ß-Terrecyclene synthase constructs the quadrane backbone in terrecyclic acid biosynthesis.

Quadrane sesquiterpenes featuring a distinctive tricyclic skeleton exhibit potent antimicrobial and anticancer activities. Although extensive studies have attempted to reveal the multistep carbocation rearrangement involved in the formation of the tricyclic quadrane scaffold, the exact biosynthetic pathway and chemical logic to generate the quadrane structure remains mysterious. Here we identified a novel sesquiterpene synthase that is capable of generating ß-terrecyclene possessing the quadrane scaffold and characterized the biosynthetic pathway of a representative fungal quadrane terrecyclic acid. Further mutagenesis coupled with isotopically sensitive branching studies of this ß-terrecyclene synthase provided insight into the mechanism involved in the formation of the quadrane scaffold.

[Research progress on chemical constituents and pharmacological activities of small molecule compounds in scorpions].

Scorpions, a group of oldest animals with wide distribution in the world, have a long history of medicinal use. Scorpio, the dried body of Buthus martensii, is a rare animal medicine mainly used for the treatment of liver diseases, spasm, and convulsions in children in China. The venom has been considered as the active substance of scorpions. However, little is known about the small molecules in the venom of scorpions. According to the articles published in recent years, scorpions contain amino acids, fatty acids, steroids, and alkaloids, which endow scorpions with antimicrobial, anticoagulant, metabolism-regulating, and antitumor activities. This paper summarizes the small molecule chemical components and pharmacological activities of scorpions, with a view to providing valuable information for the discovery of new active molecules and the clinical use of scorpions.

Deciphering the Glycosylation Steps in the Biosynthesis of P-1894B and Grincamycin Isolated from Marine-Derived Streptomyces lusitanus SCSIO LR32.

Recently, we re-isolated the glycosylated angucycline antibiotics P-1894B (1) and grincamycin (1') from the marine-derived Streptomyces lusitanus SCSIO LR32 as potent antitumor agents and identified their biosynthesis gene cluster gcn. Both P-1894B (1) and grincamycin (1') possess a trisaccharide and a disaccharide moiety comprised of five deoxysugars. In this work, three genes encoding glycosyltransferases (GcnG1, GcnG2, and GcnG3) responsible for the assembly of deoxysugars into angucycline aglycone were identified from the biosynthesis gene cluster gcn. Gene inactivations of gcnG1, gcnG2, gcnG3, and gcnG1G2 by lambda-RED-mediated gene replacements led to the construction of four mutants, in which the glycosyltransferase genes were disrupted, respectively. The metabolites from the mutants were purified and identified, including two new analogues designated as grincamycin U (3a) and V (3'). The sequential glycosylation steps in the biosynthesis of P-1894B (1) and grincamycin (1') catalyzed by GcnG3, GcnG1, and GcnG2 were elucidated.

Sulfoxanthicillin from the deep-sea derived Penicillium sp. SCSIO sof101: an antimicrobial compound against Gram-positive and -negative pathogens.

Natural products along with their analogs have been intensively explored for their antimicrobial potential against 'ESKAPE' pathogens. Herein, we report a new natural product with strong antibacterial activity, sulfoxanthocillin (1), along with its decomposed product peniformamide (2), and the known compound xanthocillin X (3) from the deep-sea derived Penicillium sp. SCSIO sof101. The structures of compounds 1 and 2 were determined by extensive spectroscopic analysis. Compound 1 showed significant activity against series pathogens with MIC values ranging 0.06-8.0 µg mL-1. As an artificial unnatural product during the isolation process, compound 2 had lower antimicrobial activity than that of compound 1, which could be attributed to a change in structural modification from an isonitrile group in compound 1 to a formamide group in compound 2. In terms of cytotoxicity, 1 showed relatively low cytotoxicity against human tumor cell lines compared with xanthocillin X (3), suggesting that the sulfate group present in 1 should be a determinant of cytotoxic activities. Overall, sulfoxanthocillin (1) merits further attention as a potential lead compound for anti-infective interventions against Gram-negative and Gram-positive bacterial pathogens.

OSMAC-Based Discovery and Biosynthetic Gene Clusters Analysis of Secondary Metabolites from Marine-Derived Streptomyces globisporus SCSIO LCY30.

