Naphthacemycins, novel circumventors of ß-lactam resistance in MRSA, produced by Streptomyces sp. KB-3346-5. II. Structure elucidation.

Seventeen new compounds, naphthacemycins A1-A11, B1-B4 and C1-C2, were isolated from a cultured broth of Streptomyces sp. KB-3346-5 during screening for circumventors of ß-lactam resistance in methicillin-resistant Staphylococcus aureus. Their structures were elucidated by spectroscopic studies, including NMR and X-ray crystallographic analysis. The naphthacemycin A series has a new skeleton displaying a 7-phenylnaphthacene-5,6,11(12H)-trione. In contrast, the quinone moiety of the A series is changed to dehydroxyquinol in the B series and to a semiquinone-like structure in the C series.

Naphthacemycins, novel circumventors of ß-lactam resistance in MRSA, produced by Streptomyces sp. KB-3346-5. I. The taxonomy of the producing strain, and the fermentation, isolation and antibacterial activities.

Screening for circumventors of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) led us to find 17 novel antibiotics, naphthacemycins A1-A11, B1-B4 and C1-C2. The naphthacemycins were isolated from a cultured broth of Streptomyces sp. KB-3346-5 by repeated silica gel column chromatography and HPLC. Naphthacemycins enhanced imipenem activity 100-500 times against MRSA at 0.5 µg ml-1, and naphthacemycins A4-A11 themselves showed MIC50 values of 1-4 µg ml-1 against 22 MRSA strains.

Synthesis and Structural Revision of Cyslabdan.

Cyslabdan was isolated from the culture broth of Streptomyces sp. K04-0144 as a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus. We accomplished the synthesis of cyslabdan according to a previously reported structure. However, we subsequently found that this structure was incorrect; our analysis of natural cyslabdan showed that it possessed R stereochemistry at the C8 position, not S, as had previously been reported. Thus, we completed the protecting-group-free synthesis of the correct structure of cyslabdan, which is described herein.

In vitro and in vivo anti-MRSA activities of nosokomycins.

The anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of nosokomycins A to D discovered in the silkworm-MRSA infection screening was investigated. The minimum inhibitory concentration (MIC) values of nosokomycins for authentic MRSA and S. aureus strains were calculated to be 0.06 to 2.0 µg/mL. They also showed potent inhibitory activity against 54 clinically isolated MRSA strains. Furthermore, nosokomycin A proved effective in the mouse-MRSA infection model.

Inhibitory effect of erythraline on Toll-like receptor signaling pathway in RAW264.7 cells.

Erythraline, isolated from the bark of Erythrina crista-galli which are used as Brazilian medicine plant for the treatment of inflammation diseases, suppressed nitric oxide (NO) production and induction of inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells stimulated by lipopolysaccharide (LPS). Because of Toll-like receptor (TLR) 4 and its signal transduction are indispensable to the production of NO and iNOS expression by LPS, we investigated the effects of erythraline on TLR signaling molecules. Western blot analysis revealed that the degradation of inhibitor of nuclear factor (NF)-κB (IκB) by LPS was suppressed by erythraline. Moreover, erythraline inhibited not only LPS-induced phosphorylation of IκB kinase (Ikk) but also phosphorylation of mitogen-activated protein kinases (MAPKs). However, it showed no effect on LPS -induced phosphorylation of transforming growth factor (TGF)-ß-activated kinase (TAK) 1 that exists upstream of Ikk and MAPKs, and is required for the activation of these signaling molecules on TLR signaling pathway. These results suggested that erythraline might have inhibited the kinase activity of TAK1. Furthermore, these results were supported from the inhibitory pattern of erythraline on TLR signaling molecules when the cells were stimulated by TLR2 ligand, peptidoglycan which activates the same pathway as LPS on TLR signal transduction.

Anti-inflammatory effect of berkeleyacetal C through the inhibition of interleukin-1 receptor-associated kinase-4 activity.

