New polycyclic tetramate macrolactams with antimycobacterial activity produced by marine-derived Streptomyces sp. KKMA-0239.

During our screening for anti-mycobacterial agents against Mycobacterium avium complex (MAC), two new polycyclic tetramate macrolactams (PTMs), named hydroxycapsimycin (1) and brokamycin (2), were isolated along with the known PTM, ikarugamycin (3), from the culture broth of marine-derived Streptomyces sp. KKMA-0239. The relative structures of 1 and 2 were elucidated by spectroscopic data analyses, including 1D and 2D NMR. Furthermore, the absolute configuration of 1 was confirmed by a single-crystal X-ray diffraction analysis. Compounds 2 and 3 exhibited moderate antimycobacterial activities against MAC, including clinically isolated drug-resistant M. avium.

Mavintramycin A is a promising antibiotic for treating Mycobacterium avium complex infectious disease.

Mycobacterium avium complex (MAC) is a serious disease that is mainly caused by infection with the non-tuberculous mycobacteria (NTM), Mycobacterium avium and Mycobacterium intracellulare. Seven new compounds, designated mavintramycins A-G (1-7), were isolated along with structurally related compounds, including amicetin (9) and plicacetin (10), from the culture broth of Streptomyces sp. OPMA40551 as anti-MAC compounds that were active against M. avium and M. intracellulare. Among them, mavintramycin A showed the most potent and selective inhibition of M. avium and M. intracellulare. Furthermore, mavintramycin A was active against more than 40 clinically isolated M. avium, including multidrug-resistant strains, and inhibited the growth of M. avium in a persistent infection cell model using THP-1 macrophages. Mavintramycin A also exhibited in vivo efficacy in silkworm and mouse infection assays with NTM. An experiment to elucidate its mechanism of action revealed that mavintramycin A inhibits protein synthesis by binding to 23S ribosomal RNA in NTM. Mavintramycin A, with a different chemical structure from those of clinically used agents, is a promising drug candidate for the treatment of MAC infectious disease.

Synthesis and biological evaluation of nectriatide derivatives, potentiators of amphotericin B activity.

Nectriatide 1a, a naturally occurring cyclic tetrapeptide, has been reported to a potentiator of amphotericin B (AmB) activity. In order to elucidate its structure-activity relationships, we synthesized nectriatide derivatives with different amino acids in solution-phase synthesis and evaluated AmB-potentiating activity against Candida albicans. Among them, C-and N-terminal protected linear peptides were found to show the most potent AmB-potentiating activity.

Haneummycin, a new 22-membered macrolide lactam antibiotic, produced by marine-derived Streptomyces sp. KM77-8.

A new antibiotic named haneummycin (1) was isolated from a culture broth of marine-derived Streptomyces sp. KM77-8 by solvent extraction and HPLC using a C4 column. The structure of 1 was elucidated including relative stereochemistry as a new 22-membered macrolide lactam associated with a cyclopentanone and three sugars by various spectroscopic analyses, such as MS and NMR. Compound 1 displayed significant antibacterial activities against Gram-positive bacteria including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) with both MIC values of 8.0 µg ml-1.

Astaxanthin Attenuates Nonalcoholic Steatohepatitis with Downregulation of Osteoprotegerin in Ovariectomized Mice Fed Choline-Deficient High-Fat Diet.

BACKGROUND: Postmenopausal estrogen decline increases the risk of developing nonalcoholic steatohepatitis (NASH), and it might accelerate progression to cirrhosis and hepatocellular carcinoma. AIMS: This study aimed to investigate a novel therapy for postmenopausal women who are diagnosed with NASH. METHODS: Seven-week-old female C57BL/6 J mice were divided into three experimental groups as follows: (1) sham operation (SHAM group), (2) ovariectomy (OVX group), and (3) ovariectomy + 0.02% astaxanthin (OVX + ASTX group). These three groups of mice were fed a choline-deficient high-fat (CDHF) diet for 8 weeks. Blood serum and liver tissues were collected to examine liver injury, histological changes, and hepatic genes associated with NASH. An in vitro study was performed with the hepatic stellate cell line LX-2. RESULTS: The administration of ASTX significantly improved pathological NASH with suppressed steatosis, inflammation, and fibrosis, in comparison with those in the OVX-induced estrogen deficiency group. As a result, liver injury was also attenuated with reduced levels of alanine aminotransferase and aspartate transaminase. In addition, our study found that ASTX supplementation decreased hepatic osteoprotegerin (OPG) in vivo, a possible factor that contributes to NASH development. In vitro, this study further confirmed that ASTX has an inhibitory effect on the secretion of OPG in LX-2 human hepatic stellate cells. CONCLUSIONS: Our findings suggest that ASTX alleviates CDHF-OVX-induced pathohistological NASH with downregulated OPG, possibly via suppression of the transforming growth factor beta pathway. ASTX could has promise for use in postmenopausal women diagnosed with NASH.

