Abstract Arthropods use chemical defenses against predators by releasing toxic or repellent compounds from specialized exocrine glands. Oribatulid mites (Oribatida: Oribatulidae) contain mandelonitrile hexanoate (MNH); when they are subjected to physical stimuli, MNH decomposes to release hydrogen cyanide (HCN), suggesting that cyanogenesis is an effective defense strategy in these mites. However, the underlying process remains unclear. Here, we investigated the defensive secretions of the oribatid mites, Phauloppia adjecta and Oribatula sakamorii, and identified MNH and mandelonitrile octanoate (MNO), along with the sesquiterpenes β-selinene and calarene. Chiral gas chromatography analysis determined the absolute configurations of MNH and MNO as the (R)-isomers, consistent with the configuration of mandelonitrilea known defensive compound in polydesmid millipedes. (R)-MNO did not undergo spontaneous hydrolysis in water at pH 3−8, suggesting that a mite-derived enzyme is required for HCN production. In vitro experiments showed that a crude mite enzyme extract incubated with (R)-MNO generated over 200 nmol of HCN, whereas negligible production occurred in the controls. These findings support an enzyme-dependent cyanogenic defense mechanism wherein mandelonitrile esters stored in the oil glands release HCN upon enzyme interaction during disturbance, potentially protecting conspecifics from predation.
Abstract Industrial processing of hemp (Cannabis sativa L.) produces side-streams, such as CBD-depleted “mother liquor” (CBD-ML), that are rich in structurally diverse but underexplored minor cannabinoids. To accelerate the discovery of bioactive cannabinoids within this complex matrix, we applied a computational pipeline integrating loop-injection high-resolution time-of-flight mass spectrometry (HR-TOF MS) chemical profiling with anti-inflammatory bioassay data. Using an ensemble of random forest models, we correlated mass spectrometric features with the inhibition of nitric oxide (NO) production in LPS-stimulated RAW264.7 macrophages. This predictive model identified an ion at m/z 357.2071 as a top predictor of bioactivity. Targeted isolation and structural elucidation by HRMS and NMR identified the bioactive compound as cannabidiolic acid ethyl ester (CBDA-EE), which likely arises as a byproduct during industrial processing of hemp. Validation studies confirmed that CBDA-EE modulates oxylipin production in stimulated macrophage cells, significantly reducing proinflammatory prostaglandins, including prostaglandin E2, while maintaining levels of prostaglandin D2 (PGD2) and its downstream metabolite 15-deoxy-PGD2. These findings demonstrate the utility of machine learning in streamlining the discovery of unstudied bioactive natural products from complex botanical mixtures.
Abstract Genome mining is an efficient strategy for natural product discovery, yet its application to terpene synthases frequently results in the repeated identification of identical products. Cembranoids, a class of coral-derived natural products featuring 14-membered carbocyclic scaffolds, present formidable challenges for chemical synthesis. To date, only two biosynthetic precursors have been synthesized across fifteen coral enzymes from six independent studies. To address these limitations, we developed Ariadne, an integrated platform for genome-wide targeted mining of terpene synthases from corals. Using this platform, we identified five cembrene synthases, including two with high sequence similarity to known cembrene B synthases and three low-similarity enzymes experimentally validated through heterologous expression in yeast, achieving a prediction accuracy of 80% from ninety terpene synthase homologs. Leveraging the rapid identification of novel cembrene synthases, phylogenetic analysis further uncovered the evolutionary trajectory of this enzyme family from coral, providing valuable guidance for the engineering of an early-diverging enzyme, which is capable of synthesizing an unreported cembrene scaffold by the coral enzyme. Collectively, this work establishes a new paradigm for terpene synthase discovery and substantially advances the genome mining in marine animals as well as the combinatorial biosynthesis of marine-derived natural products.
Abstract The enantiomeric distribution of α- and β-santalol in various Santalum and Osyris species of different origins was determined by the combination of enantioselective gas chromatography and chiral tagging molecular rotational resonance spectroscopy, supported by the preparation of (+)- and (−)-α-santalol standards by total synthesis and isolation of (−)-β-santalol from Santalum album essential oil. (−)-β-Santalol was largely dominant in all Santalum essential oil samples (>99%) except for the mixtures of santalols produced by biotechnology. The enantiomeric distribution of α-santalol was much less in favor of the levorotatory enantiomer, contained in percentages up to 5 and 45% in some Santalum and Osyris samples, respectively. The olfactory evaluations of synthetic (+)- and (−)-α-santalol samples by a panel of 56 assessors showed that they were very weak to odorless for most panelists.