Berkeleyacetal C (BAC) isolated from Penicillium sp. which had isolated from a soil sample collected in Fukushima, inhibited NO production and induction of iNOS protein in RAW264.7 cells stimulated by the Toll-like receptor (TLR) 2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS). The other inflammatory mediator production by these stimulators was also suppressed by BAC in a concentration-dependent manner. BAC inhibited LPS- or PGN-activated nuclear translocation of nuclear factor (NF)-κB and MyD88-dependent signaling molecules. However, it showed no effect on LPS-induced nuclear translocation of interferon regulatory factor (IRF)-3, a MyD88-independent signaling molecule. To clarify the mechanistic basis for BAC ability to inhibit translocation of NF-κB and activated MyD88-dependent signaling molecules, we examined interleukin-1 receptor-associated kinase (IRAK)-4, existing to the most upstream on MyD88-dependent signaling molecules, in vitro kinase assay. BAC suppressed IRAK-4 kinase activity in a concentration-dependent manner. These findings suggest that BAC inhibits LPS- and PGN- induced NO production and iNOS expression by decreasing the level of the translocating of NF-κB in nuclear through inhibiting the kinase activity of IRAK-4 in inflammatory cells.

Nosokomycins, new antibiotics discovered in an in vivo-mimic infection model using silkworm larvae. II: Structure elucidation.

The structures of nosokomycins A, B, C and D, new anti-methicillin-resistant Staphylococcus aureus antibiotics produced by Streptomyces sp. K04-0144, were elucidated by spectroscopic studies including various NMR experiments. Nosokomycins A, B, C and D are new members of the moenomycin family consisting of an oligosaccharide moiety, a 2,3-dihydroxypropionic acid and an unusual sesterterpenoid moiety. All nosokomycins lack the cyclopentenone moiety in the oligosaccharide moiety of moenomycin A.

Nosokomycins, new antibiotics discovered in an in vivo-mimic infection model using silkworm larvae. I: Fermentation, isolation and biological properties.

The in vivo-mimic assay system using silkworm larvae was used as a screening tool to discover antibiotics against methicillin-resistant Staphylococcus aureus (MRSA). Microbial culture broths were screened in this in vivo-mimic assay system and a culture broth of Streptomyces sp. K04-0144 was selected. New antibiotics, designated nosokomycins A-D, were isolated from the culture broth by HP-20 and ODS column chromatography and HPLC. Nosokomycins inhibited the growth of MRSA with MIC values of 0.125 microg ml(-1) using the liquid microdilution method. Furthermore, MRSA-infected silkworms survived when nosokomycin A or B was injected at a dose of 50 microg per larva.

Effects of hyperin, isoquercitrin and quercetin on lipopolysaccharide-induced nitrite production in rat peritoneal macrophages.

The extract of the root of Acanthopanax chiisanensis Nakai is used for the treatment of inflammation. To analyse the action mechanism of this extract, the effect of hyperin (quercetin-3-O-beta-d-galactose) isolated from the ethyl acetate fraction of the root of A. chiisanensis on nitrite production and induction of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS, 1 microg/mL)-stimulated rat peritoneal macrophages were examined. The effect of the structurally related compounds, isoquercitrin (quercetin-3-O-beta-d-glucose) and quercetin (an aglycone of the two compounds) isolated from the extract of the leaves of Vaccinium koreanum Nakai was also examined to compare the effect. It was shown that hyperin inhibited the LPS-induced iNOS expression and nitrite production. Of the three compounds, quercetin showed the most potent inhibitory activity. The phosphorylation of p44/42 mitogen activated protein kinase (MAPK), p38 MAPK and c-Jun N-terminal kinase (JNK) were also inhibited by these compounds. These findings suggested that hyperin in the extract of the root of A. chiisanensis inhibits nitric oxide (NO) production through inhibition of the expression of iNOS by attenuation of p44/p42 MAPK, p38 MAPK and JNK, and thus participates in the antiinflammatory activity of the extract.

Guadinomines, Type III secretion system inhibitors, produced by Streptomyces sp. K01-0509. II: physico-chemical properties and structure elucidation.