Detection of Kita-Kyushu Lung Cancer Antigen-1, a Cancer/Testis Antigen, in the Stomach Close to a Cancerous Condition.

Background: Kita-Kyushu lung cancer antigen-1 (KK-LC-1), encoded by CT83, is a cancer/testis antigen (CTA) and an attractive target for immunotherapy. Our previous study demonstrated frequent CT83 expression in gastric cancers (GCs) and non-tumor sites of the stomach with tumors. Additionally, there was a correlation with Helicobacter pylori (Hp) infection. Since it currently remains unclear whether KK-LC-1 is expressed in the stomach without GC, this study investigated KK-LC-1 expression in non-GC stomach. Methods: We investigated differences in CT83 gene expression at non-tumor sites of stomachs with or without tumors in 118 GC patients and 115 non-GC patients. Fisher's exact test was used for statistical analyses. Results: CT83 expression was detected in 77% of non-tumor sites in stomachs with tumors, which was significantly higher than in stomachs without tumors (7%, p < 0.0001). All patients with CT83 expression at non-tumor sites of their stomachs without tumors carried Hp. Conclusion: CT83 appears to be rarely expressed in the atrophic stomach, and furthermore, a part of patients positive for its expression will develop GC in the future, suggesting that CT83 expression is a useful marker for predicting GC.

Screening for microbial potentiators of neutral lipid degradation in CHO-K1 cells.

A cell-based assay was conducted to screen microbial culture broths for potentiators of neutral lipid degradation in Chinese Hamster Ovary K1 cells. A total of 5,363 microbial cultures from fungi and actinomycetes were screened in this assay. Brefeldin A (1) from fungal cultures was found to promote the degradation of triacylglycerol (TG) with an EC50 of 2.6 µM. Beauveriolides I (2), III (3), beauverolides A (4), B (5), and K (6) from fungal cultures showed potentiating effect on cholesteryl ester (CE) degradation with EC50s ranging from 0.02 to 0.13 µM. Among these compounds, 2 and 6 exhibited the strongest activities (EC50, 0.02 µM). From actinomycete cultures, oxohygrolidin (7) (EC50 for TG and CE, > 1.7 and 0.8 µM, respectively) and hygrolidin (8) (EC50 for TG and CE, 0.08 and 0.004 µM, respectively) promoted degradation of CE more preferably than TG.

Binding of phenochalasin A, an inhibitor of lipid droplet formation in mouse macrophages, on G-actin.

Phenochalasin A, a unique phenol-containing cytochalasin produced by the marine-derived fungus Phomopsis sp. FT-0211, was originally discovered in a cell morphological assay of observing the inhibition of lipid droplet formation in mouse peritoneal macrophages. To investigate the mode of action and binding proteins, phenochalasin A was radio-labeled by 125I. Iodinated phenochalasin A exhibited the same biological activity as phenochalasin A. [125I]Phenochalasin A was found to be associated with an approximately 40 kDa protein, which was identified as G-actin. Furthermore, detail analyses of F-actin formation in Chinese hamster ovary cells (CHO-K1 cells) indicated that phenochalasin A (2 µM) caused elimination of F-actin formation on the apical site of the cells, suggesting that actin-oriented specific function(s) in cytoskeletal processes are affected by phenochalasin A.

Discovery of prescopranone, a key intermediate in scopranone biosynthesis.

