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Many prenylated indole derivatives are widely distributed in nature. Recently, two Streptomyces prenyltransferases, IptA and its homolog SCO7467, were identified in the biosynthetic pathways for 6-dimethylallylindole (DMAI)-3-carbaldehyde and 5-DMAI-3-acetonitrile, respectively. Here, we isolated a novel prenylated indole derivative, 3-hydroxy-6-dimethylallylindolin (DMAIN)-2-one, based on systematic purification of metabolites from a rare actinomycete, Actinoplanes missouriensis NBRC 102363. The structure of 3-hydroxy-6-DMAIN-2-one was determined by HR-MS and NMR analyses. We found that A. missouriensis produced not only 3-hydroxy-6-DMAIN-2-one but also 6-dimethylallyltryptophan (DMAT) and 6-DMAI when grown in PYM (peptone-yeast extract-MgSO4) medium. We searched the complete genome of A. missouriensis for biosynthesis genes of these compounds and found a gene cluster composed of an iptA homolog (AMIS_22580, named iptA-Am) and a putative tryptophanase gene (AMIS_22590, named tnaA-Am). We constructed a tnaA-Am-deleted (ΔtnaA-Am) strain and found that it produced 6-DMAT but did not produce 6-DMAI or 3-hydroxy-6-DMAIN-2-one. Exogenous addition of 6-DMAI to mutant ΔtnaA-Am resulted in the production of 3-hydroxy-6-DMAIN-2-one. Furthermore, in vitro enzyme assays using recombinant proteins produced by Escherichia coli demonstrated that 6-DMAI was synthesized from tryptophan and dimethylallyl pyrophosphate in the presence of both IptA-Am and TnaA-Am, and that IptA-Am preferred tryptophan to indole as the substrate. From these results, we concluded that the iptA-Am-tnaA-Am gene cluster is responsible for the biosynthesis of 3-hydroxy-6-DMAIN-2-one. Presumably, tryptophan is converted into 6-DMAT by IptA-Am and 6-DMAT is then converted into 6-DMAI by TnaA-Am. 6-DMAI appears to be converted into 3-hydroxy-6-DMAIN-2-one by the function of some unknown oxidases in A. missouriensis.

To construct a natural product library for drug screening, we isolated secondary metabolites from a wide variety of actinomycetes cultured from marine sponges. The results suggested that marine sponges are a promising source of actinomycetes with the potential to produce new metabolites. Furthermore, we evaluated the chemical space occupied by our natural product library (CB library) by multidimensional principal component analysis and compared it with a commercially available compound library (ZINC library), which was randomly selected from the ZINC library (approximately 30 000 000 compounds). The CB library occupied a wider chemical space than the ZINC library. Bioactive compounds in the CB library possessed a wide chemical space that was not covered by ZINC library. These results indicate that the CB library mainly comprises secondary metabolites from actinomycetes, and it has great potential as a source of compounds for drug screening.

In the course of our screening program for anticancer agents from natural sources, five new angucyclines, JBIR-90 (1), -116 (2), -91 (3), -92 (4) and -93 (5), were isolated from the fermentation broth of Streptomyces sp. RI33. The structures of 1-5 were elucidated on the basis of 1D and 2D NMR spectroscopy and MS analyses. Compounds 2-4 showed cytotoxic activities against malignant pleural mesothelioma cells at IC(50) values of 20-50 μM., (© 2011 Japan Antibiotics Research Association All rights reserved)

A new 1,1-dichlorocyclopropane-containing angucycline, JBIR-88 (1), and a new butenolide, JBIR-89 (2), respectively, were isolated from the fermentation broths of two lichen-derived actinomycetes identified as a new species of Streptomyces (strains RI104-LiC106 and RI104-LiB101) using phylogenetic methods. The structures of 1 and 2 were elucidated on the basis of 1D and 2D NMR spectroscopy and MS analyses. Compound 1 showed cytotoxic activities against cancer cells and antimicrobial activity against Micrococcus luteus.

Two peptides, tumescenamides A and B, were isolated from the fermentation broth of a marine bacterium, Streptomyces tumescens YM23-260. The structure of tumescenamide A was determined to be a cyclic depsipeptide consisting of α-amino-2-butenoic acid, tyrosine, valine, leucine and threonine, substituted with a 2,4-dimethylheptanoyl residue at the α-NH(2) position. Tumescenamide B possesses a 2,4,6-trimethylnonanoyl residue in place of the 2,4-dimethylheptanoyl substituent in tumescenamide A. Tumescenamide A induced reporter gene expression under the control of the insulin-degrading enzyme promoter.

In the course of our screening program for inhibitors of hepatic glucose production in rat hepatoma H4IIE-C3 cells, which were used as model liver cells, five naphtoquinone derivatives-javanicin, solaniol, 9-O-methylfusarubin, 5,10-dihydroxy-1,7-dimethoxy-3-methyl-1H-naphtho[2,3-c]pyran-6,9-dione, 9-O-methylbostrycoidin-and vanillin were selected from our natural product library. These naphtoquinone derivatives inhibited hepatic glucose production at IC(50) values of 3.8-29 microM, but showed cytotoxicity against hepatic cells after incubation for 48 h. However, vanillin showed an IC(50) value of 32 microM without exhibiting cytotoxicity at 50 microM. Therefore, we examined 12 vanillin derivatives to investigate their inhibitory activities against glucose production. Among these analogs, 4-hydro-3-methoxyacetophenone and 5-nitrosalicylaldehyde exhibited stronger inhibition than the other compounds at IC(50) values of 25 and 24 microM, respectively, with no cytotoxicity at a concentration of 50 microM. Hence, 4-hydro-3-methoxyacetophenone and 5-nitrosalicylaldehyde may be useful as a lead compound of anti-type 2 diabetic drugs.

