Secondary Metabolites with Agricultural Antagonistic Potential from Aspergillus sp. ITBBc1, a Coral-Associated Marine Fungus.

A marine-derived fungal strain, Aspergillus sp. ITBBc1, was isolated from coral collected from the South China Sea in Hainan province. Intensive chemical investigation of the fermentation extract of this strain afforded four new secondary metabolites (1-4), named megastigmanones A-C and prenylterphenyllin H, along with four known compounds (5-8). Their structures were elucidated by extensive spectroscopic analysis including one-and two-dimensional (1D and 2D) NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The modified Mosher's method was undertaken to determine the absolute configurations of new compounds. The phytotoxic activity test showed that compounds 6-8 exhibited significant antagonistic activity against the germination of Triticum aestivum L. and Oryza sativa L. seeds with a dose-dependent relationship.

Phosphodiesterase 5 and Arginase Inhibitory Activities of the Extracts from Some Members of Nelumbonaceae and Nymphaeaceae Families.

The objectives of this study were (1) to investigate the effect of extracts from some plants in the families Nelumbonaceae and Nymphaeaceae on phosphodiesterase 5 (PDE5) and arginase, which have been used in erectile dysfunction treatment, and (2) to isolate and identify the compounds responsible for such activities. The characterization and quantitative analysis of flavonoid constituents in the active extracts were performed by HPLC. Thirty-seven ethanolic extracts from different parts of plants in the genus Nymphaea and Victoria of Nymphaeaceae and genus Nelumbo of Nelumbonaceae were screened for PDE5 and arginase inhibitory activities. The ethanolic extracts of the receptacles and pollens of Nelumbo nucifera Gaertn., petals of Nymphaea cyanea Roxb. ex G.Don, Nymphaea stellata Willd., and Victoria amazonica (Poepp.) Sowerby and the petals and receptacles of Nymphaea pubescens Willd. showed IC50 values on PDE5 of less than 25 µg/mL while none of the extracts showed effects on arginase. The most active extract, N. pubescens petal extract, was fractionated to isolate and identify the PDE5 inhibitors. The results showed that six flavonoid constituents including quercetin 3'-O-ß-xylopyranoside (1), quercetin 3-methyl ether 3'-O-ß-xylopyranoside (2), quercetin (3), 3-O-methylquercetin (4), kaempferol (5) and 3-O-methylkaempferol (6) inhibited PDE5 with IC50 values at the micromolar level.

Zn-dependent bifunctional proteases are responsible for leader peptide processing of class III lanthipeptides.

Lanthipeptides are an important subfamily of ribosomally synthesized and posttranslationally modified peptides, and the removal of their N-terminal leader peptides by a designated protease(s) is a key step during maturation. Whereas proteases for class I and II lanthipeptides are well-characterized, the identity of the protease(s) responsible for class III leader processing remains unclear. Herein, we report that the class III lanthipeptide NAI-112 employs a bifunctional Zn-dependent protease, AplP, with both endo- and aminopeptidase activities to complete leader peptide removal, which is unprecedented in the biosynthesis of lanthipeptides. AplP displays a broad substrate scope in vitro by processing a number of class III leader peptides. Furthermore, our studies reveal that AplP-like proteases exist in the genomes of all class III lanthipeptide-producing strains but are usually located outside the biosynthetic gene clusters. Biochemical studies show that AplP-like proteases are universally responsible for the leader removal of the corresponding lanthipeptides. In addition, AplP-like proteases are phylogenetically correlated with aminopeptidase N from Escherichia coli, and might employ a single active site to catalyze both endo- and aminopeptidyl hydrolysis. These findings solve the long-standing question as to the mechanism of leader peptide processing during class III lanthipeptide biosynthesis, and pave the way for the production and bioengineering of this class of natural products.

Submerged fermentation of Streptomyces uncialis providing a biotechnology platform for uncialamycin biosynthesis, engineering, and production.