The one strain many compounds (OSMAC) strategy is an effective method for activating silent gene clusters by cultivating microorganisms under various conditions. The whole genome sequence of the marine-derived strain Streptomyces globisporus SCSIO LCY30 revealed that it contains 30 biosynthetic gene clusters (BGCs). By using the OSMAC strategy, three types of secondary metabolites were activated and identified, including three angucyclines, mayamycin A (1), mayamycin B (2), and rabolemycin (3); two streptophenazines (streptophenazin O (4) and M (5)); and a macrolide dimeric dinactin (6), respectively. The biosynthetic pathways of the secondary metabolites in these three families were proposed based on the gene function prediction and structural information. The bioactivity assays showed that angucycline compounds 1-3 exhibited potent antitumor activities against 11 human cancer cell lines and antibacterial activities against a series of Gram-positive bacteria. Mayamycin (1) selectively exhibited potent cytotoxicity activity against triple-negative breast cancer (TNBC) cell lines such as MDA-MB-231, MDA-MB-468, and Bt-549, with IC50 values of 0.60-2.22 µM.

Characterization of the Aminosugar Biosynthetic and Regulatory Genes of Vicenistatin in Monodonata labio-Associated Streptomyces parvus SCSIO Mla-L010.

Vicenistatin (1) is a potent polyketide antitumor antibiotic composed of a 20-membered macrolactam core appended to a unique aminosugar, vicenisamine. In this study, vicenistatin was isolated and its biosynthetic gene cluster identified from Monodonata labio-associated Streptomyces parvus SCSIO Mla-L010. A set of five genes, vicC, vicD, vicE, vicF, and vicG, was confirmed to be involved in the biosynthesis of the aminosugar by gene inactivations. VicG was characterized as an N-methyltransferase that catalyzes the methylation of the 4'-amino group in the last step of the aminosugar biosynthetic pathway; the N-demethyl intermediate 4'-N-demethylvicenistatin (2) was isolated from the ΔvicG mutant strain. In addition, vicR1 was characterized as a positive pathway-specific regulatory gene. Notably, N-demethyl compound 2 was found to exert impressive antibacterial activities, with MIC values spanning 0.06-4 µg/mL, against a panel of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus, Gram-negative Helicobacter pylori, and mycobacterium Mycobacterium smegmatis and the fungal pathogen Candida albicans. Compound 2 was also found to display reduced cytotoxicities relative to vicenistatin, especially against noncancerous human cell lines.

Discovery, Structure Correction, and Biosynthesis of Actinopyrones, Cytotoxic Polyketides from the Deep-Sea Hydrothermal-Vent-Derived Streptomyces sp. SCSIO ZS0520.

Three new actinopyrone derivatives, actinopyrones E-G (1, 3, and 4), together with three known analogues, PM050463 (2), actinopyrone D (5), and PM050511 (6), were isolated from Streptomyces sp. SCSIO ZS0520 derived from a deep-sea hydrothermal vent. Their structures, complete with absolute configurations, were elucidated using extensive spectroscopic analyses combined with Mosher's method, ECD calculations, and bioinformatics analyses. These findings corrected the absolute configurations of previously reported actinopyrone analogues 2, 5, and 6 at C-3, C-9, and C-10. Notably, compound 6 displayed notable cytotoxicity against six human cell lines with IC50 values of 0.26-2.22 µM. A likely biosynthetic pathway and annotations of protein function are proposed on the basis of bioinformatics analyses. Genes coding for methyltransferase and glycosyltransferase tailoring chemistries needed to generate final structures were notably absent from the biosynthetic gene cluster. Taken together, these results enable further bioengineering of the actinopyrones and related congeners as potential antitumor agents.

Identification and Heterologous Expression of the Kendomycin B Biosynthetic Gene Cluster from Verrucosispora sp. SCSIO 07399.

Verrucosispora sp. SCSIO 07399, a rare marine-derived actinomycete, produces a set of ansamycin-like polyketides kendomycin B-D (1-3) which possess potent antibacterial activities and moderate tumor cytotoxicity. Structurally, kendomycin B-D contain a unique aliphatic macrocyclic ansa scaffold in which the highly substituted pyran ring is connected to the quinone moiety. In this work, a type I/type III polyketide synthase (PKS) hybrid biosynthetic gene cluster coding for assembly of kendomycin B (kmy), and covering 33 open reading frames, was identified from Verrucosispora sp. SCSIO 07399. The kmy cluster was found to be essential for kendomycin B biosynthesis as verified by gene disruption and heterologous expression. Correspondingly, a biosynthetic pathway was proposed based on bioinformatics, cluster alignments, and previous research. Additionally, the role of type III PKS for generating the precursor unit 3,5-dihydroxybenzoic acid (3,5-DHBA) was demonstrated by chemical complementation, and type I PKS executed the polyketide chain elongation. The kmy cluster was found to contain a positive regulatory gene kmy4 whose regulatory effect was identified using real-time quantitative PCR (RT-qPCR). These advances shed important new insights into kendomycin B biosynthesis and help to set the foundation for further research aimed at understanding and exploiting the carbacylic ansa scaffold.

