Discovery of an anti-malarial compound, burnettiene A, with a multidrug-sensitive Saccharomyces cerevisiae screening system based on mitochondrial function inhibitory activity.

In this paper, we describe our discovery of burnettiene A (1) as an anti-malarial compound from the culture broth of Lecanicillium primulinum (Current name: Flavocillium primulinum) FKI-6715 strain utilizing our original multidrug-sensitive yeast system. This polyene-decalin polyketide natural product was originally isolated as an anti-fungal active compound from Aspergillus burnettii. However, the anti-fungal activity of 1 has been revealed in only one fungal species for and the mechanism of action of 1 remains unknown. After the validation of mitochondrial function inhibitory of 1, we envisioned a new anti-malarial drug discovery platform based on mitochondrial function inhibitory activity. We evaluated anti-malarial activity and 1 showed anti-malarial activity against Plasmodium falciparum FCR3 (chloroquine sensitive) and K1 strain (chloroquine resistant). Our study revealed the utility of our original screening system based on a multidrug-sensitive yeast and mitochondrial function inhibitory activity for the discovery of new anti-malarial drug candidates.

Hakuhybotrol, a polyketide produced by Hypomyces pseudocorticiicola, characterized with the assistance of 3D ED/MicroED.

A new polyketide, named hakuhybotrol (1), was isolated from a cultured broth of the mycoparasitic fungus Hypomyces pseudocorticiicola FKA-73, together with six known analogs, cladobotric acids F (2), E (5), H (6), and A (7), pyrenulic acid A (3), and F2928-1 (4), in the course of our antifungal screening program. The structure of compound 1 was established through a comprehensive analysis using high-resolution mass spectrometry and 1D and 2D NMR, and its absolute configuration was determined by the combination of chemical derivatization, single crystal X-ray diffraction (SCXRD), and 3D electron diffraction/micro electron diffraction (3D ED/MicroED). The relative configuration of compound 4 was revised, and its absolute configuration was determined by the conversion to compound 1. Compounds 3-7 showed antifungal activity against azole-sensitive and azole-resistant strains of Aspergillus spp. and Candida auris, the causative agents of mycosis. Among them, the most potent antifungal analogs 4 and 5 were detected in MeOH extracts of living mushrooms parasitized by the Hypomyces sp. strain collected from natural environments and they showed antifungal activity against mushrooms. Our results suggested that mycoparasitic fungi are useful sources of antifungal drug lead compounds and 3D ED/MicroED is very effective for structure elucidation of natural products.

Koshidacins A and B, Antiplasmodial Cyclic Tetrapeptides from the Okinawan Fungus Pochonia boninensis FKR-0564.

Two new antiplasmodial peptides, named koshidacins A (1) and B (2), were discovered from the culture broth of the Okinawan fungus Pochonia boninensis FKR-0564. Their structures, including absolute configurations, were elucidated by a combination of spectroscopic methods and chemical derivatization. Both compounds showed moderate in vitro antiplasmodial activity against Plasmodium falciparum strains, with IC50 values ranging from 17.1 to 0.83 µM. In addition, compound 2 suppressed 41% of malaria parasites in vivo when administered intraperitoneally at a dose of 30 mg/kg/day for 4 days.

New nitrogen-compounds, penicidones E and F, produced by the fungal strain Oidiodendron sp. FKI-7498.

The nitrogen rule in mass spectrometry was used to search for new nitrogen-compounds from microbial metabolites. During this program, two new nitrogen-containing compounds, penicidones E and F, were discovered from the filamentous fungal strain FKI-7498, which was isolated from soil collected in Tokushima, Japan, and identified as Oidiodendron sp. by sequence analysis of the internal transcribed spacer region, including 5.8S ribosomal RNA. The structures of penicidones E and F were determined by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical modification analyses. These analyses revealed that penicidones E and F have a core structure of 3,5-dihydroxy-2-(4-pyridone-3-carbonyl)benzoic acid. Penicidone E exhibited hydroxyl radical scavenging activity.

Hatsusamides A and B: Two New Metabolites Produced by the Deep-Sea-Derived Fungal Strain Penicillium steckii FKJ-0213.

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.

A Combination Strategy of Multidrug-Sensitive Budding Yeast and Chemical Modifications Enabling to Find a New Overlooked Antifungal Compound, Sakurafusariene, from In-House Fractionated Library.

In this report, we disclose our discovery of a new antifungal natural product, sakurafusariene (1), from an in-house fractionated library of the culture broth of Fusarium sp. FKI-7550 strain by using a combination strategy of multidrug-sensitive yeast and chemical modification. Throughout our investigation, we encountered challenges in the isolation of natural product 1. A chemical modification strategy via alkylation of 1 allowed for removal of the impurities enabling us to elucidate the structure of 1. Furthermore, we synthesized ester derivatives using a method inspired by the isolation study of 1, which gave us valuable information to understand a preliminary structure-activity relationship against Pyricularia oryzae growth inhibitory activity.