The structures of guadinomines, new inhibitors of a bacterial Type III secretion system produced by Streptomyces sp. K01-0509, were elucidated by spectroscopic studies including various NMR experiments. Guadinomines A, B, C(1), C(2) and D consist of a carbamoylated cyclic guanidinyl moiety, an alkyl chain moiety and an L-Ala-L-Val moiety in common, while guadinomic acid is a smaller molecule consisting of a carbamoylated cyclic guanidinyl moiety and a hydroxyl hexanoate moiety.

Cyslabdan, a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus, produced by Streptomyces sp. K04-0144. I. Taxonomy, fermentation, isolation and structural elucidation.

Cyslabdan, a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus, was isolated from the culture broth of Streptomyces sp. K04-0144 by Diaion HP-20 and ODS column chromatographies and preparative HPLC. The structure of cyslabdan was elucidated by spectroscopic analyses including NMR. The compound has a labdane-type diterpene skeleton connecting with an N-acetylcysteine via thioether linkage.

Cyslabdan, a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus, produced by Streptomyces sp. K04-0144. II. Biological activities.

Cyslabdan produced by Streptomyces sp. K04-0144 was found to potentiate imipenem activity against methicillin-resistant Staphylococcus aureus (MRSA). The MIC value of imipenem against MRSA was reduced from 16 to 0.015 microg/ml in combination with cyslabdan. Study on anti-MRSA activity of other typical antibiotics in combination with cyslabdan showed that the potentiating activity was limited to beta-lactam antibiotics. Furthermore, among beta-lactam antibiotics, the activity of carbapenems was most remarkably potentiated by cyslabdan.

Structure elucidation of fungal sespendole, an inhibitor of lipid droplet synthesis in macrophages.

A new fungal metabolite named sespendole was isolated as an inhibitor of lipid droplet synthesis in mouse macrophages from the culture broth of the fungal strain Pseudobotrytis terrestris FKA-25. The structure and stereochemistry of sespendole were elucidated by spectroscopic studies including various NMR spectral analyses, exciton chirality experiments and the modified Mosher method. Sespendole was found to possess a new indolosesquiterpene skeleton modified with two isoprenes.

Sespendole, a new inhibitor of lipid droplet synthesis in macrophages, produced by Pseudobotrytis terrestris FKA-25.

Sespendole was isolated as an inhibitor of lipid droplet formation in macrophages from the culture broth of a fungal strain Pseudobotrytis terrestris FKA-25. The compound inhibited the synthesis of cholesteryl ester and triacylglycerol by mouse macrophages with IC50 values of 4.0 and 3.2 microM, respectively.

Nine constituents including six xanthone-related compounds isolated from two ascomycetes, Gelasinospora santi-florii and Emericella quadrilineata, found in a screening study focused on immunomodulatory activity.

Five metabolites tentatively called GS-1 (1)-5 (5) from Gelasinospora santi-florii, and four tentatively called EQ-4 (6), EQ-6 (7)-8 (9) together with 1-4 from Emericella quadrilineata have been isolated in a screening study on immunomodulatory fungal constituents. Among these nine metabolites, EQ-7 and 8 have been unknown. This time, the structures of GS-4 which has previously been isolated, EQ-7, and -8 have been determined to be (4R,4aS,9aR)-1,9a-dihydronidulalin A (4), (4S,4aR,9aR)-4a-carbomethoxy-1,4,4a,9a-tetrahydro-4,8-dihydroxy-6-methylxanthone (8), and 9-hydroxymicroperfuranone (9), respectively, and the six other metabolites have been identified. On bioassay, a dihydroxanthone, nidulalin A (1), a hexaketide, sordarial (5), and a xanthone, pinselin (7) have displayed significant immunosuppressive activities. The structure-activity relationships of these constituents have also been discussed.

Terpenoid tetrahydroisoquinoline alkaloids emetine, klugine, and isocephaeline inhibit the activation of hypoxia-inducible factor-1 in breast tumor cells.

