Synthesis and biological evaluation of burnettiene A derivatives enabling discovery of novel fungicide candidates.

An antifungal polyene-decalin polyketide natural product, burnettiene A (1) has been re-discovered 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 from Aspergillus burnettii. The antifungal activity of 1 against Candida albicans has been reported. However, only one fungal species for the antifungal activity of 1 has been revealed, and details of the antifungal activity against other pathogenic fungus remain unknown. After extensive screening for antifungal activity, we found that 1 exhibits broad antifungal activity against pathogenic plant fungi, including Colletotrichum gloeosporioides, Botrytis cinerea, Pyricularia oryzae, Leptosphaeria maculans, and Rhizoctonia solani. Furthermore, we synthesized 12 derivatives from 1 and evaluated their antifungal activity to reveal the detailed structure-activity relationship. The methyl ester derivative showed antifungal activity against Saccharomyces cerevisiae 12geneΔ0HSR-iERG6 100-fold more potent than that of 1. Our research indicates that 1 would be a promising natural product as a new fungicidal candidate and the methyl ester derivative especially has great potential.

The isolation of sclerotinin A as an anti-malarial compound by utilization of a global secondary metabolism regulator, laeA gene.

Previously, we successfully introduced laeA gene into a fungal strain in order to significantly increase the production of a bioactive compound, allowing use to discover novel biological activity. To demonstrate the universal applicability of the laeA gene introduction strategy for taping the potential of fungal secondary metabolism, in this present study, we created a library of microorganisms which we had the laeA gene inserted, and from that library we aimed to isolate compounds which are produced at significantly greater quantities compared to the respective wild type strains. From this investigation, we were able to isolate sclerotinin A (1) from Pochonia sp. KTF-0504 strain. We revealed that 1 showed anti-malarial activity against Plasmodium falciparum parasite strains. On the other hands, 1 showed no anti-fungal activity against multidrug-sensitive budding yeast. Our study implies that the utilization of the laeA gene in fungi is a versatile method for the discovery of drug candidates.

Isolation of microorganisms from the feces of Kitasato Yakumo beef cattle as bioactive natural product producers.

We envisioned that the rumen of Kitasato Yakumo beef cattle would contain unique microorganisms which produce bioactive compounds as their defense response to the external environment. The variety of microorganisms were collected from the feces of Kitasato Yakumo beef cattle. We evaluated the biological activity of the culture broth of the isolated strains, proving the utility of our approach.

Discovery of an antimalarial 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 antimalarial 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 antifungal active compound from Aspergillus burnettii. However, the antifungal activity of 1 has been revealed in only one fungal species, and the mechanism of action of 1 remains unknown. After the validation of mitochondrial function inhibitory of 1, we envisioned a new antimalarial drug discovery platform based on mitochondrial function inhibitory activity. We evaluated antimalarial activity and 1 showed antimalarial activity against Plasmodium falciparum FCR3 (chloroquine sensitive) and the 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 antimalarial drug candidates.

In vitro and in vivo activities of KSP-1007, a broad-spectrum inhibitor of serine- and metallo-ß-lactamases, in combination with meropenem against carbapenem-resistant Gram-negative bacteria.

KSP-1007 is a novel bicyclic boronate-based broad-spectrum ß-lactamase inhibitor and is being developed in combination with meropenem (MEM) for the treatment of infections caused by carbapenem-resistant Gram-negative bacteria, a global health concern, and here, we describe its characteristics. KSP-1007 exhibited low apparent inhibition constant (Ki app) values against all classes of ß-lactamase, including imipenemase types and oxacillinase types from Acinetobacter baumannii. Against 207 Enterobacterales and 55 A. baumannii, including carbapenemase producers, KSP-1007 at fixed concentrations of 4, 8, and 16 µg/mL dose-dependently potentiated the in vitro activity of MEM in broth microdilution MIC testing. The MIC90 of MEM/KSP-1007 at 8 µg/mL against Enterobacterales was lower than those of MEM/vaborbactam, ceftazidime/avibactam, imipenem/relebactam, and colistin and similar to those of aztreonam/avibactam, cefiderocol, and tigecycline. The in vitro activity of MEM/KSP-1007 at ≥4 µg/mL against Enterobacterales harboring metallo-ß-lactamase was superior to that of cefepime/taniborbactam. MEM/KSP-1007 showed excellent activity against Escherichia coli with PBP3 mutations and New Delhi metallo-ß-lactamase compared to aztreonam/avibactam, cefepime/taniborbactam, and cefiderocol. MEM/KSP-1007 at 8 µg/mL showed greater efficacy against A. baumannii than these comparators except for cefiderocol, tigecycline, and colistin. A 2-fold reduction in MEM MIC against 96 Pseudomonas aeruginosa was observed in combination with KSP-1007. MEM/KSP-1007 demonstrated bactericidal activity against carbapenemase-producing Enterobacterales, A. baumannii, and P. aeruginosa based on minimum bactericidal concentration/MIC ratios of ≤4. KSP-1007 enhanced the in vivo activity of MEM against carbapenemase-producing Enterobacterales, A. baumannii, and P. aeruginosa in murine systemic, complicated urinary tract, and thigh infection models. Collectively, MEM/KSP-1007 has a good profile for treating carbapenem-resistant Gram-negative bacterial infections.

