Masato Iwatsuki

The global increase in multidrug-resistant tuberculosis strains poses a major threat; consequently, there is an urgent need to identify and develop novel drugs. We have previously presented a simple fibroblast assay (SFA) screening method that enables the simultaneous assessment of antibacterial activity and cytotoxicity toward host cells. This method was developed based on our finding that live (but not dead) Mycobacterium tuberculosis bacilli exhibit cytotoxic activity against human lung fibroblasts, and that this cytotoxicity, which is proportional to the number of viable bacterial cells, is inhibited by known antimycobacterial drugs. While conventional methods to measure M. tuberculosis growth require 2–4 weeks to detect antibacterial activity; the SFA method enables detection within 2–3 days and can detect compounds that are active within host cells. Here, we applied high-throughput screening, based on assessment of cytotoxicity, to identify candidate antimycobacterial agents. In parallel, by comparing the direct antibacterial activity of compounds in liquid medium (using the broth dilution test) with that observed via SFA, we developed a new SFA-based screening system to select compounds with low cytotoxicity that are active outside or inside the host cells. Using this dual system, we screened 742 compounds from the Ōmura Natural Compound Library (Kitasato University, Japan), with 62 hits. Of these, 19 were excluded owing to their toxicity, and six exhibited antibacterial activity only via SFA screening. Four promising antimycobacterial candidates with no cytotoxicity at the 10 μg/mL threshold—aridicin A, lankamycin, streptovaricin, and lariatin A—were identified. This novel system supports the rapid identification of low-cytotoxicity antimycobacterial compounds that are active inside or outside the host cells. IMPORTANCE: This screening system, comprising the simple fibroblast assay (SFA) and broth dilution test (BDT), effectively identifies low-cytotoxicity compounds that exhibit antimycobacterial activity inside or outside the host cells.

Macrocyclic formation in natural products is typically understood to proceed through direct macrocyclization reactions. Here we demonstrate that complex macrocycles can instead be constructed through stepwise ring expansion of a pre-formed smaller scaffold. Genetic and biochemical analyses of luminamicin (1) biosynthesis revealed that a 10-membered ring is progressively transformed into a 14-membered macrocycle through two mechanistically distinct ring expansion steps. This process involves a cofactor-independent intramolecular acyl transfer and a radical-mediated oxidative rearrangement, which together reorganize a pre-assembled intermediate into the final macrocyclic framework. This stepwise remodeling strategy contrasts with canonical macrocyclization logic and provides a solution to the entropic and conformational constraints associated with the formation of large rings. More broadly, these findings establish stepwise ring expansion as a previously unrecognized biosynthetic strategy for constructing complex macrocycles and suggest new strategies for their chemical synthesis.

Macrocyclic formation in natural products is typically understood to proceed through direct macrocyclization reactions. Here we demonstrate that complex macrocycles can instead be constructed through stepwise ring expansion of a pre-formed smaller scaffold. Genetic and biochemical analyses of luminamicin (1) biosynthesis revealed that a 10-membered ring is progressively transformed into a 14-membered macrocycle through two mechanistically distinct ring expansion steps. This process involves a cofactor-independent intramolecular acyl transfer and a radical-mediated oxidative rearrangement, which together reorganize a pre-assembled intermediate into the final macrocyclic framework. This stepwise remodeling strategy contrasts with canonical macrocyclization logic and provides a solution to the entropic and conformational constraints associated with the formation of large rings. More broadly, these findings establish stepwise ring expansion as a previously unrecognized biosynthetic strategy for constructing complex macrocycles and suggest new strategies for their chemical synthesis.

Tropolones are non-benzenoid aromatics with broad bioactivity, yet their inherent planarity and metal-chelating properties pose challenges to a medicinal chemistry campaign. We developed a liquid-phase peptide synthesis strategy that leveraged a hydrophobic TAG carrier to streamline condensation, deprotection, and purification steps, overcoming challenges that hinder traditional solution-phase derivatization of tropolones. Among the synthesized tropolone-peptide hybrid derivatives, the lead analogue exceeded antimalarial potency of puberulic acid and artemisinin, highlighting the advantage of three-dimensional peptide hybridization.

Two new dimeric diketopiperazines, piperasagamines A (1) and B (2), were discovered from the cultured material of a deep-sea-derived Aspergillus sp. FKJ-0404 strain. The planar structures of 1 and 2 were elucidated by spectroscopic analysis as dimeric diketopiperazines featuring an α, β-dehydroproline moiety. The absolute configurations of 1 and 2 were determined by advanced Marfey’s analysis combined with the analysis of their reduced derivatives. Among them, 2 showed moderate antimalarial activity with an IC₅₀ value of 9.9 µg ml⁻¹ against Plasmodium falciparum FCR3 strain.