Abstract A soil-derived Mycoarthris sp. isolate yielded a combination of eight new and known secondary metabolites consisting of four cyclic tetrapeptides, omnipolyphilins A−D (1−4), and four lipopeptides, mycoarpeptins A−D (5−8). The structures of natural products 1−8 were established based on analysis of the HRESIMS, MS/MS fragmentation, and 1D and 2D NMR data. Their absolute configurations were determined using Marfey’s analysis, chemical synthesis, and conformational analysis. Omnipolyphilins A−D (1−4) stand as a unique group of fungal cyclopeptides characterized by the presence of a hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (HPIC) and 3-hydroxytyrosine (DOPA). Mycoarpeptins A−D (5−8) are acyclic lipopeptides featuring non-proteinogenic β-aminoisobutyric acid (BAIBA) residues. A survey of the metabolite profiles indicated the likelihood of regional and phylogenetic variation in the production of omnipolyphilins and mycoarpeptins.
Abstract As part of our continuing search for antifungal natural products, we investigated the secondary metabolites of Streptomyces sp. SID10815, an actinomycete isolated from a ground-nesting bee-associated environment that exhibited notable antifungal activity in preliminary screenings. Herein, we report the discovery of four new N-acetylcysteine-modified ansamycins (1−4) and three new hydroxymycotrienin analogues (5−7), together with six known ansamycins (8−13), produced by this strain. These metabolites were detected and prioritized through a metabolomics-guided dereplication strategy integrating LC−MS profiling and bioactivity correlation. Their structures were elucidated by comprehensive spectroscopic analyses, including 1D and 2D nuclear magnetic resonance (1H, 13C, COSY, HSQC, HMBC, and NOESY) in combination with high-resolution mass spectrometry. The newly identified metabolites expand the structural diversity of the ansamycin family and provide a basis for future structure−activity relationship studies of N-acetylcysteine-conjugated ansamycins. These findings define a distinctive ansamycin profile of Streptomyces sp. SID10815 that may serve as a useful chemical signature for future comparative studies of insect-associated actinomycetes.
Abstract Multinuclear non-heme iron-dependent oxidases (MNIOs) constitute an emerging family of post-translational modifying enzymes that catalyze atypical modifications. Despite their widespread distribution across bacteria, only a small number of MNIO family members have been biochemically characterized, and the catalytic capabilities, product structures, and physiological functions of the vast majority remain unexplored. Here, we report the discovery and characterization of aen gene cluster, a previously uncharacterized MNIO-associated gene cluster in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We demonstrate that the AenBC enzyme complex catalyzes four-electron oxidation of all five cysteine residues in the precursor peptide AenA, yielding a mature product, aeruginin, whose cysteine-derived modifications are most consistent with five 5-thiooxazole motifs. Under competitive metal incubation conditions, HR-MS analysis revealed that aeruginin preferentially associates with Fe3+ rather than copper. Functional analyses reveal that deletion of aenA results in a highly specific growth defect under chlorite stress, accompanied by significant upregulation of the periplasmic disulfide isomerase DsbG. Together, these findings expand the known structural diversity of MNIO-derived RiPPs and suggest a potential role for aeruginin in chlorite responsive periplasmic stress adaptation.
Abstract Engineered Aspergillus oryzae strains possessing four biosynthetic genes (AOL_s00215g276−279) from carnivorous fungus Arthrobotrys oligospora enabled the isolation of 17 toluquinol-derived meroterpenoids. Thirteen were previously undescribed, including (±)-spinulinoid G (1a/1b), (±)-kuhistanol H (2a/2b), (±)-anthoponoid J (3a/3b), (±)-anthoponoid K (4a/4b), (+)-nitrosporeunol E (5a), (E)-2-methyl-3′-methoxyfarnesyl toluquinol (6), methylfarnesylquinone B (7), methylfarnesylquinone C (8), and didehydroconicol B (9), along with four known analogues (5b, 10−12). Their structures were elucidated by extensive spectroscopic analyses, and the absolute configurations were assigned by TDDFT-ECD calculations and/or comparison of optical rotation data with those of structurally related compounds. Several compounds (2a, 2b, 4a, 10, and 12) exhibited cytotoxic activity against human cancer cell lines BGC803, HCT116, MIA PaCa-2, and HeLa. Compound 2b showed prominent cytotoxicity against HCT116 and HeLa cells, with IC50 values of 7.69 μM and 5.60 μM, respectively. Furthermore, network pharmacology highlighted compounds 2b and 4a and suggested HIF1A and PIK3CA as candidate targets, for which molecular docking predicted plausible binding modes. These findings expand meroterpenoid structural diversity and provide promising small-molecule scaffolds for anticancer lead development.
