Target Discovery of Dhilirane-Type Meroterpenoids by Biosynthesis Guidance and Tailoring Enzyme Catalysis.

Dhilirane-type meroterpenoids (DMs) featuring a 6/6/6/5/5 ring system represent a rare group of fungal meroterpenoids. To date, merely 11 DMs have been isolated or derived, leaving their chemical diversity predominantly unexplored. Herein, we leverage an understanding of biosynthesis to develop a workflow for discovery of DMs by genome mining, metabolite analysis, and tailoring enzyme catalysis. Twenty-three new DMs, including seven unprecedented scaffolds, were consequently identified. An α-ketoglutarate (α-KG)-dependent oxygenase DhiD was found to catalyze the stereodivergent ring contraction of dhilirolide D to form the dhilirane skeleton; while the cytochrome P450 DhiH reshaped the structural diversity by establishing diverse C-C bonds and oxidation. Crystallographic and mutagenesis experiments provide a molecular basis for the DhiD reaction and its stereodivergent products. Notably, DhiD exhibits substrate-controlled catalytic versatility in the chemical expansion of DMs through ring contraction, hydroxylation, dehydrogenation, epoxidation, isomerization, epimerization, and α-ketol cleavage. Bioassay results demonstrated that the obtained meroterpenoids exhibited anti-inflammatory and insecticidal activities. Our work provides insight into nature's arsenal for DM biosynthesis and the functional versatility of α-KG-dependent oxygenase and P450, which can be applied for target discovery and diversification of DM-type natural products.

Nucleobase-Coupled Xanthones with Anti-ROS Effects from Marine-Derived Fungus Aspergillus sydowii.

A MS/MS-based molecular networking approach compared to the Global Natural Product Social Molecular Networking library, in association with genomic annotation of natural product biosynthetic gene clusters within a marine-derived fungus, Aspergillus sydowii, identified a suite of xanthone metabolites. Chromatographic techniques applied to the cultured fungus led to the isolation of 11 xanthone-based alkaloids, dubbed sydoxanthones F-M. The structures of these alkaloids were elucidated using extensive spectroscopic data, including electronic circular dichroism and single-crystal X-ray diffraction data for configurational assignments. Among these analogues, sydoxanthones F-K exhibit structure features typical of nucleobase-coupled xanthones, with sydoxanthone H being an N-bonded xanthone dimer. Notably, (±)sydoxanthones F (1a/1b), (±)sydoxanthones H (3b/3a), and (±)sydoxanthones J (5b/5a) are enantiomeric pairs, while sydoxanthones G (2), I (4), and K (6) are stereoisomers of 1, 3, and 5, respectively. Furthermore, (+)sydoxanthone H (3a) demonstrated significant rescue of cell viability in H2O2-injuried SH-SY5Y cells by inhibiting reactive oxygen species production, suggesting its potential for neuroprotection.

Irpetones A and B, Anti-Osteoclastic Heptaketides from a Marine Mesophotic Zone Ircinia Sponge-Associated Fungus Irpex sp. NBUF088.

Chemical investigation of Irpex sp. NBUF088, associated with an Ircinia sp. sponge located at an 84 m deep mesophotic zone, led to the discovery of two new heptaketides, named irpetones A (1) and B (2). Their structures were identified by analysis of spectroscopic data and quantum-chemical calculations. Compound 1 exhibited inhibition against the receptor activator of NF-κB ligand-induced osteoclastogenesis in bone marrow monocytes with an IC50 of 6.3 ± 0.2 µM, causing no notable cytotoxicity. It was also determined that 1 inhibited the phosphorylation of ERK1/2-JNK1/2-p38 MAPKs and the nuclear translocation of NF-κB, consequently suppressing the activation of MAPK and NF-κB signaling pathways induced by the NF-κB ligand.