Herein, the total synthesis of the antimalarial natural products, koshidacins A and B, is disclosed. We developed an inverse peptide elongation sequence to elaborate the linear tetrapeptide by leveraging a soluble hydrophobic tag (TCbz group) for liquid-phase peptide synthesis. The key advances involve head-to-tail macrocyclization to provide the carrier-supported cyclic tetrapeptide and photoinduced deaminative alkylation to edit the native peptide residue that enabled us to construct the requisite alkyl chain in this unique natural product family.

Two novel antimalarials, named ufisonitriles A (1) and B (2), were isolated by bioassay-guided fractionation on mitochondrial function inhibitory activity using multidrug-sensitive budding yeast from the cultured broth of the Okinawan rare actinomycete Amycolatopsis sp. OK19-0009 strain. Their structures were comprehensively elucidated using HR-ESI-MS and 1D/2D NMR analyses and the modified Mosher's method. Structurally, 1 and 2 possess a novel skeleton with an isocyano group. In vitro antimalarial evaluation against Plasmodium falciparum strains revealed that 1 and 2 exhibited moderate activity with IC₅₀ values ranging from 28.6 to 1.12 μM. Furthermore, 1 and 2 reduced the malaria parasites by approximately 45% in vivo on intraperitoneal administration (30 mg/kg/day for 4 days) with no observed toxicity. We also provide a plausible biosynthesis pathway based on the genome sequence analysis.

Hygromycin B, a well-known antibiotic agent, was recently reported as an inducer of secondary metabolites for a fungal strain of Fusarium sp. In this study, we verified the versatility and usefulness of hygromycin B as an inducer of fungal secondary metabolite production. We demonstrate that hygromycin B induces the production of various fungal secondary metabolites, including polyketides, peptides, and terpenes, in 71% of 28 fungal strains belonging to the Ascomycota phylum, specifically within the Eurotiomycetes, Sordariomycetes, and Dothideomycetes classes. We also demonstrate the discovery of novel compounds, named hannocateol (1) and shirazines A (2) and B (3), from the hygromycin B-supplemented cultured materials. The production of the novel compounds increased with hygromycin B-supplementation up to 100 μg/g (maximum >100-fold) but not by HDAC inhibitors, protein synthesis inhibitors against prokaryotes, and antifungal agents. Notably, protein synthesis inhibitors against eukaryotes similar to hygromycin B, such as 5-fluorouracil and paromomycin, could induce the production of 1 and 2, suggesting that its mechanism would be associated with protein synthesis inhibition. We proved that hygromycin B is a highly versatile inducer of secondary metabolite production applicable across diverse fungal taxa, making it a simple and powerful tool for uncovering cryptic biosynthetic pathways and discovering novel bioactive compounds.

Actinomycetes, rich in biosynthetic gene clusters (BGCs), are important sources of natural products (NPs). However, the rate of discovering novel NPs from actinomycetes has declined, indicating the need for a new strategy to obtain NPs. Herein, we present a strategy for the efficient discovery of novel NPs. First, we performed a comprehensive analysis of BGCs in actinomycetes, evaluating the average number and types of BGCs per genus to identify prolific NP producers. Our analysis revealed that certain actinomycete strains, such as those in the family Pseudonocardiaceae, possess a greater number of BGCs than Streptomyces. In addition, these strains tend to possess strain‐specific BGCs compared with others. To facilitate the identification of strain‐specific compounds, we developed a comparative metabolic analysis method using molecular networking. Applying this method to eight Pseudonocardiaceae strains, we successfully discovered two novel peptides, lentindoles A (1) and B (2), featuring a 6/5/6 and 6/5/5 tricyclic ring system, respectively, along with their possible biosynthetic precursor, lentindole C (3). Our result demonstrates the effectiveness of our developed strategy, which is expected to accelerate the discovery of novel NPs.

Seven γ-pyrone-type diterpenoids, including four new compounds (candelalides D-G (1-4)) and three known compounds (candelalides A-C (5-7)), were isolated from the solid culture material of the fungus Mariannaea sp. strain FKR-1011, obtained from Zamami Island, Okinawa, Japan. The chemical structures and relative configurations of 1-4 were determined by spectroscopic analysis such as FT-IR, NMR, and HR-ESI-MS. The absolute configurations of 1-4 were determined through extensive spectroscopic data analysis and TDDFT-ECD calculations. The isolated compounds were tested for biological activity, revealing antibacterial activity against methicillin-resistant Staphylococcus aureus in combination with β-lactam antibiotics and antimalarial activity against Plasmodium falciparum K1 and FCR3 strains.