In the course of our screening program for novel metabolites from tunicate-derived fungi, novel sesquiterpenoids, named JBIR-27 (1) and -28 (2), together with known sporogen-AO1 and phomenone, were isolated from the culture broth of Penicillium sp. SS080624SCf1. The structures of 1 and 2 were determined to be eremophilane analogs on the basis of extensive NMR and MS analyses. Sporogen-AO1, phomenone and 2 showed cytotoxicity against human cervical carcinoma cell line HeLa at IC(50) values of 8.3, 19 and 92 microM, respectively, whereas 1 was inactive at a concentration of 80 microM.

In the course of our screening program for active compounds from fungal metabolites, we isolated JBIR-12 (1) as a free radical scavenger from the culture broth of Penicillium sp. NBRC 103941. Structure elucidation of 1 was carried out using methylated and/or acetylated derivatives of 1. As a consequence, the structure of 1 was determined to be a novel highly oxygenated tetrahydronaphthalene species attached to an acyl chain moiety on the basis of NMR and other spectroscopic data. It was interesting that 1 was spontaneously methylated when left in methanol. Furthermore, isomerization or rearrangements could occur during the derivatization of 1. Compound 1 exhibited potent radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radical with an IC(50) value of 75 microM.

In the course of our screening program for active compounds that induce cell morphological changes of Saccharomyces cerevisiae, the culture broth of an entomopathogenic fungus Metarhizium sp. fE61 exhibited a unique morphological phenotype. We conducted an activity-guided isolation from the fermentation broth of Metarhizium sp. fE61 to yield two new macrolide compounds named JBIR-19 (1) and -20 (2) as active substances. Their structures were determined to be 24-membered macrolide analogs containing a 2-aminoethyl phosphate ester on the basis of NMR and other spectroscopic data. Compounds 1 and 2 induced striking elongated morphology of S. cerevisiae at concentrations of 3.1 and 13 microM, but showed weak antiyeast activity at MICs of 200 and >200 microM, respectively.

Ammocidins B, C and D were isolated from the culture broth of Saccharothrix sp. AJ9571, an ammocidin A-producing strain. Their structures were determined by a detailed spectroscopic analysis and by a comparison of their NMR data with those of ammocidin A. Ammocidins A and B showed potent anti-proliferative activities against human cancer cell lines.

In the course of our screening program for regulators of circadian clock system (circadian rhythms), we isolated a new natural compound JBIR-26 (1) from Streptomyces sp. AK-AH76. The structure was determined to be 2-hydroxy-3,6-dimethylbenzoic acid on the basis of spectroscopic data. Compound 1 lengthened the period-length of mammalian clocks for 1.5 hours at a concentration of 10 microg/ml.

A novel versipelostatin (1) analogue, versipelostatin F (2) was isolated from Streptomyces versipellis 4083-SVS6. The structure of 2 was determined by the analyses of the spectroscopic data. Compound 2 inhibited the expression of GRP78 induced by 2-deoxyglucose with an IC(50) value of 0.3 muM, which is 10-times more potent compared with that of 1.

A stawamycin analog, JBIR-11 (1) was isolated from mycelium of Streptomyces viridochromogenes subsp. sulfomycini NBRC 13830. The structure was determined on the basis of the spectroscopic data. Compound 1 exhibited growth inhibitory effect against human fibrosarcoma HT1080 cells with an IC50 value of 25 microM.

A novel compound of antimycin family, JBIR-06 (1), was isolated from Streptomyces sp. ML55. The structure of 1 was established as a twelve-membered macrocyclic skeleton with a 3-(formylamino)-2-hydroxybenzamide based on the spectroscopic data. Compound 1 inhibited the expression of GRP78 induced by 2-deoxyglucose at the IC50 value of 250 nM.

A new indole-diterpene, JBIR-03 (1), was isolated from the fungus Dichotomomyces cejpii var. cejpii NBRC 103559 and its structure was determined based on the spectroscopic data. 1 exhibited anti-MRSA (methicillin-resistant Staphylococcus aureus) activity and antifungal activity against apple Valsa canker-causing fungus, Valsa ceratosperma, while it exhibited no toxicity towards human cancer cells.

In the course of our screening program for regulators of the expression of GRP78 molecular chaperone, JBIR-04 (1) and -05 (2) were isolated from Streptomyces violaceoniger 4541-SVS3 as congeners of prunustatin A (3). The structures of 1 and 2 were determined by the analyses of the spectroscopic data. These compounds mainly consist of an amino acid and amino acid derived alpha-hydroxy acid residues. 1 and 2 inhibited the expression of GRP78 induced by 2-deoxyglucose in human fibrosarcoma HT1080 cells, but their activities were highly reduced compared with those of 3 and SW-163A.

A new member of the piericidin family, JBIR-02, was isolated from mycelium of Streptomyces sp. ML55 together with two known piericidin derivatives, piericidin A(1) and IT-143-B. The structure was determined on the basis of spectroscopic data. JBIR-02 inhibited nuclear export of beta-arrestin in HeLa cells at the concentration of 20 microM.