Benastatin K, a chlorinated benastatin-related antibiotic from Streptomyces sp. HGTA384.

Benastatin K (1), a new chlorinated benastatin derivative, was isolated from the culture broth of the actinomycete Streptomyces sp. HGTA384. The structure of 1 was determined on the basis of spectroscopic analysis, including 1D and 2D NMR, as well as HRESI-MS, UV and IR, and comparison with data reported in the literature. Compound 1 and benastatins A and B exhibited inhibitory activity against Micrococcus luteus (MIC 7.8, 31.3, and 3.9 µM, respectively), and IgE-mediated ß-hexosaminidase release in RBL-2H3 cells with IC50 values of 42, 79, and 19 µM, respectively.

A new linear peptide, higapeptin, isolated from the mud flat-derived fungus Acremonium persicinum inhibits mitochondrial energy metabolism.

A combination of LC-MS/MS and feature-based molecular networking analyses led to the isolation of a new adenopeptin analog, higapeptin (1), and four known peptides, adenopeptin (2), adenopeptins B and C (3 and 4), and acremopeptin (5), from the rice culture of the fungus Acremonium persicinum (18F04103) isolated from a mud flat of the Ariake Sea in Kyushu, Japan. The structure of 1 was determined by NMR and MS/MS fragmentation analyses. The absolute configuration of the constituent amino acids was determined by Marfey's analysis after acid hydrolysis. The C-terminal residue was synthesized, and its absolute configuration was established by Marfey's analysis. Compounds 1 and 2 were found to inhibit mitochondrial energy metabolism, similar to efrapeptin D (6), a known mitochondrial ATPase inhibitor.

A monoacylglyceryltrimethylhomoserine, 21F121-A, containing a branched acyl group from Penicillium glaucoroseum.

A new monoacylglyceryltrimethylhomoserine, 21F121-A (1), was isolated from the culture of Penicillium glaucoroseum (21F00121) by LCMS-guided purification. The structure was elucidated by NMR and mass spectrometries. The absolute configuration of the homoserine moiety was analyzed by the ECD spectrum after acid hydrolysis, and the S-configuration of the glycerol moiety was determined based on the spectrum of the 1,2-dibenzoyl derivative after acid hydrolysis. Although a variety of diacylglyceryltrimethylhomoserine is distributed in lower plants and fungi, a limited number of studies on monoacyl derivatives have been reported. This is the fourth sample of monoacylglyceryltrimethylhomoserine discovered from a natural source, and the second sample isolated from a fungus. Compound 1 contains an unusual branched pentaene chain attached at the sn-1 position of glycerol and weakly inhibited the growth of HCT116 cells.

Amamine, an isoquinoline alkaloid from the Kitasatospora sp. HGTA304.

Amamine (1), a new isoquinoline alkaloid, was isolated from the culture extract of an actinomycete Kitasatospora sp. HGTA304. The structure of 1 was determined by NMR and MS analyses in combination with UV data. Compound 1 displayed α-glucosidase inhibitory potential (IC50 value of 56 µM) compared with acarbose (IC50 value of 549 µM) as standard.

Isolation, Synthesis, and Structure-Activity Relationship Study on Daphnane and Tigliane Diterpenes as HIV Latency-Reversing Agents.

Three new diterpenes, stellejasmins A (1) and B (2) and 12-O-benzoylphorbol-13-heptanoate (3), were isolated from the roots of Stellera chamaejasme L. The structures of 1-3 were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 are the first derivatives containing a hydroxy group at C-2 in the family of daphnane and tigliane diterpenes. The presence of a chlorine atom in 1 is unique in the plant metabolite. Compound 3 has an odd-number acyl group, which is biosynthetically notable. Human immunodeficiency virus (HIV) LTR-driven transcription activity was tested with 1-3 and 17 known diterpenes isolated from S. chamaejasme L. and Wikstroemia retusa A.Gray. Among these, gnidimacrin (4), stelleralide A (5), and wikstroelide A (20) were highly potent, with EC50 values of 0.14, 0.33, and 0.39 nM, respectively. The structure-activity relationship (SAR) was investigated using 20 natural and eight synthetic diterpenes. This is the first SAR study on natural daphnane and tigliane diterpenes.

