PurA is the main target of aurodox, a type III secretion system inhibitor.

Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.

Binding Mode-Based Physicochemical Screening Method Using d-Ala-d-Ala Silica Gel and Chemical Modification Approach to Facilitate Discovery of New Macrolactams, Banglactams A and B, from Nonomuraea bangladeshensis K18-0086.

Utilizing a binding mode-based physicochemical screening method using d-Ala-d-Ala silica gel, two new macrolactams, named banglactams A (1) and B (2), were discovered from the culture broth of Nonomuraea bangladeshensis K18-0086. In the course of our investigation, we found that d-Ala-d-Ala silica gel precisely differentiated the chemical structures of banglactams and separated them. However, we were not able to obtain enough of 1 to elucidate the structure due to its instability and insolubility. To overcome this challenge, we chemically modified 1 to improve solubility, enabling us to obtain a sufficient material supply for the indirect determination of the structure. Antibacterial activity evaluation of banglactams revealed that 1 binding to d-Ala-d-Ala silica gel exhibited antibacterial activity against Staphylococcus aureus; however, this was not the case with 2. This research indicates the utility of our original binding mode-based PC screening method, and the combination strategy of PC and chemical modifications led us to discover novel antibacterial compounds.

New antimalarial iromycin analogs produced by Streptomyces sp. RBL-0292.

Two new antimalarial compounds, named prenylpyridones A (1) and B (2), were discovered from the actinomycete cultured material of Streptomyces sp. RBL-0292 isolated from the soil on Hamahiga Island in Okinawa prefecture. The structures of 1 and 2 were elucidated as new iromycin analogs having α-pyridone ring by MS and NMR analyses. Compounds 1 and 2 showed moderate in vitro antimalarial activity against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains, with IC50 values ranging from 80.7 to 106.7 µM.

Virgaricins C and D, new pramanicin analogs produced by Apiospora sp. FKI-8058.

Two new pramanicin analogs, named virgaricins C (1) and D (2), were discovered by physicochemical screening from a static cultured material of Apiospora sp. FKI-8058. Their structures were elucidated by MS and NMR analyses and chemical derivatization. Compounds 1 and 2 showed moderate antimalarial activity and cytotoxicity.

Akedanones A-C, In Vitro and In Vivo Antiplasmodial 2,3-Dihydronaphthoquinones Produced by Streptomyces sp. K20-0187.

Three new antiplasmodial compounds, named akedanones A (1), B (2), and C (3), were discovered from the cultured material of Streptomyces sp. K20-0187 isolated from a soil sample collected at Takeda, Kofu, Yamanashi prefecture in Japan. The structures of compounds 1-3 were elucidated as new 2,3-dihydronaphthoquinones having prenyl and reverse prenyl groups by mass spectrometry and nuclear magnetic resonance analyses. Compound 1 and the known furanonaphthoquinone I (4) showed potent in vitro antiplasmodial activity against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains, with half-maximal inhibitory concentration values ranging from 0.06 to 0.3 µM. Compounds 1 and 4 also displayed potent in vivo antiplasmodial activity against drug-sensitive rodent malaria Plasmodium berghei N strain, with inhibition rates of 47.6 and 43.1%, respectively, on intraperitoneal administration at a dose of 5 mg kg-1 day-1 for 4 days.

The potentiation activity of ß-lactam by phomoidrides and oxasetin against methicillin-resistant Staphylococcus aureus.

Antimicrobial resistance (AMR) causes a global health threat and enormous damage for humans. Among them, Methicillin-resistant Staphylococcus aureus (MRSA) resistant to first-line therapeutic ß-lactam drugs such as meropenem (MEPM) is problematic. Therefore, we focus on combination drug therapy and have been seeking new potentiators of MEPM to combat MRSA. In this paper, we report the isolation of phomoidrides A-D and its new analog, phomoidride H along with a polyketide compound, oxasetin from the culture broth of Neovaginatispora clematidis FKI-8547 strain as potentiators of MEPM against MRSA.

Total Synthesis of Fusaramin, Enabling Stereochemical Elucidation, Structure-Activity Relationship, and Uncovering the Hidden Antimicrobial Activity against Plant Pathogenic Fungi.