Pestanoid A, a Rearranged Pimarane Diterpenoid Osteoclastogenesis Inhibitor from a Marine Mesophotic Zone Chalinidae Sponge-Associated Fungus, Pestalotiopsis sp. NBUF145.

One novel rearranged pimarane diterpenoid, pestanoid A (1), and two reported molecules, nodulisporenones A (2) and B (3), were discovered from Pestalotiopsis sp. NBUF145 fungus associated with a 62 m deep mesophotic ("twilight") zone Chalinidae sponge. The structures of 1-3 were identified by spectrometry, spectroscopy, quantum-chemical calculations, and X-ray crystallography. Compounds 1 and 2 inhibited bone marrow monocyte osteoclastogenesis in vitro with the IC50 values 4.2 ± 0.2 µM and 3.0 ± 0.4 µM, respectively, without observed cytotoxicity. Both 1 and 2 suppressed the receptor activator of NF-kB ligand-induced MAPK and NF-κB signaling by inhibiting the phosphorylation of ERK1/2-JNK1/2-p38 MAPKs and NF-κB nuclear translocation.

Brominated Depsidones with Antibacterial Effects from a Deep-Sea-Derived Fungus Spiromastix sp.

Eleven new brominated depsidones, namely spiromastixones U-Z5 (1-11) along with five known analogues (12-16), were isolated from a deep-sea-derived fungus Spiromastix sp. through the addition of sodium bromide during fermentation. Their structures were elucidated by extensive analysis of the spectroscopic data including high-resolution MS and 1D and 2D NMR data. Compounds 6-10 and 16 exhibited significant inhibition against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) with MIC values ranging from 0.5 to 2.0 µM. Particularly, tribrominated 7 displayed the strongest activity against MRSA and VRE with a MIC of 0.5 and 1.0 µM, respectively, suggesting its potential for further development as a new antibacterial agent.

New Diterpenes and Diterpene Glycosides with Antibacterial Activity from Soft Coral Lemnalia bournei.

Five new biflorane-type diterpenoids, biofloranates E-I (1-5), and two new bicyclic diterpene glycosides, lemnaboursides H-I (6-7), along with the known lemnabourside, were isolated from the South China Sea soft coral Lemnalia bournei. Their chemical structures and stereochemistry were determined based on extensive spectroscopic methods, including time-dependent density functional theory (TDDFT) ECD calculations, as well as a comparison of them with the reported values. The antibacterial activities of the isolated compounds were evaluated against five pathogenic bacteria, and all of these diterpenes and diterpene glycosides showed antibacterial activities against Staphylococcus aureus and Bacillus subtilis, with MICs ranging from 4 to 64 µg/mL. In addition, these compounds did not exhibit noticeable cytotoxicities on A549, Hela, and HepG2 cancer cell lines, at 20 µM.

New Meroterpenes from South China Sea Soft Coral Litophyton brassicum.

A chemical investigation of the extracts from the soft coral Litophyton brassicum led to the isolation and identification of four new meroterpenes, brassihydroxybenzoquinone A and B (1 and 2) and brassinaphthoquinone A and B (3 and 4), along with two known related meroterpenes (5 and 6). Their structures were elucidated using high-resolution electrospray ionization mass spectrometry (HRESIMS), nuclear magnetic resonance (NMR) spectroscopy, and a comparison with the literature data. All compounds were evaluated for antibacterial activity against six pathogenic bacterial strains and for cytotoxic activity against three cancer cell lines. In the cytotoxic assay, all compounds were inactive at 10 µM against the A549, HeLa, and MDA-MB-231 cell lines. In the antibacterial assay, compounds 1 and 2 exhibited moderate inhibitory activity with minimum inhibitory concentrations (MIC) ranging from 8 to 64 µg/mL.

Indoloquinazoline alkaloids suppress angiogenesis and inhibit metastasis of melanoma cells.