Mutasynthesis of Antibacterial Halogenated Actinomycin Analogues.

Through precursor-directed biosynthesis, feeding halogenated (F-, Cl-, Br-, I-) or methoxy-substituted 4-methyl-3-hydroxyanthranilic acid (4-MHA) analogues to the acnGHLM-deleted mutant strain of Streptomyces costaricanus SCSIO ZS0073 led to the production of ten new actinomycin analogues (4-13). Several of the actinomycin congeners displayed impressive antimicrobial activities, with MIC values spanning 0.06-64 µg/mL to clinically derived antibiotic resistant pathogens, including Staphylococcus aureus, Enterococcus faecium, and Candida albicans, with low cytotoxicity.

Identification and characterization of isocitrate dehydrogenase 1 (IDH1) as a functional target of marine natural product grincamycin B.

Grincamycins (GCNs) are a class of angucycline glycosides isolated from actinomycete Streptomyces strains that have potent antitumor activities, but their antitumor mechanisms remain unknown. In this study, we tried to identify the cellular target of grincamycin B (GCN B), one of most dominant and active secondary metabolites, using a combined strategy. We showed that GCN B-selective-induced apoptosis of human acute promyelocytic leukemia (APL) cell line NB4 through increase of ER stress and intracellular reactive oxygen species (ROS) accumulation. Using a strategy of combining phenotype, transcriptomics and protein microarray approaches, we identified that isocitrate dehydrogenase 1(IDH1) was the putative target of GCN B, and confirmed that GCNs were a subset of selective inhibitors targeting both wild-type and mutant IDH1 in vitro. It is well-known that IDH1 converts isocitrate to 2-oxoglutarate (2-OG), maintaining intracellular 2-OG homeostasis. IDH1 and its mutant as the target of GCN B were validated in NB4 cells and zebrafish model. Knockdown of IDH1 in NB4 cells caused the similar phenotype as GCN B treatment, and supplementation of N-acetylcysteine partially rescued the apoptosis caused by IDH1 interference in NB4 cells. In zebrafish model, GCN B effectively restored myeloid abnormality caused by overexpression of mutant IDH1(R132C). Taken together, we demonstrate that IDH1 is one of the antitumor targets of GCNs, suggesting wild-type IDH1 may be a potential target for hematological malignancies intervention in the future.

Efficient, green, and rapid strategy for separating actinomycin D and X2 using supercritical fluid chromatography.

Actinomycin D has long been used as a first-line antitumor drug in clinical practice. Actinomycin X2, a new drug lead, is often isolated along with actinomycin D. The minor differences between the two actinomycin analogs pose a daunting challenge in separation. In this study, supercritical fluid chromatography (SFC) was successfully utilized for the purification of actinomycin X2 and actinomycin D from a marine derived Streptomyces sp. DQS-5. After one-step SFC purification, the purities of these two compounds were determined to be 97.3 % and 97.8 %, respectively. This method provides a green alternative for the separation of these pharmacologically important actinomycin antibiotics. This study also demonstrated the development of a simple and rapid method for the separation of natural products based on SFC.

Isolation and Characterization of New Anti-Inflammatory and Antioxidant Components from Deep Marine-Derived Fungus Myrothecium SP. Bzo-l062.

In the present study, four new compounds including a pair of 2-benzoyl tetrahydrofuran enantiomers, namely, (-)-1S-myrothecol (1a) and (+)-1R-myrothecol (1b), a methoxy-myrothecol racemate (2), and an azaphilone derivative, myrothin (3), were isolated along with four known compounds (4-7) from cultures of the deep-sea fungus Myrothecium sp. BZO-L062. Enantiomeric compounds 1a and 1b were separated through normal-phase chiral high-performance liquid chromatography. The absolute configurations of 1a, 1b, and 3 were assigned by ECD spectra. Among them, the new compound 1a and its enantiomer 1b exhibited anti-inflammatory activity, inhibited nitric oxide formation in lipopolysaccharide-treated RAW264.7 cells, and exhibited antioxidant activity in the 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and oxygen radical absorbance capacity assays.