Cell-based screening of extracts of natural sources to search for inhibitors of the ubiquitin-proteasome system and identification of proteasome inhibitors from the fungus Remotididymella sp.

The ubiquitin-proteasome system (UPS) regulates selective protein degradation to maintain protein homeostasis. Small molecules that inhibit the UPS-dependent protein degradation are promising anti-tumor agents. We report a cell-based luminescent assay using HeLa cells expressing luciferase-fused oxygen-dependent destruction domain (ODD) of hypoxia-inducible factor 1 α (HIF-1 α). ODD is degraded by the UPS and this assay system can aid in the identification of natural products that inhibit either process of the UPS, including ubiquitination/deubiquitination and proteasomal degradation. This reporter assay can exclude the influences of coloring or fluorescent compounds in extracts, thereby leading to effective high-throughput processing. The screening of 15,025 extracts of natural sources identified the culture extract of the fungus Remotididymella sp. (18F02908). Bioassay-guided isolation yielded two new polyketides, mellains A (1) and B (2), together with leptosphaerodione (3) and its acetone adduct 4. Compound 1 was revealed to have an unprecedented benzo[g]isoquinoline-8,10-dione skeleton. Evaluation of the biological activities demonstrated that these polyketides inhibit the proteasomal proteolysis. This is the first report of the identification of proteasome inhibitors from natural sources using a cell-based reporter assay targeting UPS inhibitors.

JBIR-155, a Specific Class D ß-Lactamase Inhibitor of Microbial Origin.

We discovered JBIR-155 as a novel specific class D ß-lactamase inhibitor from Streptomyces polymachus SoB100815Hv02. JBIR-155 consists of a 6-oxabicyclo[3.2.0]heptan-7-one skeleton and a long unsaturated alkyl chain moiety of which absolute configuration was determined by spectroscopic data, modified Mosher's method, and analyses of the relative configuration of chemically modified derivative. JBIR-155 specifically exhibited inhibitory activity against the class D ß-lactamase, with an IC50 value of 0.36 µM.

Rhusflavanone and mesuaferrone B: tyrosinase and elastase inhibitory biflavonoids extracted from the stamens of Mesua ferrea L.

Chemical isolation and bioactivity studies were conducted on the stamens of Mesua ferrea L., which are being used in a traditional skincare formulation in Myanmar. Rhusflavanone and mesuaferrone B were obtained as the main biflavonoids together with lupeol, five common flavonoids, and five phenolic compounds. After being identified by NMR and other spectroscopic analyses, these compounds were evaluated for their 1,1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging, human leukocyte elastase inhibitory, and mushroom tyrosinase inhibitory activities. The two biflavonoids exhibited strong inhibitory activities against elastase and tyrosinase, but low DPPH-radical scavenging activities. The contents of rhusflavanone and mesuaferrone B in the stamens were 0.35 ± 0.04% and 0.55 ± 0.06%, respectively. Moreover, lupeol was considered to be a cosmetically important component of the stamens because of its high content and strong elastase inhibitory activity. Rhusflavanone was reported to be isolated from M. ferrea for the first time.

New phenylspirodrimane metabolites MBJ-0030, MBJ-0031, and MBJ-0032 isolated from the soil fungal strain Stachybotrys sp. f23793.

Chemical screening of culture medium from the soil fungus Stachybotrys sp. resulted in the isolation of the three new phenylspirodrimanes MBJ-0030 (1), MBJ-0031 (2) and MBJ-0032 (3). Their structures were determined by detailed analysis of spectroscopic data. The absolute configurations of 1-3 were determined by modified Mosher's and Marfey's methods. In addition, cytotoxic and antimicrobial evaluations of the compounds were conducted.