Metastasis is the leading cause of cancer-related mortality, targeting angiogenesis emerges as a therapeutic strategy for the treatment of melanoma metastasis. Discovery of new antiangiogenic compounds with specific mechanism of action is still desired. In present study, a bioassay-guidance uncovers the EtOAc extract of a marine-derived fungus Aspergillus clavutus LZD32-24 with significant inhibitory activity against the angiogenesis in Tg (fli1a: EGFP) zebrafish model. Extensive chromatographic fractionation led to the isolation of 48 indoloquinazoline alkaloids, including 21 new analogues namely clavutoines A-U (1-21). Their structures were determined by the spectroscopic data, including the ECD, single crystal X-ray diffraction and quantum chemical calculation for the configurational assignments. Among the bioactive analogues, quinadoline B (QB) showed the most efficacy to suppress the zebrafish vascular outgrowth in zebrafish embryos. QB markedly inhibited the migration, invasion and tube formation with weak cytotoxicity in human umbilical vein endothelial cells (HUVECs). Investigation of the mode of action revealed QB suppressed the ROCK/MYPT1/MLC2/coffin and FAK /Src signaling pathways, and subsequently disrupted actin cytoskeletal organization. In addition, QB reduced the number of new vessels sprouting from the ex vivo chick chorioallantoic membrane (CAM), and inhibited the metastasis of B16F10 melanoma cells in lung of C57BL/6 mice through suppressing angiogenesis. These findings suggest that QB is a potential lead for the development of new antiangiogenic agent to inhibit melanoma metastasis.

Bio-Active Products from Mangrove Ecosystems.

Mangrove communities represent the coastal habitats located in intertidal zones or brackish waters of tropical and subtropical coastal areas in over 118 countries [...].

Discovery of cyclohexadepsipeptides with anti-Zika virus activities and biosynthesis of the nonproteinogenic building block (3S)-methyl-l-proline.

The fungal cyclohexadepsipeptides destruxins (DTXs), isaridins (ISDs), and isariins (ISRs) are nonribosomal peptides whose structures include a 19-membered ring composed of five amino acid residues and one α- or ß-hydroxy acid residue. These cyclohexadepsipeptides contain unusual nonproteinogenic amino acid-building blocks and possess a range of antiviral, antibacterial, and other activities. The biosynthetic gene clusters for ISDs and ISRs have not been identified, and the biosynthesis of the nonproteinogenic (3S)-methyl-l-proline residue, which is found in DTXs, ISDs, and many other natural products, lacks full characterization. In an ongoing effort to identify compounds that can inhibit the Zika virus (ZIKV), we examined the extract of marine-derived fungus Beauveria felina SX-6-22 and discovered 30 DTXs, ISDs, and ISRs (1-30) including seven new compounds (1-7). The anti-ZIKV assays showed that 9-12 and 16-18 possess inhibitory activities against ZIKV RNA replication and NS5 (nonstructural protein 5) production in ZIKV-infected A549 cells. We sequenced the genome of B. felina SX-6-22 and identified three biosynthetic gene clusters detx, isd and isr, which are responsible for the biosynthesis of DTXs, ISDs, and ISRs, respectively. Comparative analyses of the three gene clusters clarified the biosynthetic relationships among these cyclohexadepsipeptides. Finally, we characterized the entire biosynthesis of nonproteinogenic building block (3S)-methyl-l-proline. The Δ1-pyrroline-5-carboxylate reductases (P5CRs), also used in the biosynthesis of l-proline, were demonstrated to catalyze the final reduction step in (3S)-methyl-l-proline formation, suggesting potential cross talk between primary and secondary metabolisms. These results provide opportunities for biosynthetic pathway engineering to generate new anti-ZIKV cyclohexadepsipeptides.

Sinuscalide A: An Antiviral Norcembranoid with an 8/8-Fused Carbon Scaffold from the South China Sea Soft Coral Sinularia scabra.