Grincamycin B Functions as a Potent Inhibitor for Glioblastoma Stem Cell via Targeting RHOA and PI3K/AKT.

Glioblastoma multiforme (GBM) is the most malignant form of glioma, and the overall survival time of patients with GBM is usually less than 14 months. Therefore, it is urgent to find new and effective medicine for GBM. Recently, marine natural products have been shown to exhibit strong inhibitory effects on cancer cells, providing a new avenue for exploring novel drugs for GBM treatment. In this study, we investigated the inhibitory effect of the Grincamycin (GCN) B-F, newly isolated from marine-derived Streptomyces Lusitanus SCSIO LR32, on GBM cells, and evaluated the mechanism of GCN B on GBM. The results, for the first time, showed that GCN B acted as a potent inhibitor to suppress growth and invasion of two human GBM cell lines U251 and 091214 in vitro. In addition, GCN B could effectively target GSCs in GBM evidenced by attenuated formation of tumor spheres and decrease of several markers of GSCs. Furthermore, we performed gene expression microarray followed by Signal-Net analysis. The result revealed that RHOA and PI3K/AKT axis played critical roles for a GCN B-mediated inhibitory effect on GSCs. Altogether, our findings highlighted GCN B as a promising inhibitor for GSCs via targeting RHOA and PI3K/AKT.

Targeted isolation of new polycyclic tetramate macrolactams from the deepsea-derived Streptomyces somaliensis SCSIO ZH66.

With a combined strategy of bioinformatics analysis, gene manipulation coupled with variation of growth conditions, the targeted activation of polycyclic tetramate macrolactams (PTMs) in the deepsea-derived Streptomyces somaliensis SCSIO ZH66 was conducted, which afforded a new (1) PTM, named somamycin A, along with three enol-type tetramic acid tautomers (2-4, somamycins B-D) of 10-epi-hydroxymaltophilin, 10-epi-maltophilin and 10-epi-HSAF, respectively. The structures of compounds 1-4 were elucidated by extensive spectroscopic analyses together with ECD calculations. Compound 1 exhibited notable growth inhibition against plant pathogenic fungi Fusariumoxysporum MHKW and Alternariabrassicae BCHB with the MIC values of 1.6 and 3.1 µg/mL, respectively, which were more potent than those of the positive control nystatin; and compounds 3 and 4 displayed moderate antifungal activities. Moreover, compounds 1-4 exhibited moderate cytotoxicity against the human cancer cell lines of HCT116 and K562.

Chlorinated bis-indole alkaloids from deep-sea derived Streptomyces sp. SCSIO 11791 with antibacterial and cytotoxic activities.

Two new chlorinated bis-indole alkaloids, dionemycin (1) and 6-OMe-7',7″-dichorochromopyrrolic acid (2), along with seven known analogs 3-9, were isolated from the deep-sea derived Streptomyces sp. SCSIO 11791. Their structures were elucidated by extensive HRESIMS, and 1D and 2D NMR data analysis. In vitro antibacterial and cytotoxic assays revealed that, compound 1, shows anti-staphylococcal activity with an MIC range of 1-2 µg/mL against six clinic strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated from human and pig. Additionally, compound 1 displayed cytotoxic activity against human cancer cell lines NCI-H460, MDA-MB-231, HCT-116, HepG2, and noncancerous MCF10A with an IC50 range of 3.1-11.2 µM. Analysis of the structure-activity relationship reveals that the chlorine atom at C-6″ could be pivotal for conferring their bioactivities, thus providing hints on chemical modifications on bis-indole alkaloid scaffold in drug design.

Antitubercular Ilamycins from Marine-Derived Streptomyces atratus SCSIO ZH16 ΔilaR.

Tuberculosis (TB) ranks as the leading cause of death from a single infectious agent (ranking more lethal than HIV/AIDS) over the course of the past decade. More concerning is that reports of multi-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of TB have been dramatically increasing. It continues to become ever more clear that novel anti-TB drugs with improved efficacies and reduced toxicities are urgently needed. We report here the discovery of 12 new ilamycin analogues, ilamycins G-R (1-12), bearing various nonproteinogenic amino acids, along with ilamycins E1 (13) and F (14), from a 200 L scale culture of the marine-derived mutant actinomycete Streptomyces atratus SCSIO ZH16 ΔilaR. Importantly, bioassays against Mycobacterium tuberculosis H37Rv revealed that all 12 new agents displayed antitubercular activities with MIC values ranging from 0.0096 to 10 µM. The structures of 1-12 were elucidated on the basis of HRESIMS, 1D and 2D NMR, and X-ray single-crystal diffraction studies. In addition, compound 10 was found to be moderately cytotoxic against a panel of tumor human cell lines. From these data we can formulate tentative structure-activity relationships for the antitubercular and antitumor activities of the ilamycins.