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.

Comparative genomics reveals insight into the phylogeny and habitat adaptation of novel Amycolatopsis species, an endophytic actinomycete associated with scab lesions on potato tubers.

A novel endophytic actinomycete, strain MEP2-6T, was isolated from scab tissues of potato tubers collected from Mae Fag Mai Sub-district, San Sai District, Chiang Mai Province, Thailand. Strain MEP2-6T is a gram-positive filamentous bacteria characterized by meso-diaminopimelic acid in cell wall peptidoglycan and arabinose, galactose, glucose, and ribose in whole-cell hydrolysates. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and hydroxy-phosphatidylethanolamine were the major phospholipids, of which MK-9(H6) was the predominant menaquinone, whereas iso-C16:0 and iso-C15:0 were the major cellular fatty acids. The genome of the strain was 10,277,369 bp in size with a G + C content of 71.7%. The 16S rRNA gene phylogenetic and core phylogenomic analyses revealed that strain MEP2-6T was closely related to Amycolatopsis lexingtonensis NRRL B-24131T (99.4%), A. pretoriensis DSM 44654T (99.3%), and A. eburnea GLM-1T (98.9%). Notably, strain MEP2-6T displayed 91.7%, 91.8%, and 87% ANIb and 49%, 48.8%, and 35.4% dDDH to A. lexingtonensis DSM 44653T (=NRRL B-24131T), A. eburnea GLM-1T, and A. pretoriensis DSM 44654T, respectively. Based on phenotypic, chemotaxonomic, and genomic data, strain MEP2-6T could be officially assigned to a novel species within the genus Amycolatopsis, for which the name Amycolatopsis solani sp. nov. has been proposed. The type of strain is MEP2-6T (=JCM 36309T = TBRC 17632T = NBRC 116395T). Amycolatopsis solani MEP2-6T was strongly proven to be a non-phytopathogen of potato scab disease because stunting of seedlings and necrotic lesions on potato tuber slices were not observed, and there were no core biosynthetic genes associated with the BGCs of phytotoxin-inducing scab lesions. Furthermore, comparative genomics can provide a better understanding of the genetic mechanisms that enable A. solani MEP2-6T to adapt to the plant endosphere. Importantly, the strain smBGCs accommodated 33 smBGCs encoded for several bioactive compounds, which could be beneficially applied in the fields of agriculture and medicine. Consequently, strain MEP2-6T is a promising candidate as a novel biocontrol agent and antibiotic producer.

Expression of Syo_1.56 SARP Regulator Unveils Potent Elasnin Derivatives with Antibacterial Activity.

Actinomycetes are prolific producers of natural products, particularly antibiotics. However, a significant proportion of its biosynthetic gene clusters (BGCs) remain silent under typical laboratory conditions. This limits the effectiveness of conventional isolation methods for the discovery of novel natural products. Genetic interventions targeting the activation of silent gene clusters are necessary to address this challenge. Streptomyces antibiotic regulatory proteins (SARPs) act as cluster-specific activators and can be used to target silent BGCs for the discovery of new antibiotics. In this study, the expression of a previously uncharacterized SARP protein, Syo_1.56, in Streptomyces sp. RK18-A0406 significantly enhanced the production of known antimycins and led to the discovery of 12 elasnins (1-12), 10 of which were novel. The absolute stereochemistry of elasnin A1 was assigned for the first time to be 6S. Unexpectedly, Syo_1.56 seems to function as a pleiotropic rather than cluster-specific SARP regulator, with the capability of co-regulating two distinct biosynthetic pathways, simultaneously. All isolated elasnins were active against wild-type and methicillin-resistant Staphylococcus aureus with IC50 values of 0.5-20 µg/mL, some of which (elasnins A1, B2, and C1 and proelasnins A1, and C1) demonstrated moderate to strong antimalarial activities against Plasmodium falciparum 3D7. Elasnins A1, B3, and C1 also showed in vitro inhibition of the metallo-ß-lactamase responsible for the development of highly antibiotic-resistant bacterial strains.

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.

Re-discovery of MS-347a as a fungicide candidate through a new drug discovery platform with a multidrug-sensitive Saccharomyces cerevisiae screening system and the introduction of a global secondary metabolism regulator, laeA gene.