Building on the 2012 study "Analysis and Purification of Bioactive Natural Products", this work further explores the essential aspects that impact the purity and utility of contemporary natural product (NP) isolates. Recent advancements in FAIR data sharing enabled a secondary ("meta") analysis of the raw NMR spectra of 100 NPs selected randomly to assess their purity using quantitative NMR (qNMR). First, all 100 NPs were analyzed via rapid integration-based qNMR. Second, a detailed qNMR analysis of ten representative NPs involving impurity assignments and spreadsheet annotation was performed and coverage probabilities generated using the NIST uncertainty machine. The average purity of the isolates was ∼84% (±5%), equivalent to impurity levels of 16 (11-21)%. Outcomes show that 1H NMR spectra acquired primarily for structure determination are adequate for purity analysis. Consideration of all observed peak patterns made purity determinations cohesive and permitted an estimation of impurity identities. With proper instructions in place, the methodology became robust to the point that different participants generated congruent results. Guidelines for purity determination with relative (100%-based) qNMR methodology are presented. Further reflection on the results indicates the need for critical scrutiny of current practices of extraction, isolation, and purification involved in bioactive NP discovery.
Elucidating natural products encoded by cryptic biosynthetic gene clusters (BGCs) in agriculturally important biocontrol fungi such as Trichoderma afroharzianum T22 (ThT22) could lead to the discovery of new compounds that may be explored as potential agrochemicals. Herein, we discovered new lipopeptide natural products, trichopeptide A-D, via genome mining and heterologous reconstitution of the tep BGC from ThT22. Comprehensive NMR, high-resolution mass spectrometry (HRMS), tandem mass spectrometry (MS/MS), Marfey's analysis, and chemical synthesis experiments established the structures of trichopeptides as linear pentapeptides acylated with a conjugated triene acid at the N-terminus. A notable structural feature of trichopeptides is the presence of a nonproteinogenic amino acid residue, (Z)-dehydrobutyrine, in the linear peptide. The residue is proposed to be derived from threonine by the action of a noncanonical dehydrating condensation domain (DhC) in the nonribosomal peptide synthetase (NRPS) TepB. This represents only the second example in which a fungal NRPS is responsible for the biosynthesis of dehydroamino acids in a peptide product. Our results expand the chemical inventory of an agriculturally important fungus and pave the way for the future investigation of possible plant-ThT22 interactions mediated by lipopeptides.
Marine cyanobacteria are a rich source of diverse bioactive natural products, targeting proteins involved in many diseases. Here, we combined metagenomic analysis to enhance the structure elucidation process of two new cyclodepsipeptides named dapalides D (1) and E (2) from a collection of a cyanobacterial mat containing multiple Dapis species from Guam. Dapalides D/E are composed of 11 amino acids, including multiple identical units with different configurations. Enantioselective amino acid identification of the acid hydrolyzate established the identity of amino acids, including the configuration of α/β-stereogenic centers. Identification and analysis of the dapalides D/E biosynthetic gene cluster from a metagenome-assembled genome aided the elucidation of α-configuration and establishment of the order of individual building blocks, collectively revealing the total structure. Phylogenomic analysis indicates that the dapalides D/E producer belongs to Dapis sp. (Dapis sp. VPG23-80 MAG-2), which shares a 95.2% average nucleotide identity with Dapis sp. VPG23-80 MAG-1, the producer of dapalides A-C that cooccurs in the same assemblage. Dapalide D (1) showed moderate growth inhibitory activity against various cancer cell lines. This work expands the dapalide structure class and further highlights the use of combined chemical and metagenomic analyses for natural product structure elucidation.