Fusaramin (1) was isolated as a mitochondrial inhibitor. However, the fungal producer stops producing 1, which necessitates us to supply 1 by total synthesis. We proposed the complete stereochemical structure based on the biosynthetic pathway of sambutoxin. We have established concise and robust total synthesis of 1, enabling us to determine the complete stereochemical structure and to elucidate the structure-activity relationship, and uncover the hidden antiplant pathogenic fungal activity.

Antifungal profile against Candida auris clinical isolates of tyroscherin and its new analog produced by the deep-sea-derived fungal strain Scedosporium apiospermum FKJ-0499.

A new antifungal compound, named N-demethyltyroscherin (1), was discovered from the static fungal cultured material of Scedosporium apiospermum FKJ-0499 isolated from a deep-sea sediment sample together with a known compound, tyroscherin (2). The structure of 1 was elucidated as a new analog of 2 by MS and NMR analyses. The absolute configuration of 1 was determined by chemical derivatization. Both compounds showed potent in vitro antifungal activity against clinically isolated Candida auris strains, with MIC values ranging from 0.0625 to 4 µg ml-1.

Synergistic effect of secondary metabolites isolated from Pestalotiopsis sp. FKR-0115 in overcoming ß-lactam resistance in MRSA.

Six aromatic secondary metabolites, pestalone (1), emodin (2), phomopsilactone (3), pestalachlorides B (4), C (5), and D (6), were isolated from Pestalotiopsis sp. FKR-0115, a filamentous fungus collected from white moulds growing on dead branches in Minami Daito Island. The efficacy of these secondary metabolites against methicillin-resistant Staphylococcus aureus (MRSA) with and without meropenem (ß-lactam antibiotic) was evaluated using the paper disc method and broth microdilution method. The chemical structures of the isolated compounds (1-6) were characterised using spectroscopic methods, including nuclear magnetic resonance and mass spectrometry. All six isolated compounds exhibited synergistic activity with meropenem against MRSA. Among the six secondary metabolites, pestalone (1) overcame bacterial resistance in MRSA to the greatest extent.

Design and Synthesis of d-Ala-d-Ala Silica Gel for a Binding Mode-Based Physicochemical Screening Method.

Vancomycin is a potent and broad-spectrum antibiotic that binds to the d-Ala-d-Ala moiety of the growing bacterial cell wall and kills bacteria. This fascinating binding model prompted us to design and synthesize d-Ala-d-Ala silica gels for the establishment of a new physicochemical (PC) screening method. In this report, we confirmed that vancomycin binds to d-Ala-d-Ala silica gel and can be eluted with MeOH containing 50 mM TFA. Finally, d-Ala-d-Ala silica gel enables to purify vancomycin from the culture broth of a vancomycin-producing strain, Amycolatopsis orientalis.

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.

Medermycin Inhibits TNFα-Promoted Inflammatory Reaction in Human Synovial Fibroblasts.

Synovial inflammation plays a crucial role in the destruction of joints and the experience of pain in osteoarthritis (OA). Emerging evidence suggests that certain antibiotic agents and their derivatives possess anti-inflammatory properties. Medermycin (MED) has been identified as a potent antibiotic, specifically active against Gram-positive bacteria. In this study, we aimed to investigate the impact of MED on TNFα-induced inflammatory reactions in a synovial cell line, SW-982, as well as primary human synovial fibroblasts (HSF) using RNA sequencing, rtRT-PCR, ELISA, and western blotting. Through the analysis of differentially expressed genes (DEGs), we identified a total of 1478 significantly upregulated genes in SW-982 cells stimulated with TNFα compared to the vehicle control. Among these upregulated genes, MED treatment led to a reduction in 1167 genes, including those encoding proinflammatory cytokines such as IL1B, IL6, and IL8. Pathway analysis revealed the enrichment of DEGs in the TNF and NFκB signaling pathway, further supporting the involvement of MED in modulating inflammatory responses. Subsequent experiments demonstrated that MED inhibited the expression of IL6 and IL8 at both the mRNA and protein levels in both SW982 cells and HSF. Additionally, MED treatment resulted in a reduction in p65 phosphorylation in both cell types, indicating its inhibitory effect on NFκB activation. Interestingly, MED also inhibited Akt phosphorylation in SW982 cells, but not in HSF. Overall, our findings suggest that MED suppresses TNFα-mediated inflammatory cytokine production and p65 phosphorylation. These results highlight the potential therapeutic value of MED in managing inflammatory conditions in OA. Further investigations utilizing articular chondrocytes and animal models of OA may provide valuable insights into the therapeutic potential of MED for this disease.