Uncialamycin (UCM) belongs to the anthraquinone-fused subfamily of 10-membered enediyne natural products that exhibits an extraordinary cytotoxicity against a wide spectrum of human cancer cell lines. Antibody-drug conjugates, utilizing synthetic analogues of UCM as payloads, are in preclinical development. UCM is exclusively produced by Streptomyces uncialis DCA2648 on solid agar medium with low titers (∼0.019 mg/l), limiting its supply by microbial fermentation and hampering its biosynthetic and engineering studies by in vivo pathway manipulation. Here, we report cultivation conditions that enable genetic manipulation of UCM biosynthesis in vivo and allow UCM production, with improved titers, by submerged fermentation of the engineered S. uncialis strains. Specifically, the titer of UCM was improved nearly 58-fold to ∼1.1 mg/l through the combination of deletion of biosynthetic gene clusters encoding unrelated metabolites from the S. uncialis wild-type, chemical mutagenesis and manipulation of pathway-specific regulators to generate the engineered S. uncialis strains, and finally medium optimization of the latter for UCM production. Genetic manipulation of UCM biosynthesis was demonstrated by inactivating selected genes in the engineered S. uncialis strains, one of which afforded a mutant strain accumulating tiancimycin B, a common biosynthetic intermediate known for the anthraquinone-fused subfamily of enediyne natural products. These findings highlight a biotechnology platform for UCM biosynthesis, engineering, and production that should facilitate both its fundamental studies and translational applications.

Discovery of Alternative Producers of the Enediyne Antitumor Antibiotic C-1027 with High Titers.

The potent cytotoxicity and unique mode of action make the enediyne antitumor antibiotic C-1027 an exquisite drug candidate for anticancer chemotherapy. However, clinical development of C-1027 has been hampered by its low titer from the original producer Streptomyces globisporus C-1027. Here we report three new C-1027 alternative producers, Streptomyces sp. CB00657, CB02329, and CB03608, from The Scripps Research Institute actinomycetes strain collection. Together with the previously disclosed Streptomyces sp. CB02366 strain, four C-1027 alternative producers with C-1027 titers of up to 11-fold higher than the original producer have been discovered. The five C-1027 producers, isolated from distant geographic locations, are distinct Streptomyces strains based on morphology and taxonomy. Pulsed-field gel electrophoresis and Southern analysis of the five C-1027 producers reveal that their C-1027 biosynthetic gene clusters (BGCs) are all located on giant plasmids of varying sizes. The high nucleotide sequence similarity among the five C-1027 BGCs implies that they most likely have evolved from a common ancestor.

New Antibacterial Phenone Derivatives Asperphenone A-C from Mangrove-Derived Fungus Aspergillus sp. YHZ-1.

Marine fungi are a promising source of novel bioactive natural products with diverse structure. In our search for new bioactive natural products from marine fungi, three new phenone derivatives, asperphenone A-C (1-3), have been isolated from the ethyl acetate extract of the fermentation broth of the mangrove-derived fungus, Aspergillus sp. YHZ-1. The chemical structures of these natural products were elucidated on the basis of mass spectrometry, one- and two-dimensional NMR spectroscopic analysis and asperphenone A and B were confirmed by single-crystal X-ray crystallography. Compounds 1 and 2 exhibited weak antibacterial activity against four Gram-positive bacteria, Staphylococcus aureus CMCC(B) 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus, with the MIC values higher than 32.0 µM.

Novel chaetospirolactone and orsellide F from an endophytic fungus Chaetomium sp.

Chaetospirolactone (1), a novel spiro-lactone bearing a rare 1-oxaspiro [4.4] non-7-ene-2,6-dione skeleton, and orsellide F (2), together with six known compounds (3-8), were isolated from an endophytic fungus Chaetomium sp. NF00754. Their structures were determined by interpretation of spectroscopic data. The absolute configurations of 1 and 2 were established by analysis of single X-ray crystallographic data and CD spectra. Compounds 3, 4, and 6 showed moderate acetylcholinesterase inhibitory activity with IC50 values of 7.34, 5.19, and 7.67 µM, respectively.

Strepchazolins A and B: Two New Alkaloids from a Marine Streptomyces chartreusis NA02069.