Abstract Liver X receptors (LXRα/β) are members of the nuclear receptor superfamily that have garnered attention over the last two decades. LXRα/β regulate myriad biological functions, including metabolism, cholesterol homeostasis, and immune responses, suggesting they may be effective drug targets for a range of diseases. Kuwanon G, a botanical-origin flavonoid, has previously been shown to regulate LXR-driven biological pathways, but the exact target of this compound has not been fully demonstrated. We conducted LXRα/β radioligand-binding and co-transfection reporter assays, as well as direct examination of LXRα/β target gene expression in HepG2 cells. Kuwanon G displayed potent LXRα binding activity as well as agonist activity in cell-based assays. Based on the activity of kuwanon G, we also examined other kuwanons with relevant structures (kuwanon C and kuwanon K). While kuwanon C showed insignificant activity in the radioligand binding assay, kuwanon K, a constitutional isomer of kuwanon G, displayed high-affinity binding to LXRα/β, but intriguingly, kuwanon K functioned as an LXRα/β inverse agonist in the cotransfection and gene expression assays. This study provides critical structural insights into the chemical diversity that drives agonism vs inverse agonism as essential knowledge for opportunities in LXRα/β ligands from botanical sources.
Abstract The total synthesis of epimedonins A and B, two prenylated 2-phenoxychromones isolated from Epimedium koreanum, was accomplished in this study. Since their initial discovery in 2014, the structures of these natural products have been ambiguous, and structural revisions based on spectroscopic reanalysis were proposed in 2018. To resolve this structural ambiguity, an efficient and divergent synthetic strategy was developed to achieve agreement between the synthesized spectroscopic data and the original report. Furthermore, their biological potential was evaluated, revealing that epimedonin A significantly promotes neurite outgrowth in Neuro2a cells, suggesting its therapeutic potential for neuronal differentiation and neurodegeneration.
Abstract Cavenderia subdiscoidea and Cavenderia ungulata are dictyostelid social amoebae found in northern Thailand. Genomic analysis identified two type III polyketide synthase genes, designated cspks and cupks. Heterologous expression of both genes in Aspergillus oryzae NSAR1 yielded a variety of acylphloroglucinols derived from different starter units. Among these were cavenderic acids A−E (1−5) and two related ester derivatives (6−7). Compounds 1 and 2 are reported here for the first time as natural products, whereas compounds 3−7 are new compounds. Structural analysis suggested that compounds 1−7 are biosynthetically derived from C4−C10 α,ω-dicarboxylic acid CoA thioesters, representing a putative and unprecedented starter-unit class for type III PKSs. Additionally, four known acylphloroglucinols (8−10 and 12) and one new derivative (11), derived from saturated and unsaturated fatty acid starter units, were identified. These findings demonstrate the broad starter-unit promiscuity of Cavenderia type III PKSs and their ability to generate structurally diverse acylphloroglucinols when expressed in a heterologous host. Compounds 1, 3−4, and 8−11 were evaluated for antimicrobial activity. Compound 8 (jambone C) exhibited the strongest antibacterial activity against several Gram-positive bacteria. Comparison of the tested compounds further suggested that terminal polar functional groups reduced antibacterial activity, providing preliminary structure−activity relationship insights for acylphloroglucinols.
Six new nor-terpene endoperoxides, diacarnusins A-F (1-6), along with five known ones (7-11), were isolated from the extract of the Red Sea sponge Diacarnus erythraeanus, which presented cytotoxic activity against the colon carcinoma cell line. The structure elucidation was performed by analysis of mass spectrometric and 1D and 2D NMR data. The relative and absolute configurations of the stereocenters of the compounds were deduced based on the Capon and Macleod empirical rule and comparison of the sign of their specific rotations with those of endoperoxides with proven absolute configurations. The cytotoxicity of the pure compounds was evaluated against a panel of cancer cell lines. The structure elucidation and biological activity of the compounds are discussed.