A key intermediate in scopranone biosynthesis, prescopranone, accumulated in the mycelium of Streptomyces avermitilis SUKA carrying the biosynthetic gene cluster for scopranone lacking the sprT encoding the monooxygenase. The structure of prescopranone was elucidated by NMR and other spectral data. Prescopranone consists of a 2-pyranone ring with two atypical scoop-like moieties (1-ethyl-1-propenyl and 2-ethylbutyl groups), which was deduced as a product of the modular polyketide syntheses encoded by sprA, sprB, and sprC. Prescopranone inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity in a BMP receptor mutant cell line.

Conditioned medium from stem cells derived from human exfoliated deciduous teeth ameliorates NASH via the Gut-Liver axis.

Non-alcoholic steatohepatitis (NASH) occurrence has been increasing and is becoming a major cause of liver cirrhosis and liver cancer. However, effective treatments for NASH are still lacking. We examined the benefits of serum-free conditioned medium from stem cells derived from human exfoliated deciduous teeth (SHED-CM) on a murine non-alcoholic steatohepatitis (NASH) model induced by a combination of Western diet (WD) and repeated administration of low doses of carbon tetrachloride intraperitoneally, focusing on the gut-liver axis. We showed that repeated intravenous administration of SHED-CM significantly ameliorated histological liver fibrosis and inflammation in a murine NASH model. SHED-CM inhibited parenchymal cell apoptosis and reduced the activation of inflammatory macrophages. Gene expression of pro-inflammatory and pro-fibrotic mediators (such as Tnf-α, Tgf-ß, and Ccl-2) in the liver was reduced in mice treated with SHED-CM. Furthermore, SHED-CM protected intestinal tight junctions and maintained intestinal barrier function, while suppressing gene expression of the receptor for endotoxin, Toll-like receptor 4, in the liver. SHED-CM promoted the recovery of Caco-2 monolayer dysfunction induced by IFN-γ and TNF-α in vitro. Our findings suggest that SHED-CM may inhibit NASH fibrosis via the gut-liver axis, in addition to its protective effect on hepatocytes and the induction of macrophages with unique anti-inflammatory phenotypes.

New Terpendole Congeners, Inhibitors of Sterol O-Acyltransferase, Produced by Volutella citrinella BF-0440.

New terpendoles N-P (1-3) were isolated along with 8 structurally related known compounds including terpendoles and voluhemins from a culture broth of the fungus Volutella citrinella BF-0440. The structures of 1-3 were elucidated using various spectroscopic experiments including 1D- and 2D-NMR. All compounds 1-3 contained a common indole-diterpene backbone. Compounds 2 and 3 had 7 and 6 consecutive ring systems with an indole ring, respectively, whereas 1 had a unique indolinone plus 4 consecutive ring system. Compounds 2 and 3 inhibited both sterol O-acyltransferase 1 and 2 isozymes, but 1 lost the inhibitory activity. Structure-activity relationships of fungal indole-diterpene compounds are discussed.

The Anti-atherogenic Activity of Beauveriolide Derivative BVD327, a Sterol O-Acyltransferase 2-Selective Inhibitor, in Apolipoprotein E Knockout Mice.

The fungal 13-membered cyclodepsipeptides, beauveriolides I and III, were previously reported to be atheroprotective activity in mouse models via inhibiting sterol O-acyltransferase (SOAT) activity. A total of 149 beauveriolide derivatives (BVDs) synthesized combinatorially were evaluated in in silico absorption, distribution, metabolism and excretion (ADME) analysis and inhibitory activity toward the two SOAT isozymes, SOAT1 and SOAT2. Hence, only 11 BVDs exhibited SOAT2-selective inhibition. Among these, we chose BVD327, which had the highest ADME score, for further evaluation. BVD327 administration (50 mg/kg/d, per os (p.o.)) significantly decreased atherosclerotic lesions in the aorta and heart (25.4 ± 6.9 and 20.6 ± 2.9%, respectively) in apolipoprotein E knockout (Apoe-/-) mice fed a cholesterol-enriched diet (0.2% cholesterol and 21% fat) for 12 weeks. These findings indicate that beauveriolide derivatives can be used as anti-atherosclerotic agents.

Voluhemins, new inhibitors of sterol O-acyltransferase, produced by Volutella citrinella BF-0440.

