Kenichi Nonaka

Marine actinomycetes are prolific microorganisms; however, knowledge of their diversity, distribution, and secondary metabolites is limited. Marine actinomycetes represent an untapped source of novel bioactive compounds. In this study, we investigated shrimp shell as substrates for model production bioactive metabolites from actinomycetes under solid state fermentation (SSF) conditions. A total of fifteen actinomycetes were isolated from six sponges and one tunicate. The isolated actinomycetes were grown on solid shrimp shells. Cultures of actinomycetes were extracted with ethyl acetate (EtOAc) and extracts were bioassayed for activity against Staphylococcus aureus. One isolate 18A13O1 from the sponge, Rhabdastrella globostellata, exhibited antibacterial activity on primary screening compared to the other samples and was chosen for further study. Visualization using SEM showed aerial and substrate mycelia. Through phylogenetic analysis, it was confirmed that isolate 18A13O1 is a Pseudonocardia carboxydivorans. Purification of an EtOAc extract yielded A13B2, which showed a minimum inhibition concentration against S. aureus at 15.6 μg/mL. It can be concluded that this basic information is very important for further studies related to the development of the production of bioactive secondary metabolites through the solid state fermentation process.

Two new nitrogen-containing metabolites, designated hatsusamide A (1) and B (2), were isolated from a culture broth of Penicilliumsteckii FKJ-0213 together with the known compounds tanzawaic acid B (3) and trichodermamide C (4) by physicochemical (PC) screening. The structures of 1 and 2 were determined as a tanzawaic acid B-trichodermamide C hybrid structure and a new analog of aspergillazines, respectively. The absolute configuration of 1 was determined by comparing the values of tanzawaic acid B and trichodermamide C in the literatures, such as ¹H-nuclear magnetic resonance (¹H-NMR) data and optical rotation, after hydrolysis of 1. Compounds 1–4 were evaluated for cytotoxicity and anti-malarial activities. Compounds 1 and 3 exhibited weak anti-malarial activity at half-maximal inhibitory concentration (IC₅₀) values of 27.2 and 78.5 µM against the K1 strain, and 27.9 and 79.2 µM against the FCR3 strain of Plasmodium falciparum, respectively. Furthermore, 1 exhibited cytotoxicity against HeLa S3, A549, Panc1, HT29 and H1299 cells, with IC₅₀ values of 15.0, 13.7, 12.9, 6.8, and 18.7 μM, respectively.

Two new sulfur compounds, designated thioporidiol A (1) and B (2), were discovered by the MoS-screening program from a culture broth of Trichoderma polypori FKI-7382. The structures of 1 and 2 were determined as C13 lipid structures with an N-acetylcysteine moiety. The relative configuration at the C-5 and C-6 position of 1 was determined by the derivatives of α-methoxy-α-phenylacetic acid diesters, and the absolute configuration of the N-acetylcysteine moiety was determined by advanced Marfey’s analysis. Compounds 1 and 2 were evaluated for anti-microbial, cytotoxic and anti-malarial activities. Compound 2 exhibited anti-microbial activity against Candida albicans ATCC 64548.

Saprolegnia parasitica, belonging to oomycetes, is one of virulent pathogen of fishes such as salmon and trout, and causes tremendous damage and losses in commercial aquacultures by saprolegniasis. Previously, malachite green, an effective medicine, had been used to control saprolegniasis. However, this drug has been banned around the world due to its mutagenicity. Therefore, novel anti-saprolegniasis compounds are urgently needed. As a new frontier to discover bioactive compounds, we focused on the deep-sea fungi for the isolation of anti-saprolegniasis compounds. In this paper, on the course of anti-saprolegniasis agents from 546 cultured broths of 91 deep-sea fungal strains, we report a new compound, named quellenin (1) together with three known compounds, diorcinol (2), violaceol-I (3) and violaceol-II (4), from deep-sea fungus Aspergillus sp. YK-76. This strain was isolated from an Osedax sp. annelid, commonly called bone-eating worm, collected at the São Paulo Ridge in off Brazil. Compounds 2, 3 and 4 showed anti-S. parasitica activity. Our results suggest that diorcinol and violaceol analogs and could be good lead candidates for the development of novel agents to prevent saprolegniasis.

