Insights into the structure-activity relationship of a type III secretion system inhibitor, aurodox.

Aurodox was originally isolated in 1972 as a linear polyketide compound exhibiting antibacterial activity against Gram-positive bacteria. We have since identified aurodox as a specific inhibitor of the bacterial type III secretion system (T3SS) using our original screening system for inhibition of T3SS-mediated hemolysis in enteropathogenic Escherichia coli (EPEC). In this research, we synthesized 15 derivatives of aurodox and evaluated EPEC T3SS inhibitory activity as well as antibacterial activity against EPEC. One of the derivatives was highly selective for T3SS inhibition, equivalent to that of aurodox, but without exhibiting antibacterial activity (69-fold selectivity). This work revealed the structure-activity relationship for the inhibition of T3SS by aurodox and suggests that the target of T3SS is distinct from the target for antibacterial activity.

Confluenine G, a new compound from a basidiomycetous yeast Moesziomyces sp. FKI-9540 derived from the gut of a moth Acherontia lachesis (Lepidoptera, Sphingidae).

Most natural products derived from microorganisms have been sought from actinomycetes and filamentous fungi. As an attempt to develop new microbial resources in the exploratory research for natural products, we searched for new compounds from unexploited microbial taxa presumed to have biosynthetic gene clusters. A new compound confluenine G (1) and a known compound (2Z)-2-octyl-2-pentenedioic acid (2) were isolated from a cultured broth of basidiomycetous yeast, Moesziomyces sp. FKI-9540, derived from the gut of a moth Acherontia lachesis (Lepidoptera, Sphingidae). Based on the results of HR-ESI-MS and NMR analyses, the planar structure of 1 was elucidated. Confluenine G (1) was a new analog of nitrogen-oxidized isoleucine and had rare substructures with oxime and hydroxamic acid in molecule.

Traminines A and B, produced by Fusarium concentricum, inhibit oxidative phosphorylation in Saccharomyces cerevisiae mitochondria.

Two new tetramic acid derivatives, traminines A (1) and B (2), were isolated from a culture broth of Fusarium concentricum FKI-7550 by bioassay-guided fractionation using multidrug-sensitive Saccharomyces cerevisiae 12geneΔ0HSR-iERG6. The chemical structures of 1 and 2 were elucidated by NMR studies. Compounds 1 and 2 inhibited the growth of the multidrug-sensitive yeast strain on nonfermentable medium containing glycerol, but not on fermentable medium containing glucose. These results strongly suggest that they target mitochondrial machineries presiding over ATP production via oxidative phosphorylation. Throughout the assay monitoring overall ADP-uptake/ATP-release in yeast mitochondria, 1 and 2 were shown to inhibit one or more enzymes involving oxidative phosphorylation. Based on biochemical characterization, we found that the interference with oxidative phosphorylation by 1 is attributable to the dual inhibition of complex III and FoF1-ATPase, whereas that by 2 is solely due to the inhibition of complex III.

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.

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.

An efflux pump deletion mutant enabling the discovery of a macrolide as an overlooked anti-P. aeruginosa active compound.

Antimicrobial resistance is a serious, worldwide problem. Pseudomonas aeruginosa (P. aeruginosa) is the pathogen that poses a major threat to human health. However, resistance-nodulation-cell division type multidrug efflux pump systems defend P. aeruginosa from many antibiotics. Therefore, only limited therapeutic drugs are available. In this regard, we screened overlooked anti- P. aeruginosa compounds from the Omura Natural Compound library using an efflux pump deletion P. aeruginosa mutant strain, YM64, which led us to find a semisynthetic macrolide, 5-O-mycaminosyltylonolide, whose anti- P. aeruginosa activity against a standard laboratory adapted strain, PAO1, was enhanced by an efflux pump inhibitor, phenylalanine-arginine beta-naphthylamide.

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 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.

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.

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.

Involvement of leukotriene B4 in itching in a mouse model of ocular allergy.