Sinuscalide A (1), featuring an uncommon 8/8-fused carbon scaffold, three new norditerpenes, sinuscalides B-D (2-4), and sinuscatone A (5), with a 5/4/9 tricyclic carbon ring system, along with four known compounds were isolated from the South China Sea soft coral Sinularia scabra. The structures of the new compounds were established by extensive spectroscopic analysis, X-ray diffraction, and electronic circular dichroism (ECD). A plausible biosynthetic pathway for 1 was proposed. In a bioassay, compound 1 showed antiviral activity against human enterovirus EV71 (IC50 = 5.0 µM) and an inhibitory effect against RANKL-induced osteoclastogenesis (92.3% inhibition at 10 µM). Compound 5 exhibited mild inhibition against Streptococcus pneumoniae and Salmonella paratyph (MIC 16 µg/mL).

Sclerotiamides C-H, Notoamides from a Marine Gorgonian-Derived Fungus with Cytotoxic Activities.

Bioassay-guided fractionation in association with LC-MS and NMR detection led to the isolation of six new alkaloids, sclerotiamides C-H (1-6), from the marine gorgonian-derived fungus Aspergillus sclerotiorum LZDX-33-4. Their structures were determined from extensive spectroscopic data, including ECD data and single-crystal X-ray diffraction analysis for configurational assignments. Sclerotiamides C (1) and D (2) are notoamide-type alkaloids with the incorporation of a unique 2,2-diaminopropane unit, and sclerotiamides E (3) and F (4) are unprecedented notoamide hybrids with a new coumarin unit. Sclerotiamide H (6) represents a new highly oxidized notoamide scaffold. Sclerotiamides C and F showed significant inhibition against a panel of tumor cell lines with IC50 values ranging from 1.6 to 7.9 µM. Sclerotiamide C induces apoptosis in HeLa cells by arresting the cell cycle, activating ROS production, and regulating apoptosis-related proteins in the MAPK signaling pathway. The present study extends the scaffold diversity of the notoamides and provides a potential lead for the development of a cytotoxic agent.

Concise Biosynthesis of Tropone-Containing Spiromaterpenes by a Sesquiterpene Cyclase and a Multifunctional P450 from a Deep-Sea-Derived Spiromastix sp. Fungus.

Spiromaterpenes are a group of rare tropone-containing sesquiterpenes with antineuroinflammatory activity. Herein, we elucidate their biosynthetic pathway in a deep-sea-derived Spiromastix sp. fungus by heterologous expression, biochemical characterization, and incubation experiments. The sesquiterpene cyclase SptA was first characterized to catalyze the production of guaia-1(5),6-diene, and a multifunctional cytochrome P450 catalyzed the tropone ring formation. These results provide important clues for the rational mining of bioactive guaiane-type sesquiterpenes and expand the repertoire of P450 activities to synthesize unique building blocks of natural products.

Versicotide G suppresses osteoclastogenesis and prevents osteolysis.

Excessive formation and function of osteoclasts cause various osteolytic bone diseases. Natural products are a potential source for the discovery of new therapeutic candidates to treat bone destruction diseases. In this study, chemical informatics and bioassay guided examination of the marine-derived Aspergillus versicolor F77 fungus chemically resulted in the isolation of seven cyclopeptides, of which versicotides G-J (1-4) are new cyclohexapeptides. Their structures were identified by spectroscopic data in association with Marfey method and single crystal X-ray diffraction data for configurational assignments. Bioassay revealed that versicotide G (1, VG) is the most active among the analogs to suppress the receptor activator of nuclear factor-KB ligand (RANKL)-induced osteoclastogenesis in bone marrow derived monocytes (BMMs) without affecting BMMs viability. VG also suppressed RANKL-induced actin-ring formation and resorbing function of osteoclast dose-dependently. Mechanistically, VG attenuated RANKL-induced intracellular calcium elevation by inhibiting PLCγ1 phosphorylation and blocking the activation of downstream phosphatase calcineurin. In addition, VG abrogated the expression and translocation of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), leading to the downregulation of the expression of osteoclast-specific genes and the abolishment of the osteoclast formation. In the in vivo test, VG suppressed osteoclast formation and bone loss in Ti-induced calvarial osteolytic mouse model.These findings imply that VG is a promising candidate for the remedy of bone destruction-related diseases.