New compounds, designated voluhemins A (1) and B (2), are isolated from the culture broth of the fungal strain Volutella citrinella BF-0440 along with structurally related known NK12838 (3). Spectroscopic data, including 1D and 2D NMR, elucidated their structures. Compounds 1-3 have a common indoline-diterpene core and two additional isoprenyl moieties. Compounds 1 and 3 contain a hemiaminal unit, while 2 is O-methylated 1. Their inhibitory activities toward sterol O-acyltransferase (SOAT) 1 and 2 isozymes in SOAT1- and SOAT2-expressing Chinese hamster ovary (CHO) cells show that 2 selectively inhibits the SOAT2 isozyme.

Inhibition of cholesteryl ester synthesis by polyacetylenes from Atractylodes rhizome.

Using activity guided purification, four known compounds, sesquiterpene atractylenolide III (1), and the polyacetylenes 14-acetoxy-12-senecioyloxytetradeca-2E,8E,10E-trien-4,6-diyn-1-ol (2), 14-acetoxy-12-α-methylbutyl-2E,8E,10E-trien-4,6-diyn-1-ol (3), and 14-acetoxy-12-ß -methylbutyl-2E,8E,10E-trien-4,6-diyn-1-ol (4), were isolated from a traditional herbal medicine, Atractylodes rhizome. Structurally similar 3 and 4 (3/4 mixture) were obtained as a mixture. In intact Chinese hamster ovary (CHO) K1 cell assays, 1, 2, and a 3/4 mixture selectively inhibited cholesterol [14C]oleate synthesis from [14C]oleate with IC50 values of 73.5 µM, 35.4 µM, and 10.2 µM, respectively, without any effects on cytotoxicity. As a potential target of these inhibitors involved in cholesteryl ester (CE) synthesis, effects on sterol O-acyltransferase (SOAT) activity were investigated using microsomes prepared from CHO-K1 cells as an enzyme source. Hence, these compounds inhibit SOAT activity with IC50 values (211 µM for 1, 29.0 µM for 2, and 11.8 µM for 3/4 mixture) that correlate well with those measured from intact cell assays. Our results strongly suggest that these compounds inhibit CE synthesis by blocking SOAT activity in CHO-K1 cells.

Inhibition of neutral lipid synthesis by avarols from a marine sponge.

The effects of 14 sesquiterpene hydroquinones, including 8 marine sponge-derived avarols (1-8) and 6 semisynthetic derivatives (9-14), on lipid droplet accumulation and neutral lipid synthesis in Chinese hamster ovary (CHO) K1 cells were investigated. In intact CHO-K1 cell assays, avarol (1) markedly decreased the number and size of lipid droplets in CHO-K1 cells and exhibited the most potent inhibitory activity on the synthesis of cholesteryl ester (CE) and triglyceride (TG) with IC50 values of 5.74 and 6.80 µM, respectively. In enzyme assays, sterol O-acyltransferase (SOAT), the final enzyme involved in CE biosynthesis, and diacylglycerol acyltransferase (DGAT), the final enzyme involved in TG biosynthesis, were inhibited by 1 with IC50 values of 7.31 and 20.0 µM, respectively, which correlated well with those obtained in the intact cell assay. These results strongly suggest that 1 inhibited SOAT and DGAT activities in CHO-K1 cells, leading to a reduction in the accumulation of CE and TG in lipid droplets.

Celludinones, new inhibitors of sterol O-acyltransferase, produced by Talaromyces cellulolyticus BF-0307.

New indanones, designated celludinones A ((±)-1) and B (2), were isolated from the culture broth of the fungal strain Talaromyces cellulolyticus BF-0307. The structures of celludinones were elucidated by spectroscopic data, including 1D and 2D NMR. Celludinone A was found to be a mixture of racemic isomers ((±)-1), which were isolated by a chiral column. Compounds (+)-1 and (-)-1 inhibited the sterol O-acyltransferase (SOAT) 1 and 2 isozymes in a cell-based assay using SOAT1- and SOAT2-expressing Chinese hamster ovary (CHO) cells, while 2 selectively inhibited the SOAT2 isozyme.

A Mixture of Atropisomers Enhances Neutral Lipid Degradation in Mammalian Cells with Autophagy Induction.