Two new alkaloids, strepchazolins A (1) and B (2), together with a previously reported compound, streptazolin (3), were isolated from a marine actinomycete, Streptomyces chartreusis NA02069, collected in the Coast of Hainan Island, China. The structures of new compounds were determined by extensive NMR, mass spectroscopic and X-ray crystallographic analysis, as well as modified Mosher's method. Compound 1 showed weak anti-Bacillus subtilis activity with the MIC value of 64.0 µM, and weak inhibitory activity against acetylcholinesterase (AChE) in vitro with IC50 value of 50.6 µM, while its diastereoisomer, Compound 2, is almost inactive.

Malibatol A regulates microglia M1/M2 polarization in experimental stroke in a PPARγ-dependent manner.

BACKGROUND: Activation of microglia plays a crucial role in immune and inflammatory processes after ischemic stroke. Microglia is reported with two opposing activated phenotypes, namely, classic phenotype (M1) and the alternative phenotype (M2). Inhibiting M1 while stimulating M2 has been suggested as a potential therapeutic approach in the treatment of stroke. FINDINGS: In this study, we indicated that a novel natural anti-oxidant extracted from the Chinese plant Hopea hainanensis, malibatol A (MA), decreased the infarct size and alleviated the brain injury after mice middle cerebral artery occlusion (MCAO). MA inhibited expression inflammatory cytokines in not only MCAO mice but also lipopolysaccharide (LPS)-stimulated microglia. Moreover, treatment of MA decreased M1 markers (CD16, CD32, and CD86) and increased M2 markers (CD206, YM-1) while promoting the activation of nuclear receptor PPARγ. CONCLUSIONS: MA has anti-inflammatory effects in MCAO mice in a PPARγ-dependent manner, making it a potential candidate for stroke treatment.

Synthesis and evaluation of 8,4'-dideshydroxy-leinamycin revealing new insights into the structure-activity relationship of the anticancer natural product leinamycin.

Leinamycin (LNM, 1) is a novel antitumor antibiotic produced by Streptomyces atroolivaceus S-140 and features an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-dithiolane moiety of LNM is essential for its antitumor activity via an episulfonium ion-mediated DNA alkylation upon reductive activation in the presence of cellular thiols. We recently isolated leinamycin E1 (LNM E1, 2) from an engineered strain S. atroolivaceus SB3033, which lacks the 1,3-dioxo-1,2-dithiolane moiety. Here we report the chemical synthesis of 8,4'-dideshydroxy-LNM (5) from 2 and determination of the cytotoxicity of 5 against selected cancer cell lines in comparison with 1; 5 exhibits comparable activity as 1 with the EC50 values between 8.21 and 275 nM. This work reveals new insight into the structure-activity relationship of LNM and highlights the synergy between metabolic pathway engineering and medicinal chemistry for natural product drug discovery.

Enediynes: Exploration of microbial genomics to discover new anticancer drug leads.

The enediyne natural products have been explored for their phenomenal cytotoxicity. The development of enediynes into anticancer drugs has been successfully achieved through the utilization of polymer- and antibody-drug conjugates (ADCs) as drug delivery systems. An increasing inventory of enediynes would benefit current application of ADCs in many oncology programs. Innovations in expanding the enediyne inventory should take advantage of the current knowledge of enediyne biosynthesis and post-genomics technologies. Bioinformatics analysis of microbial genomes reveals that enediynes are underexplored, in particular from Actinomycetales. This digest highlights the emerging opportunities to explore microbial genomics for the discovery of novel enediyne natural products.

Antiviral activity of Paulownia tomentosa against enterovirus 71 of hand, foot, and mouth disease.