Klugine (1), isocephaeline (2), and emetine (4) inhibited hypoxia-inducible factor-1 (HIF-1) activation by hypoxia in T47D breast tumor cells (IC(50) values 0.2, 1.1, and 0.11 muM, respectively). Compounds 1, 2, and 4 inhibited both hypoxia- and iron chelator-induced HIF-1 activation by blocking HIF-1alpha protein accumulation.

Saururus cernuus lignans--potent small molecule inhibitors of hypoxia-inducible factor-1.

Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1alpha protein accumulation without affecting HIF-1alpha mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.

Lignans from Acanthopanax chiisanensis having an inhibitory activity on prostaglandin E2 production.

The chloroform and the ethyl acetate fractions from the roots of Acanthopanax chiisanensis exhibited the significant inhibition of TPA-induced prostaglandin E(2) (PGE(2)) production in rat peritoneal macrophages. Five lignans were isolated from the chloroform fraction and their structures were elucidated as l-sesamin, helioxanthin, savinin, taiwanin C, and 3-(3,4-dimethoxybenzyl)-2-(3,4-methylenedioxybenzyl)butyrolactone. Among the lignans tested, taiwanin C showed the most potent inhibitory activity (IC(50) = 0.12 microM) on PGE(2) production with the relative order of potency, taiwanin C >> 3-(3,4-dimethoxybenzyl)-2-(3,4-methylenedioxybenzyl)butyrolactone > savinin = helioxanthin. l-Sesamin showed no inhibitory activity at 30 microM.

Hypoxia-inducible factor-1 activation by (-)-epicatechin gallate: potential adverse effects of cancer chemoprevention with high-dose green tea extracts.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that induces oxygen-regulated genes in response to reduced oxygen conditions (hypoxia). Expression of the oxygen-regulated HIF-1alpha subunit correlates positively with advanced disease stages and poor prognosis in cancer patients. Green tea catechins are believed to be responsible for the cancer chemopreventive activities of green tea. We found that (-)-epicatechin-3-gallate (ECG, 1), one of the major green tea catechins, strongly activates HIF-1 in T47D human breast carcinoma cells. Among the green tea catechins tested, 1 demonstrated the strongest HIF-1-inducing activity, while (-)-epigallocatechin-3-gallate (EGCG, 2) was significantly less active. However, 2 is relatively unstable in the in vitro system studied. Compound 1 also increases the expression of HIF-1 target genes including GLUT-1, VEGF, and CDKN1A. In T47D cells, 1 induces nuclear HIF-1alpha protein without affecting HIF-1alpha mRNA. Both the induction of HIF-1alpha protein and activation of HIF-1 by 1 can be blocked by iron and ascorbate, indicating that 1 may activate HIF-1 through the chelation of iron. These results suggest that intended cancer chemoprevention with high-dose green tea extracts may be compromised, by the ability of tea catechins to promote tumor cell survival pathways associated with HIF-1 activation.

Mechanism targeted discovery of antitumor marine natural products.

Antitumor drug discovery programs aim to identify chemical entities for use in the treatment of cancer. Many strategies have been used to achieve this objective. Natural products have always played a major role in anticancer medicine and the unique metabolites produced by marine organisms have increasingly become major players in antitumor drug discovery. Rapid advances have occurred in the understanding of tumor biology and molecular medicine. New insights into mechanisms responsible for neoplastic disease are significantly changing the general philosophical approach towards cancer treatment. Recently identified molecular targets have created exciting new means for disrupting tumor-specific cell signaling, cell division, energy metabolism, gene expression, drug resistance and blood supply. Such tumor-specific treatments could someday decrease our reliance on traditional cytotoxicity-based chemotherapy and provide new less toxic treatment options with significantly fewer side effects. Novel molecular targets and state-of-the-art, molecular mechanism-based screening methods have revitalized antitumor research and these changes are becoming an ever-increasing component of modern antitumor marine natural products research. This review describes marine natural products identified using tumor-specific mechanism-based assays for regulators of angiogenesis, apoptosis, cell cycle, macromolecule synthesis, mitochondrial respiration, mitosis, multidrug efflux and signal transduction. Special emphasis is placed on natural products directly discovered using molecular mechanism-based screening.