Re-evaluation and a Structure-Activity Relationship Study for the Selective Anti-anaerobic Bacterial Activity of Luminamicin toward Target Identification.

Luminamicin (1) isolated in 1985, is a macrodiolide compound exhibiting selective antibacterial activity against anaerobes. However, the antibacterial activity of 1 was not fully examined. In this research, re-evaluation of the antibacterial activity of 1 revealed that 1 is a narrow spectrum and potent antibiotic againstClostridioides difficile(C. difficile) and effective against fidaxomicin resistantC. difficilestrain. This prompted us to obtain luminamicin resistantC. difficilestrains for the determination of the molecular target of 1 inC. difficile. Sequence analysis of 1-resistantC. difficileindicated that the mode of action of 1 differs from that of fidaxomicin. This is because no mutation was observed in RNA polymerase and mutations were observed in a hypothetical protein and cell wall protein. Furthermore, we synthesized derivatives from 1 to study the structure-activity relationship. This research indicated that the maleic anhydride and the enol ether moieties seem to be pivotal functional groups to maintain the antibacterial activity againstC. difficileand the 14-membered lactone may contribute to taking an appropriate molecular conformation.

Emblestatin: a new peptide antibiotic from Embleya scabrispora K20-0267.

A new peptide, emblestatin (1), was discovered from a culture broth of Embleya scabrispora K20-0267. This strain was isolated from soil using an agar medium containing lysozyme. Based on NMR and mass spectrometric analyses, 1 consists of 2-(2-hydroxyphenyl)-2-oxazoline, ß-alanine, glutamine, Nα-methyl-Nω-hydroxyornithine and 3-amino-1-hydroxy-2-piperidone moieties. Further analysis using the advanced Marfey's method revealed that all amino acids with the stereogenic α-carbon in 1 had the L configuration. Compound 1 exhibited iron chelating activity and weak antibacterial activity against Proteus vulgaris and Staphylococcus aureus.

Growth and Migration Blocking Effect of Nanaomycin K, a Compound Produced by Streptomyces sp., on Prostate Cancer Cell Lines In Vitro and In Vivo.

Since castration-resistant prostate cancer (CRPC) acquires resistance to molecularly targeted drugs, discovering a class of drugs with different mechanisms of action is needed for more efficient treatment. In this study, we investigated the anti-tumor effects of nanaomycin K, derived from "Streptomyces rosa subsp. notoensis" OS-3966. The cell lines used were LNCaP (non-CRPC), PC-3 (CRPC), and TRAMP-C2 (CRPC). Experiments included cell proliferation analysis, wound healing analysis, and Western blotting. In addition, nanaomycin K was administered intratumorally to TRAMP-C2 carcinoma-bearing mice to assess effects on tumor growth. Furthermore, immuno-histochemistry staining was performed on excised tissues. Nanaomycin K suppressed cell proliferation in all cell lines (p < 0.001) and suppressed wound healing in TRAMP-C2 (p = 0.008). Nanaomycin K suppressed or showed a tendency to suppress the expression of N-cadherin, Vimentin, Slug, and Ras in all cell lines, and suppressed the phosphorylation of p38, SAPK/JNK, and Erk1/2 in LNCaP and TRAMP-C2. In vivo, nanaomycin K safely inhibited tumor growth (p = 0.001). In addition, suppression of phospho-Erk1/2 and increased expression of E-cadherin and cleaved-Caspase3 were observed in excised tumors. Nanaomycin K inhibits tumor growth and suppresses migration by inhibiting epithelial-mesenchymal transition in prostate cancer. Its mechanism of action is related to the inhibition of phosphorylation of the MAPK signaling pathway.

A combination strategy of a semisynthetic macrolide, 5-O-mycaminosyltylonolide with polymyxin B nonapeptide for multi-drug resistance P. aeruginosa.