Inhibition of cellular inflammatory mediator production and amelioration of learning deficit in flies by deep sea Aspergillus-derived cyclopenin.

In the course of screening lipopolysaccharide (LPS)-induced nitric oxide (NO) production inhibitors, two related benzodiazepine derivatives, cyclopenol and cyclopenin, were isolated from the extract of a deep marine-derived fungal strain, Aspergillus sp. SCSIOW2. Cyclopenol and cyclopenin inhibited the LPS-induced formation of NO and secretion of IL-6 in RAW264.7 cells at nontoxic concentrations. In terms of the mechanism underlying these effects, cyclopenol and cyclopenin were found to inhibit the upstream signal of NF-κB activation. These compounds also inhibited the expression of IL-1ß, IL-6, and inducible nitric oxide synthase (iNOS) in mouse microglia cells, macrophages in the brain. In relation to the cause of Alzheimer's disease, amyloid-ß-peptide is known to induce inflammation in the brain. Therefore, the present study investigated the ameliorative effects of these inhibitors on an in vivo Alzheimer's model using flies. Learning deficits were induced by the overexpression of amyloid-ß42 in flies, and cyclopenin but not cyclopenol was found to rescue learning impairment. Therefore, novel anti-inflammatory activities of cyclopenin were identified, which may be useful as a candidate of anti-inflammatory agents for neurodegenerative diseases.

The Isolation of Pyrroloformamide Congeners and Characterization of Their Biosynthetic Gene Cluster.

Dithiolopyrrolones are microbial natural products containing a disulfide or thiosulfonate bridge embedded in a unique bicyclic structure. By interfering with zinc ion homeostasis in living cells, they show strong antibacterial activity against a variety of bacterial pathogens, as well as potent cytotoxicity against human cancer cells. In the current study, two new dithiolopyrrolones, pyrroloformamide C (3) and pyrroloformamide D (4), were isolated from Streptomyces sp. CB02980, together with the known pyrroloformamides 1 and 2. The biosynthetic gene cluster for pyrroloformamides was identified from Streptomyces sp. CB02980, which shared high sequence similarity with those of dithiolopyrrolones, including holomycin and thiolutin. Gene replacement of pyfE, which encodes a nonribosomal peptide synthetase (NRPS), abolished the production of 1-4. Overexpression of pyfN, a type II thioesterase gene, increased the production of 1 and 2. Genome neighborhood network analysis of the characterized and orphan gene clusters of dithiolopyrrolones revealed a unified mechanism for their biosynthesis, involving an iterative-acting NRPS and a set of conserved tailoring enzymes for the bicyclic core formation.

New Borrelidins from Onchidium sp. Associated Streptomyces olivaceus SCSIO LO13.

Borrelidins M-O (1-3), along with four previously known family members (4-7), were isolated from marine pulmonated mollusks Onchidium sp. associated Streptomyces olivaceus SCSIO LO13. The structures of 1-3 were elucidated by extensive spectral analyses of HR-ESI-MS, 1D and 2D NMR data. In addition, the cytotoxic and antibacterial activities of 1-7 were evaluated enabling us to propose some tentative structure-activity relationships (SARs), especially those involving modifications at C(22) and the moieties at C(7) and C(8) of the borrelidin scaffold.

CytA, a reductase in the cytorhodin biosynthesis pathway, inactivates anthracycline drugs in Streptomyces.

Antibiotic-producing microorganism can develop strategies to deal with self-toxicity. Cytorhodins X and Y, cosmomycins A and B, and iremycin, are produced as final products from a marine-derived Streptomyces sp. SCSIO 1666. These C-7 reduced metabolites show reduced antimicrobial and comparable cytotoxic activities relative to their C-7 glycosylated counterparts. However, the biosynthetic mechanisms and relevant enzymes that drive C-7 reduction in cytorhodin biosynthesis have not yet been characterized. Here we report the discovery and characterization of a reductase, CytA, that mediates C-7 reduction of this anthracycline scaffold; CytA endows the producer Streptomyces sp. SCSIO 1666 with a means of protecting itself from the effects of its anthracycline products. Additionally, we identified cosmomycins C and D as two intermediates involved in cytorhodin biosynthesis and we also broadened the substrate specificity of CytA to clinically used anthracycline drugs.