The bark, leaves, and flowers of Paulownia trees have been used in traditional Chinese medicine to treat infectious and inflammatory diseases. We investigated the antiviral effects of Paulownia tomentosa flowers, an herbal medicine used in some provinces of P. R. China for the treatment of skin rashes and blisters. Dried flowers of P. tomentosa were extracted with methanol and tested for antiviral activity against enterovirus 71 (EV71) and coxsackievirus A16 (CAV16), the predominant etiologic agents of hand, foot, and mouth disease in P. R. China. The extract inhibited EV71 infection, although no effect was detected against CAV16 infection. Bioactivity-guided fractionation was performed to identify apigenin as an active component of the flowers. The EC50 value for apigenin to block EV71 infection was 11.0 µM, with a selectivity index of approximately 9.3. Although it is a common dietary flavonoid, only apigenin, and not similar compounds like naringenin and quercetin, were active against EV71 infection. As an RNA virus, the genome of EV71 has an internal ribosome entry site that interacts with heterogeneous nuclear ribonucleoproteins (hnRNPs) and regulates viral translation. Cross-linking followed by immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that EV71 RNA was associated with hnRNPs A1 and A2. Apigenin treatment disrupted this association, indicating that apigenin suppressed EV71 replication through a novel mechanism by targeting the trans-acting factors. This study therefore validates the effects of Paulownia against EV71 infection. It also yielded mechanistic insights on apigenin as an active compound for the antiviral activity of P. tomentosa against EV71 infection.

Polyketides from mantis-associated fungus Daldinia eschscholzii IFB-TL01.

Three new polyketides, named daldinone F (1), nodulisporin G (2), and dalmanol C (3), together with five known compounds, 4-8, were isolated from cultures of Daldinia eschscholzii. The structures of the new compounds were elucidated by extensive NMR and MS analyses. Compound 1 showed moderate cytotoxic activity against SW480 cancer cells with an IC50 value of 9.59 µM, and its absolute configuration was determined by single crystal X-ray diffraction.

Drimane sesquiterpenoids from the Aspergillus oryzae QXPC-4.

Three new drimane sesquiterpenoids, astellolides C-E (1-3, resp.), four new drimane sesquiterpenoid p-hydroxybenzoates, astellolides F-I (4-7, resp.), together with two known compounds astellolides A and B (8 and 9, resp.), have been isolated from the liquid culture of Aspergillus oryzae (strain No. QXPC-4). Their structures were established by comprehensive analysis of spectroscopic data. The relative and absolute configurations were determined on the basis of NOESY and CD data, together with single-crystal X-ray diffraction analyses of compounds 1-3. The metabolites were evaluated for their cytotoxic activities, however, no compounds showed a significant cytotoxicity against the tested cell lines at a concentration of 20 µM.

Actinotetraose L, a new tetrasaccharide derivative isolated from the mycelia of Amycolatopsis sp. HCa1.

A new tetrasaccharide derivative, actinotetraose L (1), was isolated from the methanol extract of the mycelia of the grasshopper-associated rare actinobacterium Amycolatopsis sp. HCa1. The structure of the new compound was elucidated by a combination of 1D, 2D NMR (HMQC, HMBC, COSY, and NOESY), and HR-ESI-MS analyses as O-{3,4-di-O-[(E)-2-ethyl-2-butenoyl]- ß-D-glucopyranosyl-(1 â†’ 2)}-O-{3,4-di-O-[(E)-2-methyl-2-butenoyl]-ß-D-glucopyranosyl-(1 â†’ 2)}-6,6-di-O-[(E)-2-methyl-2-butenoyl]-α-D-glucopyranosyl-(1 â†’ 1)-α-D-glucopyranoside. The immunosuppressive activity and cytotoxicity of the compound were evaluated by T-cell viability and MTT assays. Nonetheless, no significant activity was observed.

New tricycloalternarenes from fungus Alternaria sp.

Two new tricycloalternarenes I (1) and J (2), together with five known derivatives (3-7), were isolated from the culture of marine fungus Alternaria sp. The structures were elucidated by a combination of spectroscopic approach ((1)H, (13)C NMR, HMBC, COSY, and NOESY) and the low-temperature (100 K) single-crystal X-ray crystallography analysis. The antimicrobial assays of tricycloalternarenes I (1) and J (2) were tested.

Cytotoxic and antimicrobial flavonoids from Cryptocarya concinna.