The emergence and spread of antimicrobial resistant pathogens continue to threaten our ability to combat several infections. Among them, Pseudomonas aeruginosa (P. aeruginosa) poses a major threat to human health. P. aeruginosa has intrinsic resistance to many antibiotics due to the impermeability of its outer membrane and a resistance-nodulation-cell division type multidrug efflux pump system. Therefore, only limited therapeutic drugs are effective against the pathogen. To address this problem, we have recently discovered an overlooked anti- P. aeruginosa compound, 5-O-mycaminosyltylonolide (OMT) from the Omura Natural Compound library using an efflux pump deletion P. aeruginosa mutant strain, YM64. In this report, we aim to demonstrate the promising potential of OMT for as a novel anti- P. aeruginosa compound and performed combination assays of OMT with polymyxin B nonapeptide, an example of a permeabilizing agent, against multi-drug resistant P. aeruginosa clinical isolates.

New antimalarial fusarochromanone analogs produced by the fungal strain Fusarium sp. FKI-9521.

Two new antimalarial compounds, named deacetyl fusarochromene (1) and 4'-O-acetyl fusarochromanone (2), were discovered from the static fungal cultured material of Fusarium sp. FKI-9521 isolated from feces of a stick insect (Ramulus mikado) together with three known compounds fusarochromanone (3), 3'-N-acetyl fusarochromanone (4), and 5 (fusarochromene or banchromene). The structures of 1 and 2 were elucidated as new analogs of 3 by MS and NMR analyses. The absolute configurations of 1, 2, and 4 were determined by chemical derivatization. All five compounds showed moderate in vitro antimalarial activity against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains, with IC50 values ranging from 0.08 to 6.35 µM.

Studies on the Catechin Constituents of Bark of Cinnamomum sieboldii.

Screening for bioactivity related to anti-infective, anti-methicillin-resistant Staphylococcus aureus (MRSA) and anti-viral activity, led us to identify active compounds from a methanol extract of Litsea japonica (Thub.) Juss. and the hot water extract of bark of Cinnamomum sieboldii Meisn (also known as Karaki or Okinawa cinnamon). The two main components in these extracts were identified as the catechin trimers (+)-cinnamtannin B1 and pavetannin B5. Moreover, these extracts exhibited anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity. The structures of these catechin trimers were previously determined by chemical and spectroscopic methods. Pavetanin B5 has never been reported to be isolated as a pure form and has been obtained as a mixture with another component. Although other groups have reported the putative structure of pavetannin B5, preparation of the methylated derivative of pavetannin B5 in this study allowed us to obtain the pure form for the first time as the undecamethyl derivative and confirm its exact structure. Commercially available (+)-cinnamtannin B1 and aesculitannin B (C2'-epimer of cinnamtannin B1) both of which contained pavetannin B5 as a minor component, and C. sieboldii bark extract (approx. 5/2 mixture of (+)-cinnamtannin B1/pavetannin B5) were assessed for anti-SARS-CoV-2 activity. Both C. sieboldii bark extract and commercially available aesculitannin B showed viral growth inhibitory activity.

Rediscovery of Tetronomycin as a Broad-Spectrum and Potent Antibiotic against Drug-Resistant Gram-Positive Bacteria.

Tetronomycin (1), first isolated from a cultured broth of Streptomyces sp. by Juslen et al. in 1974, is a polycyclic polyether compound. However, the biological activity of 1 has not been thoroughly examined. In this study, we found that 1 exhibits more potent antibacterial activity than two well-known antibacterial drugs (vancomycin and linezolid) and is effective against several drug-resistant clinical isolates including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. Furthermore, we reassigned the 13C NMR spectra of 1 and performed a preliminary structure-activity relationship study of 1 to synthesize a chemical probe for target identification, which implied different targets based on its ionophore activity.

Comprehensive analysis of biosynthetic gene clusters in bacteria and discovery of Tumebacillus as a potential producer of natural products.

Limited microbial genera such as Streptomyces have served as sources of natural products (NPs), whereas most others have been less investigated. The vast accumulation of genomic data available in the NCBI database enables us to bioinformatically estimate the ability of other microbial groups to produce NPs. We analyzed 21,052 complete bacterial genome sequences using antiSMASH and compared the average numbers of biosynthetic gene clusters (BGCs) related to polyketides, non-ribosomal peptides, and/or terpenes biosynthesis at the genus level. Our bioinformatic analyses showed that Tumebacillus has 5-15 BGCs and is a promising NP producer. We searched for NPs from the culture broth of Tumebacillus permanentifrigoris JCM 14557T and found two novel compounds (tumebacin with anti-Bacillus activity and tumepyrazine) and identified two known compounds. Our results highlight the diversity of sources of NPs awaiting discovery.