Bafilomycin and concanamycin analogs (1-3) are microbial natural products with potent V-ATPase inhibitory activity. Despite their promising therapeutic potential, limited access to these plecomacrolide scaffolds has constrained their broader bioactivity evaluation. Here, we report an integrative approach combining genome mining, strain engineering, and molecular networking to expand the chemical diversity of natural concanamycin analogs. Genome analysis identified a concanamycin-like biosynthetic gene cluster in Streptomyces humidus with a unique genetic architecture, suggesting that it may encode previously uncharacterized analogs. Coupling strain engineering, untargeted metabolomics, and feature-based molecular networking of the engineered S. humidus strain expedited dereplication and visualization of an underexplored subnetwork of concanamycin-like metabolites. This prioritization strategy enabled the isolation of five known analogs (4-7, 9) and three new analogs that we termed tanamycin E, X, and F (10-12). Optimization of cultivation conditions for the engineered S. humidus strain facilitated the isolation of 93.0 mg of TAN-1323 D (9) and 24.7 mg of the newly identified compound 10, per liter of culture. Collectively, this work provides scalable strategies for accessing and prioritizing new derivatives, overcoming traditional limitations in the production and discovery of bioactive natural products.
Coccidioidomycosis, or Valley fever, is a fungal respiratory disease caused by Coccidioides immitis or Coccidioides posadasii. Clinical manifestations range from asymptomatic infection to chronic pulmonary disease. Despite the increasing endemic range and affected population, therapeutic strategies display moderate efficacy and adverse side effects. This study explores California's rich botanical diversity as a source for agents that inhibit the early stages of Coccidioides development. We established a screening protocol containing a colorimetric metabolism assay and microscopic imaging. We then applied this protocol to extracts derived from local plants in San Diego, California, and discovered a potent inhibitor, persenone A, from avocados.
Factor inhibiting HIF-1α (FIH) is a 2-oxoglutarate-dependent oxygenase that controls hypoxia signaling and metabolic homeostasis by hydroxylating HIF-1α. Although selective pharmacological inhibition of FIH represents an emerging therapeutic strategy for metabolic disorders, structurally diverse natural inhibitors remain largely unexplored. Here, we identified five natural FIH inhibitors spanning distinct phytochemical classes, including three flavonoids (wogonin, luteolin, morin), a coumarin (isofraxidin), and an anthraquinone (rhein). Co-crystal structures revealed that structurally diverse natural products converge on a common bidentate metal-chelation geometry within the FIH active site despite substantial differences in scaffold architecture. Among these inhibitors, wogonin most closely mimicked the orientation of the HIF-1α Asn803 side chain within the substrate-binding cleft, resulting in inhibitory potency comparable to that of the 2-oxoglutarate analog N-oxalylglycine. These findings establish the first structural framework for natural-product-based FIH inhibition and demonstrate that structurally distinct natural inhibitors adopt a conserved metal-chelation geometry within the FIH active site. This framework provides a basis for the future development of metabolically oriented FIH inhibitors.
Nigroepocins A-D (1-4), along with one known compound clavatol (5), were isolated from the fermentation broth of Nigrospora sp. SN56127. Their structures, including absolute configurations, were determined by NMR, MS, and single-crystal X-ray diffraction analyses. Each of the four compounds (1-4) possesses a three-membered epoxy ring, which is rare in other compounds isolated from Nigrospora. All compounds (1-5) were evaluated for their antifungal activities against Fusarium graminearum, and nigroepocin A (1) exhibited inhibition with EC50 values of 110 and 23.1 μM against mycelial growth and conidial germination, respectively.
Ansamycins with diverse ring systems play an indispensable role in the pharmaceutical industry. A novel 20-membered ester-incorporating benzenoid ansamycin, phenolmacrolide A (1), and a new naphthalenoid ansamycin, olimycin I (2), were obtained from the marine-derived Streptomyces olivaceus NBUA17 using a strategy of combining genome mining, SNAP-MS, and HSQC NMR interpretation tools. Furthermore, investigation of the putative ansamycin biosynthetic gene cluster suggested that the novel benzenoid ansamycin scaffold of compound 1 could be formed in part by a hydroxylase and luciferase-like monooxygenase. Compound 1 exhibited moderate activity inhibiting proliferation, migration, and invasion against PANC-1 cells in vitro by inducing cell apoptosis and promoting cancer cell entry into G2/M phase, and inhibiting epithelial-mesenchymal transition. Further drug affinity-responsive target stability assay, molecular docking, cell thermal shift assay, and siRNA transfection identified EEF1A2 as a molecular target of compound 1 in PANC-1 cells. This study identifies an unprecedented subtype within the benzenoid ansamycin family, broadens the structural and bioactive diversity of macrocyclic ansamycins, and underscores the potential of marine-derived actinomycetes as a prolific source of pharmacologically promising lead molecules.