Itching of ocular allergy is alleviated but not completely relieved by H(1) histamine receptor antagonists, suggesting that histamine is not the sole itch mediator in ocular allergy. We investigated whether leukotriene B(4) (LTB(4)), a mediator of cutaneous itch, is involved in the itch of ocular allergy in mice. Mice were immunized by the repeated subcutaneous injections of ragweed pollen and alum into the caudal back, and given a subconjunctival injection of ragweed pollen extract into the palpebra for allergic challenge. Challenge with ragweed pollen extract markedly elicited ocular scratching in sensitized mice. The scratching was almost abolished by mast cell deficiency. The H(1) antagonist terfenadine partially inhibited scratching at a dose that almost completely suppressed plasma extravasation. Scratching was inhibited by the glucocorticoid betamethasone and the 5-lipoxygenase inhibitor zileuton at doses that inhibited the challenge-induced production of LTB(4). A subconjunctival injection of LTB(4) at doses 1/10,000 or less than that required for histamine elicited ocular scratching in naïve mice. The LTB(4) receptor antagonist ONO-4057 inhibited the ragweed pollen challenge-induced ocular scratching at doses that suppressed LTB(4)-induced ocular scratching. In addition to histamine, LTB(4) is involved in the ocular itching of pollen allergy. H(1) receptor antagonists with an inhibitory effect on the action and/or production of LTB(4) may have more potent anti-pruritic activity than selective H(1) antagonists.

Sesquicillin F, a new insecticidal meroterpenoid produced by Mariannaea macrochlamydospora FKI-4735.

A new insecticidal meroterpenoid, named sesquicillin F (1), has been isolated from a culture broth of Mariannaea macrochlamydospora FKI-4735, together with 4-hydroxy-5,6-dimethylpyran-2-one (2). Compounds 1 and 2 were insecticidally active against Halyomorpha halys at 1 ppm.

Aldsulfin, a novel unusual anti-mannheimiosis epithiodiketopiperazine antibiotic produced by Lasiodiplodia pseudotheobromae FKI-4499.

An anti-mannheimiosis agent, aldsulfin, was isolated from a culture broth of the fungus Lasiodiplodia pseudotheobromae FKI-4499, together with a known compound, lasiodipline C, using bioassay-guided fractionation. Spectroscopic analysis of aldsulfin, using NMR, mass spectrometry, and CD analyses revealed it to be an epithiodiketopiperazine with an unstable and unusual hemithioaminal moiety. Aldsulfin showed antibacterial activity against Mannheimia haemolytica and Pasteurella multocida.

Quellenin, a new anti-Saprolegnia compound isolated from the deep-sea fungus, Aspergillus sp. YK-76.

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.

Identification of natural inhibitors of Entamoeba histolytica cysteine synthase from microbial secondary metabolites.

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.

Development of a novel polyimide hollow-fiber oxygenator.

We have developed a membrane oxygenator using a novel asymmetric polyimide hollow fiber. The hollow fibers are prepared using a dry/wet phase-inversion process. The gas transfer rates of O(2) and CO(2) through the hollow fibers are investigated in gas-gas and gas-liquid systems. The polyimide hollow fiber has an asymmetric structure characterized by the presence of macrovoids, and the outer diameter of the hollow fiber is 330 microm. It is found that the polyimide hollow-fiber oxygenator can enhance the gas transfer rates of O(2) and CO(2), and that the hollow fiber provides excellent blood compatibility in vitro and in vivo.

At least thirty-four days of animal continuous perfusion by a newly developed extracorporeal membrane oxygenation system without systemic anticoagulants.

We developed an extracorporeal membrane oxygenation (ECMO) system with high antithrombogenicity and durability characteristics for prolonged continuous cardiopulmonary support. The oxygenator consists of a special hollow-fiber-type polyolefin gas-exchange membrane, which has an ultrathin dense layer in contact with the blood, in order to prevent plasma leakage during protracted use (Platinum Cube NCVC). The centrifugal pump (RotaFlow) is free of seals. The entire blood-contacting surface of the system is coated with a newly developed heparin material (Toyobo-NCVC coating). We performed a venoarterial bypass in a goat, and the ECMO system was driven for 34 days without systemic anticoagulants. Plasma leakage from the oxygenator did not occur, and sufficient gas exchange performance was maintained. Thrombus formation was hardly observed in the ECMO system except in the casing margins of the oxygenator. This ECMO system showed potential for long-term cardiopulmonary support with minimal or no use of systemic anticoagulants.