Eremophilane-Type Sesquiterpenes from a Marine-Derived Fungus Penicillium Copticola with Antitumor and Neuroprotective Activities.

Chemical examination of a marine sponge-associated Penicillium copticola fungus resulted in the isolation of ten undescribed eremophilanes, namely copteremophilanes A-J (1-10), along with two new glycosides, 5-glycopenostatin F (11) and 5-glucopenostatin I (12). Their structures were determined by extensive spectroscopic data, in association with ECD data and chemical conversions for configurational assignments. Analogs 1, 2, and 10 represent a group of uncommon skeletons of eremophilanes with an aromatic ring and a methyl migration from C-5 to C-9, and analogs 11 and 12 are characteristic of a PKS scaffold bearing a glucose unit. The incorporation of a chlorinated phenylacetic unit in 3-9 is rarely found in nature. Analog 7 showed neuroprotective effect, whereas 8 exhibited selective inhibition against human non-small cell lung cancer cells (A549). This study enriched the chemical diversity of eremophilanes and extended their bioactivities to neuroprotection.

Equisetin Targets Intracellular Staphylococcus aureus through a Host Acting Strategy.

Mammalian cells act as reservoirs of internalized bacteria to circumvent extracellular antibacterial compounds, resulting in relapse and reinfection diseases. The intracellular persistence of Staphylococcus aureus renders most traditional antibiotics useless, due to their inadequate subcellular accumulation. To replenish our antibiotic arsenal, we found that a marine-derived compound, equisetin, efficiently eliminates intracellular S. aureus by potentiating the host autophagy and inducing mitochondrial-mediated ROS generation to clear the invading S. aureus. The remarkable anti-infection activity of equisetin was validated in a peritonitis-infected mouse model. The marine product equisetin utilizes a unique dual mechanism to modulate the host-pathogen interaction in the clearance of intracellular bacteria. Thus, equisetin is an inspiring host-acting candidate for overcoming intracellular pathogens.

Guignardones Y-Z, Antiviral Meroterpenes from Penicillium sp. NBUF154 Associated with a Crella Sponge from the Marine Mesophotic Zone.

Guignardones Y-Z (1-2), two new meroterpenoids, and six known metabolites involving guignardones A-H (3-4), gyorgy-isoflavone (5), daidzein (6), blumenol A (7) and guignardianone A (8) were isolated from the fungus Penicillium sp. NBUF154, which was obtained from a 60 m deep Crella sponge. Their structures including absolute configurations were unambiguously elucidated by exhaustive spectroscopic analysis and ECD calculations. A putative biosynthetic pathway toward guignardones (1-4) is here proposed. Biological evaluation of compounds 1-8 showed that 1 and 7 exert potent inhibitory effects towards human enterovirus 71 (EV71).

Polyketides from the marine-derived fungus Aspergillus falconensis: In silico and in vitro cytotoxicity studies.