Five new flavonoids, cryptoconones A-E (1-5), along with six known compounds (6-11), were isolated from the stems of Cryptocarya concinna. The structures of these compounds were elucidated on the basis of spectroscopic data interpretation, and the absolute configurations were determined via circular dichroism spectra and X-ray crystal analysis. The cytotoxic and antimicrobial activities of these compounds were also evaluated. Compounds 9 and 10 exhibited moderate cytotoxic activities against HCT116, HT-29, SW480, and MDA-MB-231 cell lines with IC50 values ranging from 6.25 to 9.35 µM. Compounds 8 and 11 exhibited antimicrobial activity against Fusarium moniliforme and Botrytis cinerea, respectively, with the same minimum inhibitory concentration of 5 µg/mL.

Disulfide bridge-targeted metabolome mining unravels an antiparkinsonian peptide.

Peptides are a particular molecule class with inherent attributes of some small-molecule drugs and macromolecular biologics, thereby inspiring continuous searches for peptides with therapeutic and/or agrochemical potentials. However, the success rate is decreasing, presumably because many interesting but less-abundant peptides are so scarce or labile that they are likely 'overlooked' during the characterization effort. Here, we present the biochemical characterization and druggability improvement of an unprecedented minor fungal RiPP (ribosomally synthesized and post-translationally modified peptide), named acalitide, by taking the relevant advantages of metabolomics approach and disulfide-bridged substructure which is more frequently imprinted in the marketed peptide drug molecules. Acalitide is biosynthetically unique in the macrotricyclization via two disulfide bridges and a protease (AcaB)-catalyzed lactamization of AcaA, an unprecedented precursor peptide. Such a biosynthetic logic was successfully re-edited for its sample supply renewal to facilitate the identification of the in vitro and in vivo antiparkinsonian efficacy of acalitide which was further confirmed safe and rendered brain-targetable by the liposome encapsulation strategy. Taken together, the work updates the mining strategy and biosynthetic complexity of RiPPs to unravel an antiparkinsonian drug candidate valuable for combating Parkinson's disease that is globally prevailing in an alarming manner.

Vaticaffinol, a resveratrol tetramer, exerts more preferable immunosuppressive activity than its precursor in vitro and in vivo through multiple aspects against activated T lymphocytes.

In the present study, we aimed to investigate the immunosuppressive activity of vaticaffinol, a resveratrol tetramer isolated from Vatica mangachapoi, on T lymphocytes both in vitro and in vivo, and further explored its potential molecular mechanism. Resveratrol had a wide spectrum of healthy beneficial effects with multiple targets. Interestingly, its tetramer, vaticaffinol, exerted more intensive immunosuppressive activity than resveratrol. Vaticaffinol significantly inhibited T cells proliferation activated by concanavalin A (Con A) or anti-CD3 plus anti-CD28 in a dose- and time-dependent manner. It also induced Con A-activated T cells undergoing apoptosis through mitochondrial pathway. Moreover, this compound prevented cells from entering S phase and G2/M phase during T cells activation. In addition, vaticaffinol inhibited ERK and AKT signaling pathways in Con A-activated T cells. Furthermore, vaticaffinol significantly ameliorated ear swelling in a mouse model of picryl chloride-induced ear contact dermatitis in vivo. In most of the aforementioned experiments, however, resveratrol had only slight effects on the inhibition of T lymphocytes compared with vaticaffinol. Taken together, our findings suggest that vaticaffinol exerts more preferable immunosuppressive activity than its precursor resveratrol both in vitro and in vivo by affecting multiple targets against activated T cells.

Actinotetraoses I-K: tetrasaccharide metabolites produced by an insect-derived actinobacteria, Amycolatopsis sp. HCa1.

An isolate of rare actinobacteria strain Amycolatopsis sp. HCa1 obtained from the gut of grasshopper produced seven different metabolites in vitro. The metabolites isolated from its mycelia cakes were characterized by NMR and MS analyses. Actinotetraose hexatiglate (or tigloside; 1) with nonreducing glucotetraose skeleton was isolated as a major constituent; three new tetrasaccharide derivatives actinotetraoses I-K (2-4, resp.) and three known actinotetraoses A-C (5-7, resp.) were also isolated.