntheses include oxidative C–C bond cleavage of a piperidine ring in the core structure of the obscurine intermediate and site-selective C–H borylation of a pyridine nucleus to enable cross-coupling reactions.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast
Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker’s yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.
59 BASIC BIOLOGICAL SCIENCES↗
Transcriptomics outputs and phylogenetic trees used for pathway discovery of diterpenoid alkaloids in Delphinium and Aconitum
Transcriptome assemblies, open reading frames in nucleotide and peptide sequences, clustered transcriptomes and corresponding amino acid files, and expression matrices in TPM and raw counts for RNA-seq datasets from Delphinium grandiflorum, Aconitum plicatum, Aconitum lycoctonum, Aconitum carmichaelii, Aconitum japonicum, Aconitum kusnezoffii, and Aconitum vilmorinianum. Also included are phylogenetic trees for terpene synthases and cytochromes P450 mined from these assemblies.
biosynthesis↗
Exploring the potential of applying infrared vibrational (micro)spectroscopy in ergot alkaloids determination: Techniques, current status, and challenges
Explore the source record for details and available documents.
99 GENERAL AND MISCELLANEOUS↗
A Phenotarget Approach for Identifying an Alkaloid Interacting with the Tuberculosis Protein Rv1466
In recent years, there has been a revival of interest in phenotypic-based drug discovery (PDD) due to target-based drug discovery (TDD) falling below expectations. Both PDD and TDD have their unique advantages and should be used as complementary methods in drug discovery. The PhenoTarget approach combines the strengths of the PDD and TDD approaches. Phenotypic screening is conducted initially to detect cellular active components and the hits are then screened against a panel of putative targets. This PhenoTarget protocol can be equally applied to pure compound libraries as well as natural product fractions. Here we described the use of the PhenoTarget approach to identify an anti-tuberculosis lead compound. Fractions from Polycarpa aurata were identified with activity against Mycobacterium tuberculosis H37Rv. Native magnetic resonance mass spectrometry (MRMS) against a panel of 37 proteins from Mycobacterium proteomes showed that a fraction from a 95% ethanol re-extraction specifically formed a protein-ligand complex with Rv1466, a putative uncharacterized Mycobacterium tuberculosis protein. The natural product responsible was isolated and characterized to be polycarpine. The molecular weight of the ligand bound to Rv1466, 233 Da, was half the molecular weight of polycarpine less one proton, indicating that polycarpine formed a covalent bond with Rv1466.
phenotarget approach, MRMS, polycarpine, drug disc↗
Verazine biosynthesis from simple sugars in engineered Saccharomyces cerevisiae
Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW). Finally, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.
59 BASIC BIOLOGICAL SCIENCES↗
Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme
A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer’s medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC 50 = 20 μM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4’-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.
60 APPLIED LIFE SCIENCES↗
Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins
Radiation of the plant pyridoxal 5'-phosphate (PLP)-dependent aromatic L -amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic L -amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. In this paper, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.
59 BASIC BIOLOGICAL SCIENCES↗
Antifungal and Phytotoxic Activities of Isolated Compounds from Helietta parvifolia Stems
The identification of natural and environmentally friendly pesticides is a key area of interest for the agrochemical industry, with many potentially active compounds being sourced from numerous plant species. In this study, we report the bioassay-guided isolation and identification of phytotoxic and antifungal compounds from the ethyl acetate extract of Helietta parvifolia stems. We identified eight compounds, consisting of two coumarins and six alkaloids. Among these, a new alkaloid, 2-hydroxy-3,6,7-trimethoxyquinoline-4-carbaldehyde (6), was elucidated, along with seven known compounds. The phytotoxicity of purified compounds was evaluated, and chalepin (4) was active against Agrostis stolonifera at 1 mM with 50% inhibition of seed germination and it reduced Lemna pausicotata (duckweed) growth by 50% (IC50) at 168 μM. Additionally, we evaluated the antifungal activity against the fungal plant pathogen Colletotrichum fragariae using a thin-layer chromatography bioautography assay, which revealed that three isolated furoquinoline alkaloids (flindersiamine (3), kokusagenine (7), and maculine (8)) among the isolated compounds had the strongest inhibitory effects on the growth of C. fragariae at all tested concentrations. Our results indicate that these active natural compounds, i.e., (3), (4), (7), and (8), could be scaffolds for the production of more active pesticides with better physicochemical properties.
59 BASIC BIOLOGICAL SCIENCES↗
Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (-)-Eurotiumin A
Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to > 99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