Microbial natural products possess a wide range of biological and biochemical potential. Among them, fungal secondary metabolites are one of the most important sources for discovering new drugs or lead compounds. In the present study, we explored substances produced by the strain Penicillium sp. FKI-7127 for its antiviral activity. We identified brefeldin A as a novel antiviral agent against dengue viruses. The inhibitory effect of brefeldin A was confirmed by virus titer and immunofluorescence assay. Brefeldin A inhibited dengue viruses regardless of serotypes and other related viruses including Zika virus and Japanese encephalitis virus. Time-of-addition study showed that brefeldin A exerts its antiviral effect at an early stage of the dengue virus (DENV) life cycle. These studies demonstrate that (i) brefeldin A could be used as a lead compound for drug development of anti-DENV and other related viruses and (ii) fungal metabolites are a potential and valuable source for dengue virus drug discovery.

Amebiasis is a common worldwide diarrheal disease, caused by the protozoan parasite, Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its known side effects and low efficacy against asymptomatic cyst carriers. E. histolytica is also capable of surviving sub-therapeutic levels of metronidazole in vitro. Novel drugs with different mode of action are therefore urgently needed. The sulfur assimilatory de novo L-cysteine biosynthetic pathway is essential for various cellular activities, including the proliferation and anti-oxidative defense of E. histolytica. Since the pathway, consisting of two reactions catalyzed by serine acetyltransferase (SAT) and cysteine synthase (CS, O-acetylserine sulfhydrylase), does not exist in humans, it is a rational drug target against amebiasis. To discover inhibitors against the CS of E. histolytica (EhCS), the compounds of Kitasato Natural Products Library were screened against two recombinant CS isozymes: EhCS1 and EhCS3. Nine compounds inhibited EhCS1 and EhCS3 with IC₅₀ values of 0.31–490 μM. Of those, seven compounds share a naphthoquinone moiety, indicating the structural importance of the moiety for binding to the active site of EhCS1 and EhCS3. We further screened >9,000 microbial broths for CS inhibition and purified two compounds, xanthofulvin and exophillic acid from fungal broths. Xanthofulvin inhibited EhCS1 and EhCS3. Exophillic acid showed high selectivity against EhCS1, but exhibited no inhibition against EhCS3. In vitro anti-amebic activity of the 11 EhCS inhibitors was also examined. Deacetylkinamycin C and nanaomycin A showed more potent amebicidal activity with IC₅₀ values of 18 and 0.8 μM, respectively, in the cysteine deprived conditions. The differential sensitivity of trophozoites against deacetylkinamycin C in the presence or absence of L-cysteine in the medium and the IC₅₀ values against EhCS suggest the amebicidal effect of deacetylkinamycin C is due to CS inhibition.

While isolating fungal strains from soil samples, two strains, Talaromyces siamensis FKA-61 and Phomopsis sp. FKA-62, were found to produce a black pigment near the boundary with an adjoining colony on agar plates. Moreover, strain FKA-61 inhibited the growth of strain FKA-62. Co-culture of T. siamensis FKA-61 with Phomopsis sp. FKA-62 resulted in the production of an antifungal agent, BE-31405, which proved active against Phomopsis sp. FKA-62. We screened for fungal genera that have the ability to induce production of antifungal BE-31405. Surprisingly, production of BE-31405 was induced only by co-culturing T. siamensis FKA-61 with a variety of BE-31405-sensitive fungal strains but never with BE-31405-insensitive strains. This is the first report of the induced production of a specific antibiotic active against antibiotic-sensitive fungal strains by co-culturing with them.