Mechanism of Action of Prethioviridamide, an Anticancer Ribosomally Synthesized and Post-Translationally Modified Peptide with a Polythioamide Structure.

Thioviridamide, prethioviridamide, and JBIR-140, which are ribosomally synthesized and post-translationally modified peptides (RiPPs) possessing five thioamide bonds, induce selective apoptosis in various cancer cells, especially those expressing the adenovirus oncogene E1A. However, the target protein of this unique family of bioactive compounds was previously unknown. To investigate the mechanism of action, we adopted a combined approach of genome-wide shRNA library screening, transcriptome profiling, and biochemical identification of prethioviridamide-binding proteins. An shRNA screen identified 63 genes involved in cell sensitivity to prethioviridamide, which included translation initiation factors, aminoacyl tRNA synthetases, and mitochondrial proteins. Transcriptome profiling and subsequent analysis revealed that prethioviridamide induces the integrated stress response (ISR) through the GCN2-ATF4 pathway, which is likely to cause cell death. Furthermore, we found that prethioviridamide binds and inhibits respiratory chain complex V (F1Fo-ATP synthase) in mitochondria, suggesting that inhibition of complex V leads to activation of the GCN2-ATF4 pathway. These results imply that the members of a unique family of RiPPs with polythioamide structure target mitochondria to induce the ISR.

Solid-liquid Interface Screening SystemーApplication to the Screening of Antibiotic and Cytotoxic Substance-producing Fungi.

　A useful tool for the screening of fungi producing biologically active secondary metabolites such as antibiotics and cytotoxic substances has been developed. An agar plate-organic solvent interface cultivation (A/S-IFC) system, which comprised a hydrophobic organic solvent (upper phase) , a fungal mat (middle phase) and an agar plate (lower phase) , was constructed. The metabolite profiles were compared among the A/S-IFC, a traditional submerged cultivation (SmC) and an extractive liquid surface immobilization (Ext-LSI) system consisted of a hydrophobic solvent (upper phase) , a fungal cells-ballooned microspheres (middle phase) and a liquid medium (lower phase) , with high-performance liquid chromatography-photodiode array detector (HPLC-PDA) . In the A/S-IFC, many hydrophobic metabolites vastly different from those in the SmC were accumulated in the organic phase as with the Ext-LSI. For example, a valuable azaphilone, sclerotiorin, was remarkably produced into the organic phase in the A/S-IFC. The A/S-IFC was applied to the screening of antibiotic-producing fungi. As a result of paper disk method, it was found that 321 isolated among 811 strains produced antifungal metabolites (hit rate, 39.6%) . Furthermore, 8, 23, and 30 strains also produced cytotoxic metabolites against SKOV-3 (human ovary adenocarcinoma) , MESO-1 (human malignant pleural mesothelioma) , and Jurkat cells (immortalized human T lymphocyte) .

Transthyretin Amyloid Fibril Disrupting Activities of Extracts and Fractions from Juglans mandshurica Maxim. var. cordiformis (Makino) Kitam.

Transthyretin-related amyloidosis is a slowly progressive disorder caused by deposition of insoluble amyloid plaques formed by fibrillization of mutant or defective transthyretin (TTR) monomers that leads to neurodegeneration and organ failure. Thus, any compound exhibiting TTR amyloid formation inhibitory activity or TTR amyloid fibril disrupting activity might be a potential candidate for the development of therapies for these disorders. Our aim in this study was the evaluation of the TTR amyloid fibril disrupting potential of extracts of leaves and immature fruits of two Juglans plants, i.e., Juglans mandshurica var. sachalinensis and Juglans mandshurica var. cordiformis. The TTR amyloid fibril disrupting activity was measured by Thioflavin-T (ThT) assay and PROTEOSTAT® Protein aggregation assay methods. A fifty percent acetone extract of the fruits of Juglans mandshurica var. cordiformis showed strong amyloid fibril disrupting activity, and was further fractionated using different solvents. Ethyl acetate and n-butanol fractions showed significant activity in both assays. Syringic acid was isolated and identified as main compound in both of these fractions; however, it did not show any activity. Furthermore, some of the previously reported compounds from Juglans plants including naphthoquinone derivatives and phenolic compounds were evaluated to identify the potential bioactive compounds. Among them, juglone, a naphthoquinone derivative showed promising activity. However, juglone also showed strong cytotoxicity in HEK293 cells. Thus, future studies should focus on the isolation and identification of naphthoquinone derivatives or other compounds from Juglans plan ts with potent bioactivity and low cytotoxicity.

