Search

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.

Conventional screening for novel bioactive compounds in actinomycetes often results in the rediscovery of known compounds. In contrast, recent genome sequencing revealed that most of the predicted gene clusters for secondary metabolisms are not expressed under standard cultivation conditions. To explore the potential metabolites produced by these gene clusters, we implemented a cryptic gene activation strategy by screening mutants that acquire resistance to rifampicin. The induction of rifampicin resistance in 11 actinomycete strains generated 164 rifampicin-resistant mutants (rif mutants). The comparison of the metabolic profiles between the rif mutants and their wild-type strains indicated that one mutant (TW-R50-13) overproduced an unidentified metabolite (1). During the isolation and structural elucidation of metabolite 1, an additional metabolite was found; both are unprecedented compounds featuring a C5N unit and a methylbenzene moiety. Of these partial structures, the biosynthesis of the latter has not been reported. A feeding experiment using (13)C-labeled precursors demonstrated that the methylbenzene moiety is most likely synthesized by the action of polyketide synthase. The gene deletion experiments revealed that the genes for the methylbenzene moiety are located at a different locus than the genes for the C5N unit.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an enzyme that catalyzes hydrolysis of 3'-phosphotyrosyl bonds and is involved in repair of irreversible topoisomerase I (Top1)-DNA covalent complexes. Tdp1 inhibitors are regarded as potential cancer therapeutics in combination with Top1 inhibitors, which are currently used to treat human cancers. While screening for Tdp1 inhibitors, we discovered a novel compound, JBIR-21 (1), from the culture of an anamorphic fungus, RF-13305. The structure of 1 was established by extensive NMR and MS analyses. Compound 1 showed inhibitory activity against Tdp1 (IC(50) value, 18 μM) and cytotoxic activity against cancer cell lines (IC(50) values, 3.5-13 μM). Compound 1 also exhibited antitumor activity in a mouse xenograft model without adverse effects.

Searching for metabolites from Streptomyces sp. RI051-SDHV6 resulted in the discovery of a novel peptide, JBIR-96 (1). The structure of 1 was established as an N-phenylacetylated pentapeptide involving a cysteic acid and a peptide lactone structure by extensive NMR and MS analyses. In addition, the absolute configuration of 1 was established by Marfey's and modified Mosher's methods.

Two new anthracyclines, tetracenoquinocin (1) and 5-iminoaranciamycin (2), together with the known compounds aranciamycin (3) and antibiotic SM 173B were isolated from the culture of Streptomyces sp. Sp080513GE-26 associated with a marine sponge, Haliclona sp. The structures of 1 and 2 were established on the basis of extensive NMR and MS analyses along with (13)C-labeling experiments. The compounds 1-3 were evaluated for cytotoxicity against two cancer cell lines.

In the course of our screening program for isoprenoids of marine actinobacterial origin, 523 actinobacterial strains were isolated from marine samples. Actinobacteria usually use the 2-C-methyl-d-erythritol 4-phosphate pathway for the production of primary metabolites, but some have been reported to use the mevalonate (MVA) pathway for the production of isoprenoids as secondary metabolites. 3-Hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) is a key enzyme and plays an important role in the MVA pathway. Therefore, we screened strains possessing the HMGR gene from the 523 strains mentioned above and also investigated isoprenoid compounds from cultures of strains possessing HMGR genes. As a result, Streptomyces sp. SpC080624SC-11 isolated from a marine sponge, Cinachyra sp., was shown to possess the HMGR gene and produce novel isoprenoids, JBIR-46 (1), -47 (2), and -48 (3). On the basis of extensive NMR and MS analyses, the structures of 1-3 were determined to be phenazine derivatives harboring dimethylallyl moieties. Furthermore, the isoprene units of 2 and 3 were confirmed to be synthesized via the MVA pathway in a feeding experiment using [1-(13)C]acetate.

Two new modified indole-containing peptides, JBIR-34 (1) and JBIR-35 (2), were isolated from the fermentation broth of a sponge-derived actinomycete identified by phylogenetic methods as a Streptomyces sp. (strain Sp080513GE-23). The planar structures of 1 and 2 were assigned on the basis of 1D and 2D NMR spectroscopy and MS analyses. Further, the absolute configurations of the amino acid residues were determined using Marfey's method.

New 34-membered polyol macrolides JBIR-129 (1) and JBIR-139 (2) were isolated from the culture of the terrestrial Streptomyces RK74. The planar structures of 1 and 2 were established on the basis of 1D and 2D NMR, ESI-TOF-MS, IR, and UV spectra. The relative configurations of the sugar units were determined by analyzing vicinal ¹H-¹H coupling constants and steric information. Both 1 and 2 showed cytotoxic activity against human ovarian adenocarcinoma SKOV-3 cells with IC₅₀ values of 0.3 and 0.4 μM, respectively.

Three new trichostatin analogues, JBIR-109 (1), JBIR-110 (2), and JBIR-111 (3), were isolated from the culture of the marine sponge-derived Streptomyces sp. strain RM72, together with trichostatin A (4) and trichostatic acid (5). The planar structures of 1-3 were determined on the basis of extensive NMR and MS analyses. In addition, the absolute configurations of the amino acid residues were determined by Marfey's method. The histone deacetylase inhibitory activities of 1-5 were examined, and their structure-activity relationships are discussed.

The search for metabolites of Kibdelosporangium sp. AK-AA56 resulted in the discovery of novel N-phenylacetylated peptides, JBIR-78 (1) and JBIR-95 (2). Compounds 1 and 2 were established to be N-phenylacetylated heptapeptides by extensive NMR and HRESIMS analyses. The absolute configuration of the standard amino acids including a cysteic acid moiety was determined using Marfey’s method on the acid hydrolysates of 1 and 2. The relative and absolute configurations of a nonstandard amino acid, β-hydroxyleucine, were elucidated using the J-based and modified Mosher’s methods, respectively. In an antimicrobial test, 1 showed antibacterial activity against Micrococcus luteus.

A novel naphthoquinone-like polyketide, JBIR-85 (1), with a unique skeleton and antioxidative activity was isolated from a culture of Streptomyces sp. RI-77. The planar structure of 1 was established on the basis of extensive NMR and MS analyses. The structure of 1 including the absolute configuration was established via X-ray crystallographic analysis. Since 1 exhibits a unique skeleton, we performed feeding experiments to reconfirm the structure and predict the biosynthetic pathway.

A novel compound, benthophoenin {1}, was isolated from the mycelium of Streptomyces prunicolor. The structure of 1 was determined mainly by nmr spectral analysis to be a phenazine carboxylic acid with a geranyl and two benzoyl residues

Proteasome assembling chaperone (PAC) 3 acts as a homodimer and plays an important role in proteasome formation. We screened JBIR-22 (1) as an inhibitor for protein-protein interaction (PPI) of PAC3 homodimer from our natural product library using a protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG) in vitro and found that 1 exhibited potent inhibitory activity against PAC3 homodimerization. Compound 1 showed long-term cytotoxicity against the human cervical carcinoma cell line, HeLa. This is the first report of a PPI inhibitor for proteasome assembly factors.

Two new aminocaprophenone alkaloids, ficuseptamines A (1) and B (2), and a new pyrrolidine alkaloid, ficuseptamine C (3), together with 12 known alkaloids and a known acetophenone derivative were isolated from a methanolic extract of the leaves of Ficus septica. The structures of 1−3 were determined on the basis of their spectroscopic data. The compounds obtained were evaluated for cytotoxicity against two cancer cell lines.