Fermentation of the marine-derived fungus Aspergillus falconensis, isolated from sediment collected from the Red Sea, Egypt on solid rice medium containing 3.5% NaCl yielded a new dibenzoxepin derivative (1) and a new natural isocoumarin (2) along with six known compounds (3-8). Changes in the metabolic profile of the fungus were induced by replacing NaCl with 3.5% (NH4)2SO4 that resulted in the accumulation of three further known compounds (9-11), which were not detected when the fungus was cultivated in the presence of NaCl. The structures of the new compounds were elucidated by HRESIMS and 1D/2D NMR as well as by comparison with the literature. Molecular docking was conducted for all isolated compounds on crucial enzymes involved in the formation, progression and metastasis of cancer which included human cyclin-dependent kinase 2 (CDK-2), human DNA topoisomerase II (TOP-2) and matrix metalloproteinase 13 (MMP-13). Diorcinol (7), sulochrin (9) and monochlorosulochrin (10) displayed notable stability within the active pocket of CDK-2 with free binding energy (ΔG) equals to -25.72, -25.03 and -25.37 Kcal/mol, respectively whereas sulochrin (9) exerted the highest fitting score within MMP-13 active center (ΔG = -33.83 Kcal/mol). In vitro cytotoxic assessment using MTT assay showed that sulochrin (9) exhibited cytotoxic activity versus L5178Y mouse lymphoma cells with an IC50 value of 5.1 µM and inhibition of migration of MDA-MB 231 breast cancer cells at a concentration of 70 µM.

Epigenetic Manipulation to Trigger Production of Guaiane-Type Sesquiterpenes from a Marine-Derived Spiromastix sp. Fungus with Antineuroinflammatory Effects.

Epigenetic manipulation of a deep-sea sediment-derived Spiromastix sp. fungus using suberoylanilide hydroxamic acid (SAHA) induction resulted in the activation of a terpene-related biosynthetic gene cluster, and nine new guaiane-type sesquiterpenes, spiromaterpenes A-I (1-9), were isolated. Their structures were determined using various spectroscopic techniques, in association with the modified Mosher's method, computed electronic circular dichroism (ECD) spectra, and chemical conversion for configurational assignments. Compounds 4-6 exhibited significant effects against the NO production on lipopolysaccharide (LPS)-induced microglia cells BV2, and the preliminary SAR analyses demonstrated that a 2(R),11-diol unit is favorable. The most active 5 abolished LPS-induced NF-κB translocation from the cytosol to the nucleus in BV-2 microglial cells, accompanied by the marked reduction of the transcription levels of pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α dose-dependently in both LPS-induced BV-2 and BV-2 cells, as well as the protein and mRNA levels of iNOS and COX-2. This study complements the gap in knowledge regarding the anti-neuroinflammatory activity of guaiane-type sesquiterpenoids at the cellular level and suggests that 5 is promising for further optimization as a multifunctional agent for antineuroinflammation.

Briarane-type diterpenoids suppress osteoclastogenisis by regulation of Nrf2 and MAPK/NF-kB signaling pathway.

Excess osteoclastic activity leads to an imbalance in bone remodeling and causes most adult skeletal diseases. Natural products are a promising source to attenuate the osteoporosis and relevant diseases of bone loss. Herein, a bioassay-guided detection of gorgonian corals resulted in junceellolide D (JD), a briarane-type diterpenoid from gorgonian Dichotella gemmacea, showing significant inhibition against the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation in bone marrow macrophages (BMMs) in vitro. To extend the investigation for structure-activity relationship (SAR), a total of 39 briarane-type analogues were isolated including 28 new compounds, and their structures were determined by extensive analyses of spectroscopic data. The SAR data indicated that JD is the most active to inhibit osteoclast development due to the decreased number of multinucleated tartrate-resistance acid phosphatase positive cells, suppression of the actin ring formation, blockage of bone resorption, and downregulation of osteoclast-specific marker genes. Mechanistically, JD increased the protein stability of nuclear factor (erythroid-derived 2)-related factor-2 (Nrf2) and promoted Nrf2 nuclear translocation followed by activation its downstream antioxidant enzymes, which strongly abolished RANKL-induced generation of reactive oxygen species (ROS). Furthermore, JD inhibits the RANKL-stimulated activation of NF-κB and MAPK signaling pathways. Hence, JD is considered as a promising lead compound for anti-osteoclastogenesis via activating Nrf2 and suppressing NF-κB and MAPK signaling pathways to prevent osteoclast-mediated bone destructive diseases.