Atropisomers with a biaryl dihydronaphthopyranone structure, dinapinones A1 (DPA1) (M position) and A2 (DPA2) (P position), were isolated from the fungus culture broth of Talaromyces pinophilus FKI-3864 as inhibitors of [14C]neutral lipid ([14C]triacylglycerol (TG) and [14C]cholesteryl ester (CE)) synthesis from [14C]oleic acid in Chinese hamster ovary-K1 (CHO-K1) cells. DPA2 inhibited [14C]TG and [14C]CE synthesis (IC50s, 0.65 and 5.6 µM, respectively), but DPA1 had no inhibitory activity on [14C]TG and [14C]CE synthesis even at 12 µM. However, a 1:1 mixture of DPA1 and DPA2 (DPAmix) had the most potent inhibitory activity on [14C]TG and [14C]CE synthesis (IC50s, 0.054 and 0.18 µM, respectively). The mechanism of action of DPAmix was investigated. DPAmix had no effects on the enzymes involved in neutral lipid synthesis, while DPAmix enhanced the degradation of [14C]neutral lipids with concomitant decrease in cytosolic lipid droplets accumulated in CHO-K1 cells. From analysis of autophagy marker proteins, DPAmix caused dose-dependent induction of microtubule-associated protein light chain 3-II (LC3-II) and degradation of p62. In the autophagic flux assay using bafilomycin A1, DPAmix upregulated autophagosome turnover. These results reveal that DPAmix enhances neutral lipid degradation together with induction of autophagy.

Callyspongiamides A and B, sterol O-acyltransferase inhibitors, from the Indonesian marine sponge Callyspongia sp.

Callyspongiamides A (1) and B (2), two new sterol O-acyltransferase (SOAT) inhibitors, were isolated from the Indonesian marine sponge Callyspongia sp. together with a known congener, dysamide A (3). The structures of 1 and 2 were elucidated to be polychlorine-containing modified dipeptides based on their spectroscopic data. Compounds 1-3 inhibited both of the SOAT isozymes, SOAT1 and SOAT2, in cell-based and enzyme-based assays.

Discovery of a Fungal Multicopper Oxidase That Catalyzes the Regioselective Coupling of a Tricyclic Naphthopyranone To Produce Atropisomers.

Atropisomeric dinapinones A1 and A2 (DPA1 and DPA2) were isolated from a culture of Talaromyces pinophilus FKI-3864. Monapinone coupling enzyme (MCE), which dimerizes naphthopyranone monapinone A (MPA), was purified from a cell-free extract of T. pinophilus FKI-3864. MCE regioselectively dimerizes MPA at the 8,8'-positions to synthesize the atropisomers DPA1 and DPA2 in a ratio of approximately 1:2.5 without a cofactor. The optimal pH value and temperature for MCE were 4.0 and 50 °C, and the apparent Km and Vmax values for MPA were (72.7±23.2) µm and (1.21±0.170) µmol min-1 mg-1 protein. The MCE polypeptide is significantly homologous with multicopper oxidases. Heterologous expression of MCE and functional analysis confirmed that MCE catalyzes the regioselective coupling reaction of MPA to produce DPA. No fungal multicopper oxidase has previously been reported to catalyze regioselective intermolecular oxidative phenol coupling to produce naphthopyranone atropisomers.

Design and Synthesis of A-Ring Simplified Pyripyropene†A Analogues as Potent and Selective Synthetic SOAT2 Inhibitors.

Currently, pyripyropene A, which is isolated from the culture broth of Aspergillus fumigatus FO-1289, is the only compound known to strongly and selectively inhibit the isozyme sterol O-acyltransferase 2 (SOAT2). To aid in the development of new cholesterol-lowering or anti-atherosclerotic agents, new A-ring simplified pyripyropene A analogues have been designed and synthesized based on total synthesis, and the results of structure-activity relationship studies of pyripyropene A. Among the analogues, two A-ring simplified pyripyropene A analogues exhibited equally efficient SOAT2 inhibitory activity to that of natural pyripyropene A. These new analogues are the most potent and selective SOAT2 inhibitors to be used as synthetic compounds and attractive seed compounds for the development of drug for dyslipidemia, including atherosclerotic disease and steatosis.