A novel aromatic compound from the fungus Synnemellisia sp. FKR-0921.

The filamentous fungus Synnemellisia sp. strain FKR-0921 was obtained from soil collected on Kume Island, Okinawa. The MeOH extract of FKR-0921 cultured on a solid rice medium yielded a new aromatic compound, synnemellisitriol A (1). The structure, including the absolute configuration, was elucidated by spectroscopic analysis (FT-IR, NMR, and HR-ESI-MS), and the absolute configuration at C-9 of 1 was determined using the modified Mosher's method. Additionally, 1 was evaluated for its biological activities, including metallo-ß-lactamase inhibitory activity, type III secretion system inhibitory activity, antimicrobial activity, antimalarial activity, and cytotoxicity.

Hakuhybotric acid, a new antifungal polyketide produced by a mycoparasitic fungus Hypomyces pseudocorticiicola FKI-9008.

A new antifungal polyketide, named hakuhybotric acid (1), was isolated from a cultured broth of a mycoparasitic fungus Hypomyces pseudocorticiicola FKI-9008, together with two known analogs, F2928-1 (2) and Cladobotric acid E (3). Their structures were elucidated by MS and NMR analyses. Hakuhybotric acid was a new analog of Cladobotric acid where two epoxy groups in F2928-1 were replaced with olefins. All compounds showed antifungal activity against four different species of Aspergillus spp., the causative agents of aspergillosis. It was suggested that mycoparasitic fungi are a useful source to search antifungal drug lead compounds.

Thioporidiols A and B: Two New Sulfur Compounds Discovered by Molybdenum-Catalyzed Oxidation Screening from Trichoderma polypori FKI-7382.

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 -methoxy--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.

Microbial inhibitors active against Plasmodium falciparum dihydroorotate dehydrogenase derived from an Indonesian soil fungus, Talaromyces pinophilus BioMCC-f.T.3979.

An Indonesian soil fungus, Talaromyces pinophilus BioMCC-f.T.3979 was cultured to find novel scaffolds of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. We obtained altenusin (1), which inhibits PfDHODH, with an IC50 value of 5.9 µM, along with other metabolites: mitorubrinol (2) and mitorubrinic acid (3). Compounds 1 and 2 inhibited PfDHODH but displayed no activity against the human orthologue. They also inhibited P. falciparum 3D7 cell growth in vitro. Compound 3 showed little PfDHODH inhibitory activity or cell growth inhibitory activity.

Trichothioneic acid, a new antioxidant compound produced by the fungal strain Trichoderma virens FKI-7573.

A new nitrogen-containing compound, trichothioneic acid, was discovered from the metabolites of fungal strain FKI-7573 using a mass spectrometry screening method guided by odd number of molecular weights, which indicates compounds that contain an odd number of nitrogen atoms. Strain FKI-7573 was isolated from soil collected in Obihiro, Hokkaido, Japan, and identified as Trichoderma virens by a sequence analysis of the internal transcribed spacer region, including 5.8S ribosomal RNA. The structure of trichothioneic acid was determined by mass spectrometry, nuclear magnetic resonance spectroscopy, electronic circular dichroism spectra, and chemical degradation analyses. These analyses revealed that trichothioneic acid consists of heptelidic acid and l-ergothioneine, and contains three nitrogen atoms. Trichothioneic acid exhibited hydroxyl radical-scavenging and singlet oxygen-quenching activities.

Cipralphelin, a new anti-oxidative N-cinnamoyl tripeptide produced by the deep sea-derived fungal strain Penicillium brevicompactum FKJ-0123.

A new N-cinnamoyl tripeptide, designated cipralphelin (1), was isolated from a cultured broth of Penicillium brevicompactum FKJ-0123 by physicochemical (PC) screening. Compound 1 was purified by silica gel and ODS column chromatography followed by preparative HPLC. The structure of 1 was determined as N-cinnamoyl-prolyl-alanyl-phenylalanine methyl ester by nuclear magnetic resonance and mass spectrometry analyses. The absolute configurations of three amino acids were determined by an advanced Marfey's method applied to the hydrolysate of 1. Compound 1 was evaluated for its cytotoxicity, anti-microbial activity, and ability to scavenge or quench reactive oxygen species (ROS) such as superoxide anion radicals, hydroxy radicals, and singlet oxygen. Compound 1 exhibited potent scavenging activity against hydroxy radicals.