We found that the culture broth of fungi showed anti-fungal activity against multidrug-sensitive budding yeast. However, we could not identify the anti-fungal compound due to the small quantity. Therefore, we attempted to increase the productivity of the target compound by the introduction of a global secondary metabolism regulator, laeA to the strain, which led to the successful isolation of 10-folds greater amount of MS-347a (1) than Aspergillus sp. FKI-5362. Compound 1 was not effective against Candida albicans and the detailed anti-fungal activity of 1 remains unverified. After our anti-fungal activity screening, 1 was found to inhibit the growth of broad plant pathogenic fungal species belonging to the Ascomycota. It is noteworthy that 1 showed little insecticidal activity against silkworms, suggesting its selective biological activity against plant pathogenic fungi. Our study implies that the combination strategy of multidrug-sensitive yeast and the introduction of laeA is useful for new anti-fungal drug discovery.

Efflux pump inhibitor, phenylalanine-arginine beta-naphthylamide analog potentiates the activity of 5-O-mycaminosyltylonolide for multi-drug resistant Pseudomonas aeruginosa.

The emergence and spread of antimicrobial resistance are global threats. Pseudomonas aeruginosa (P. aeruginosa) is responsible for a substantial proportion of this global health issue because of its intrinsic resistance to many antibiotics due to the impermeability of its outer membrane and its multidrug efflux pump systems. Therefore, therapeutic drugs are limited, and the development of new drugs is extremely challenging. As an alternative approach, we focused on a combinational treatment strategy and found that 5-O-mycaminosyltylonolide (OMT) showed potent antibacterial activity against P. aeruginosa in the presence of an efflux pump inhibitor, phenylalanine-arginine beta-naphthylamide (PAßN). In this report, we prepared a PAßN derivative and compared the potentiation activity of OMT by PAßNs against multidrug-resistant P. aeruginosa clinical isolates.

Correlation between the spread of IMP-producing bacteria and the promoter strength of blaIMP genes.

The first report of transmissible carbapenem resistance encoded by blaIMP-1 was discovered in Pseudomonas aeruginosa GN17203 in 1988, and blaIMP-1 has since been detected in other bacteria, including Enterobacterales. Currently, many variants of blaIMPs exist, and point mutations in the blaIMP promoter have been shown to alter promoter strength. For example, the promoter (Pc) of blaIMP-1, first reported in P. aeruginosa GN17203, was a weak promoter (PcW) with low-level expression intensity. This study investigates whether point mutations in the promoter region have helped to create strong promoters under antimicrobial selection pressure. Using bioinformatic approaches, we retrieved 115 blaIMPs from 14,529 genome data of Pseudomonadota and performed multiple alignment analyses. The results of promoter analysis of the 115 retrieved blaIMPs showed that most of them used the Pc located in class 1 integrons (n = 112, 97.4%). The promoter analysis by year revealed that the blaIMP population with the strong promoter, PcS, was transient. In contrast, the PcW-TG population, which had acquired a TGn-extended -10 motif in PcW and had an intermediate promoter strength, gradually spread throughout the world. An inverse correlation between Pc promoter strength and Intl1 integrase excision efficiency has been reported previously [1]. Because of this trade-off, it is unlikely that blaIMPs with strong promoters will increase rapidly, but the possibility that promoter strength will increase with the use of other integrons cannot be ruled out. Monitoring of the blaIMP genes, including promoter analysis, is necessary for global surveillance of carbapenem-resistant bacteria.

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.

A new tetronomycin analog, broad-spectrum and potent antibiotic against drug-resistant Gram-positive bacteria.

We discovered a new tetronomycin analog, C-32-OH tetronomycin (2) from the Streptomyces sp. K20-0247 strain, which produces tetronomycin (1). After NMR analysis of 2, we determined the planar structure. Futhermore, the absolute stereochemistry of 2 was deduced based on the biosynthetic pathway of 1 in the K20-0247 strain and a comparison of experimental electronic circular dichroism (ECD) results of 1 with 2. While 2 exihibits potent antibacterial activity aganist Gram-positive baceria including vancomycin-intermediate Staphylococcus aureus (VISA) strains and vancomycin-resistant Enterococci (VRE), the antibacterial activity of 2 shows 16-32-folds weaker than that of 1 suggesting that the C-34 methyl group in 1 is one of the very important functinal group. Moreover, we evaluated the ionophore activity of 1 and 2 and neither compound shows ionophore activity at reasonable concetrations. Our research suggests that 1 and 2 would have different target(s) from an ionophore mechanism in the antibacterial activity and tetronomycins are promising natural products for broad-spectrum antibiotics.

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.

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.

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.