Biosynthesis of Quinolidomicin, the Largest Known Macrolide of Terrestrial Origin: Identification and Heterologous Expression of a Biosynthetic Gene Cluster over 200 kb.

Quinolidomicin A1 is the largest macrolide compound from terrestrial sources, consisting of a 60-membered ring, and its biosynthetic gene cluster was revealed to be over 200 kb. The gene cluster for quinolidomicin was cloned and heterologously expressed. Confirmation of the product led to a structural revision, in which the hydroxy group in the chromophore moiety of the reported structure was replaced by an amine group.

JBIR-150, a novel 20-membered polyene macrolactam from marine-derived Streptomyces sp. OPMA00071.

During the course of constructing a natural product library for drug screening consisting of microbial culture extracts originated from marine samples, we evaluated natural product components profiles via UPLC TOF-MS and routine biological tests for cytotoxic and antibiotic activities for all of the culture extract samples. By combination of chemical screening and biological activities, we succeeded in discovering a 20-membered macrolactam antibiotic subsequently designated JBIR-150 (1) from a marine-derived actinomycete identified as Streptomyces sp. that was isolated from an Okinawan marine sediment. The chemical structure of 1 was determined by interpreting NMR spectroscopic and mass spectrometric data. Compound 1 exhibited moderate cytotoxicity against MESO-1 and Jurkat cells.

Neothioviridamide, a Polythioamide Compound Produced by Heterologous Expression of a Streptomyces sp. Cryptic RiPP Biosynthetic Gene Cluster.

During genome mining for thioviridamide-like biosynthetic gene clusters that could produce polythioamide RiPP (ribosomally synthesized and post-translationally modified peptides), we discovered a novel cryptic biosynthetic gene cluster. During efforts to express this biosynthetic gene using heterologous expression of this biosynthetic gene cluster, a novel compound designated as neothioviridamide was produced. We report herein the cloning and heterologous expression of the neothioviridamide biosynthetic gene cluster and the isolation, structure determination, and cytotoxic activity of neothioviridamide.

Characterization of Giant Modular PKSs Provides Insight into Genetic Mechanism for Structural Diversification of Aminopolyol Polyketides.

Polyketides form many clinically valuable compounds. However, manipulation of their biosynthesis remains highly challenging. An understanding of gene cluster evolution provides a rationale for reprogramming of the biosynthetic machinery. Herein, we report characterization of giant modular polyketide synthases (PKSs) responsible for the production of aminopolyol polyketides. Heterologous expression of over 150 kbp polyketide gene clusters successfully afforded their products, whose stereochemistry was established by taking advantage of bioinformatic analysis. Furthermore, phylogenetic analysis of highly homologous but functionally diverse domains from the giant PKSs demonstrated the evolutionary mechanism for structural diversification of polyketides. The gene clusters characterized herein, together with their evolutionary insights, are promising genetic building blocks for de novo production of unnatural polyketides.

Unveiling the Biosynthetic Pathway of the Ribosomally Synthesized and Post-translationally Modified Peptide Ustiloxin B in Filamentous Fungi.

The biosynthetic machinery of the first fungal ribosomally synthesized and post-translationally modified peptide (RiPP) ustiloxin B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side-chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5'-phosphate (PLP)-dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi.