A new polycyclic tetramate macrolactam from Allostreptomyces RD068384: stereochemistry and antifungal potential.

A new polycyclic tetramate macrolactam designated allostreptamide (1), together with four known congeners, were isolated from the culture extract of Allostreptomyces RD068384. The planar structure of the new compound was elucidated through interpretation of NMR and MS data. The absolute configuration was determined through ROESY and ECD analyses. The isolated compounds revealed antifungal potential against fourteen Candida albicans isolates with minimum inhibitory concentrations (MICs) ranging from 64 to 2048 µg ml-1. Compound 3 showed antibiofilm action and considerably reduced the viability of five isolates (36%) in the formed biofilm. The qRT-PCR revealed that 3 downregulated the BCR1, PLB2, ALS1, and SAP5 biofilm related gene expression. Therefore, 3 could be a promising antifungal therapy for C. albicans infections.

Molecular basis of N-glycan recognition by pradimicin a and its potential as a SARS-CoV-2 entry inhibitor.

Virus entry inhibitors are emerging as an attractive class of therapeutics for the suppression of viral transmission. Naturally occurring pradimicin A (PRM-A) has received particular attention as the first-in-class entry inhibitor that targets N-glycans present on viral surface. Despite the uniqueness of its glycan-targeted antiviral activity, there is still limited knowledge regarding how PRM-A binds to viral N-glycans. Therefore, in this study, we performed binding analysis of PRM-A with synthetic oligosaccharides that reflect the structural motifs characteristic of viral N-glycans. Binding assays and molecular modeling collectively suggest that PRM-A preferentially binds to branched oligomannose motifs of N-glycans via simultaneous recognition of two mannose residues at the non-reducing ends. We also demonstrated, for the first time, that PRM-A can effectively inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. Significantly, the anti-SARS-CoV-2 effect of PRM-A is attenuated in the presence of the synthetic branched oligomannose, suggesting that the inhibition of SARS-CoV-2 infection is due to the interaction of PRM-A with the branched oligomannose-containing N-glycans. These data provide essential information needed to understand the antiviral mechanism of PRM-A and suggest that PRM-A could serve as a candidate SARS-CoV-2 entry inhibitor targeting N-glycans.

Okichromanone, a new antiviral chromanone from a marine-derived Microbispora.

Okichromanone (1), a new chromanone, was isolated from the culture extract of a sponge-derived actinomycete Microbispora, along with known 1-hydroxyphenazine (2). Compound 1 was elucidated to exist as a mixture of two isomeric structures (1a and 1b) at a ratio of nearly 3:2. Compounds 1 and 2 showed anti HSV-I activity with IC50 values 40 and 86 µM, respectively, and anti HSV-II activity with IC50 values 59 and 123 µM, respectively.

Phytohabitans aurantiacus sp. nov., an actinomycete isolated from soil.

A novel actinomycete, designated RD004123T, was isolated from a soil sample collected in Hokkaido, Japan, and its taxonomic position was investigated by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain RD004123T fell within the cluster of the family Micromonosporaceae but did not form a reliable cluster with any member of the family. The similarity values between strain RD004123T and the type species of 29 genera in the family Micromonosporaceae were 91.7-97.7 %. Meanwhile, phylogenomic analyses indicated that strain RD004123T was closely related to members of the genus Phytohabitans. Strain RD004123T contained both meso-diaminopimelic acid and l-lysine as the diagnostic diamino acids of the peptidoglycan. The predominant isoprenoid quinones were MK-10(H8) and MK-10(H6), and the major fatty acids were anteiso-C17 :  0, iso-C16 :  0, iso-C15 :  0 and C17 :  0. The detected polar lipids were phosphatidylinositol mannosides, phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol. These chemotaxonomic features corresponded to those of the genus Phytohabitans. Meanwhile, the results of genome comparison analyses and phenotypic characterizations distinguished strain RD004123T from the other members of the genus Phytohabitans. Therefore, strain RD004123T should be assigned as representing a novel species of the genus Phytohabitans, for which the name Phytohabitans aurantiacus sp. nov. is proposed. The type strain is RD004123T (=NBRC 114997T=DSM 114330T).

Botryorhodines K and L, two new cytotoxic depsidones from a fungus of the genus Arcopilus.

Botryorhodines K (1) and L (2), two new depsidone derivatives, along with one known metabolite, 4-O-demethylbarbatic acid (3), were isolated from the culture extract of a fungus of the genus Arcopilus. The structures of 1‒3 were determined by the analysis of NMR and MS spectral data and the absolute configuration of 1 was established by single-crystal X-ray diffraction analysis. Compounds 1 and 2 showed antimicrobial activity against Gram-positive bacteria and cytotoxicity against murine leukemia P388 cells.

Streptomyces silvisoli sp. nov., a polyene producer, and Streptomyces tropicalis sp. nov., two novel actinobacterial species from peat swamp forests in Thailand.

Two novel actinobacterial strains, designated RB6PN23T and K1PA1T, were isolated from peat swamp soil samples in Thailand and characterized using a polyphasic taxonomic approach. The strains were filamentous Gram-stain-positive bacteria containing ll-diaminopimelic acid in their whole-cell hydrolysates. Phylogenetic analysis of their 16S rRNA gene sequences revealed that strain RB6PN23T was most closely related to Streptomyces rubrisoli (99.1 % sequence similarity) and Streptomyces ferralitis (98.5%), while strain K1PA1T showed 98.8 and 98.7% sequence similarities to Streptomyces coacervatus and Streptomyces griseoruber, respectively. However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were below the species-level thresholds (95-96 % ANI and 70 % dDDH). The genomes of strains RB6PN23T and K1PA1T were estimated to be 7.88 Mbp and 7.39 Mbp in size, respectively, with DNA G+C contents of 70.2 and 73.2 mol%. Moreover, strains RB6PN23T and K1PA1T encode 37 and 24 putative biosynthetic gene clusters, respectively, and in silico analysis revealed that these new species have a high potential to produce unique natural products. Genotypic and phenotypic characteristics confirmed that strains RB6PN23T and K1PA1T represented two novel species in the genus Streptomyces. The names proposed for these strains are Streptomyces silvisoli sp. nov. (type strain RB6PN23T=TBRC 17040T=NBRC 116113T) and Streptomyces tropicalis sp. nov. (type strain K1PA1T=TBRC 17041T=NBRC 116114T). Additionally, a giant linear polyene compound, neotetrafibricin A, exhibiting antifungal activity in strain RB6PN23T, was identified through HPLC and quadrupole time-of-flight MS analysis. The crude extract from the culture broth of strain RB6PN23T exhibited strong antifungal activity against Fusarium verticillioides, Fusarium fujikuroi and Bipolaris zeicola. This finding suggests that strain RB6PN23T could be a promising candidate for biological control of fungal diseases.

Saccharopolyspora oryzae sp. nov., isolated from rhizosphere soil of the wild rice species Oryza rufipogon.

A novel actinobacterium, designated as strain WRP15-2T, was isolated from rhizosphere soil of rice plant (Oryza rufipogon). The strain was Gram-stain-positive, aerobic, and non-motile. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain WRP15-2T fell into the genus Saccharopolyspora. The strain shared the highest 16S rRNA gene sequence similarity with the type strains Saccharopolyspora kobensis JCM 9109T (99.1%), Saccharopolyspora indica VRC122T (98.9%), and Saccharopolyspora antimicrobica DSM 45119T (98.7%). However, the digital DNA-DNA hybridization and average nucleotide identity values among these strains confirmed that the microorganism represented a novel member of the genus Saccharopolyspora. Chemotaxonomic data revealed that strain WRP15-2T possessed MK-9(H4) as the predominant menaquinone. It contained meso-diaminopimelic acid as the diagnostic diaminopimelic acid and arabinose, galactose, and ribose as predominant whole-cell sugars. The detected phospholipids were dominated by phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxy-phosphatidylmethylethanolamine, and phosphatidylcholine. The predominant cellular fatty acids were iso-C16:0, C16:0, and iso-C15:0. The G + C content of the genomic DNA was 69.5%. Based on these genotypic and phenotypic data, it is supported that strain WRP15-2T represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora oryzae sp. nov. is proposed. The type strain is WRP15-2T ( = TBRC 15728T = NBRC 115560T).

Iseoic acids and bisiseoate: three new naphthohydroquinone/naphthoquinone-class metabolites from a coral-derived Streptomyces.

Two new naphthohydroquinone derivatives designated iseoic acids A (1) and B (2) and a new symmetrical glycerol bisester of naphthoquinonepropanoic acid designated bisiseoate (3) were isolated from the culture extract of a marine-derived actinomycete Streptomyces sp. DC4-5. The structures of 1-3 were determined by analyzing one- and two-dimensional NMR data and MS analytical data. The absolute configurations were determined by NOESY analysis and the phenylglycine methyl ester (PGME) method for 1 and by considering the structural similarity and biosynthesis for 2 and 3. Compound 3 exhibited modest cytotoxicity against P388 murine leukemia cells with an IC50 value of 19 µM.

Development of a drug discovery approach from microbes with a special focus on isolation sources and taxonomy.

After the successful discoveries of numerous antibiotics from microorganisms, frequent reisolation of known compounds becomes an obstacle in further development of new drugs from natural products. Exploration of biological sources that can provide novel scaffolds is thus an urgent matter in drug lead screening. As an alternative source to the conventionally used soil microorganisms, we selected endophytic actinomycetes, marine actinomycetes, and actinomycetes in tropical areas for investigation and found an array of new bioactive compounds. Furthermore, based on the analysis of the distribution pattern of biosynthetic gene clusters in bacteria together with available genomic data, we speculated that biosynthetic gene clusters for secondary metabolites are specific to each genus. Based on this assumption, we investigated actinomycetal and marine bacterial genera from which no compounds have been reported, which led to the discovery of a variety of skeletally novel bioactive compounds. These findings suggest that consideration of environmental factor and taxonomic position is critically effective in the selection of potential strains producing structurally unique compounds.

Microbispofurans A-C, plant growth-promoting furancarboxylic acids from plant root-derived Microbispora sp.

Microbispofurans A-C (1-3), new alkyl/alkenyl furancarboxylic acids, were isolated from the culture extract of the plant root-derived Microbispora sp. RD004716. The planar structures of 1-3 were determined by extensive analysis of 1D and 2D NMR spectroscopic data. Although 1-3 showed no appreciable antimicrobial activity or cytotoxicity, strong plant growth-promotion activity of the germinated red leaf lettuce seeds was observed at 10 µM. Furancarboxylic acids and their methyl esters were found in actinomycetes and fungi; however, the isolation of furandicarboxylic acid was unprecedented.

Bacterial microbiome in tropical lichens and the effect of the isolation method on culturable lichen-derived actinobacteria.

Ten samples of tropical lichens collected from Doi Inthanon, Thailand, were explored for the diversity of their bacterial microbiomes through 16S rRNA-based metagenomics analysis. The five predominant lichen-associated bacteria belonged to the phyla Proteobacteria (31.84%), Planctomycetota (17.08%), Actinobacteriota (15.37%), Verrucomicrobiota (12.17%), and Acidobacteriota (7.87%). The diversity analysis metric showed that Heterodermia contained the highest bacterial species richness. Within the lichens, Ramalina conduplicans and Cladonia rappii showed a distinct bacterial community from the other lichen species. The community of lichen-associated actinobacteria was investigated as a potential source of synthesized biologically active compounds. From the total Operational Taxonomic Units (OTUs) found across the ten different lichen samples, 13.21% were identified as actinobacteria, including the rare actinobacterial genera that are not commonly found, such as Pseudonocardia, Kineosporia, Dactylosporangium, Amycolatopsis, Actinoplanes, and Streptosporangium. Evaluation of the pretreatment method (heat, air-drying, phenol, and flooding) and isolation media used for the culture-dependent actinobacterial isolation revealed that the different pretreatments combined with different isolation media were effective in obtaining several species of actinobacteria. However, metagenomics analyses revealed that there were still several strains, including rare actinobacterial species, that were not isolated. This research strongly suggests that lichens appear to be a promising source for obtaining actinobacteria.

Isolation and structure determination of allopteridic acids A-C and allokutzmicin from an unexplored actinomycete of the genus Allokutzneria.

Two classes of new polyketides, allopteridic acids A-C (1-3) and allokutzmicin (4), were isolated from the culture extract of an actinomycete of the genus Allokutzneria. The structures of 1-4 were elucidated through the interpretation of NMR and MS analytical data. Compounds 1-3 possess the same carbon skeleton with pteridic acids but their monocyclic core structures are distinct from the spiro-bicyclic acetal structures of pteridic acids. Compound 4 is a linear polyketide of an unprecedented class, featured by a guanidino-terminus and an epoxide modification. Compounds 1-3 promoted the root elongation of germinated lettuce seeds by ca. 10-40% at 1~10 µM whereas 4 retarded the seed growth. Compound 4 exhibited weak antimicrobial activity against Candida albicans with MIC 25 µg mL-1.

Species-specific secondary metabolism by actinomycetes of the genus Phytohabitans and discovery of new pyranonaphthoquinones and isatin derivatives.

To further exploit secondary metabolic potential of a minor actinomycete genus Phytohabitans within the family Micromonosporaceae, metabolite profiling by HPLC-UV analysis, combined with 16S rDNA sequence-based phylotyping were attempted on seven Phytohabitans strains available at the public culture collection. The strains were grouped into three clades and each exhibited unique and distinct metabolite profiles, which were highly conserved among strains within the same clade. These results were consistent with previous observations on two other actinomycetes genera, reconfirming species-specificity of secondary metabolite production, which were conventionally thought to be strain-specific. A strain RD003215, belonging to the P. suffuscus clade, produced multiple metabolites, some of which were presumed to be naphthoquinones. Liquid fermentation followed by chromatographic separation of the broth extract led to the discovery of three new pyranonaphthoquinones, designated habipyranoquinones A-C (1-3), and one new isatin derivative, (R)-N-methyl-3-hydroxy-5,6-dimethoxyoxindole (4), along with three known synthetic compounds, 6,8-dihydroxydehydro-α-lapachone (5), N-methyl-5,6-dimethoxyisatin (6), and 5,6-dimethoxyisatin (7). Structures of 1-4 were unequivocally elucidated by NMR, MS, and CD spectral analysis, with assistance of density functional theory-based NMR chemical shift prediction and ECD spectral calculation. Compound 2 displayed antibacterial activity against Kocuria rhizophila and Staphylococcus aureus with MIC 50 µg/mL and cytotoxicity against P388 murine leukemia cells with an IC50 value of 34 µM. Compounds 1 and 4 also showed cytotoxicity against P388 cells with IC50 values of 29 and 14 µM, respectively.

Phaeolschidin F, a new symmetrical bis(styrylpyrone) derivative with redox-catalyzing activity from the mushroom Gymnopilus aeruginosus (order Agaricales).

Phaeolschidin F (1) was isolated from fruiting bodies of the bitter and toxic mushroom Gymnopilus aeruginosus. Structure analysis by NMR and MS revealed that 1 is a new symmetrical bis(styrylpyrone). A series of anti-oxidant and pro-oxidant tests characterized that 1 is a redox catalyst having more anti-oxidant and less pro-oxidant activities than quercetin.

Nostochopcerol, a new antibacterial monoacylglycerol from the edible cyanobacterium Nostochopsis lobatus.

A new antibacterial 3-monoacyl-sn-glycerol, nostochopcerol (1), was isolated from a cultured algal mass of the edible cyanobacterium Nostochopsis lobatus MAC0804NAN. The structure of compound 1 was established by the analysis of NMR and MS data while its chirality was established by comparison of optical rotation values with synthetically prepared authentics. Compound 1 inhibited the growth of Bacillus subtilis and Staphylococcus aureus at MIC of 50 µg/mL and 100 µg/mL, respectively.

Antifungal and antiaflatoxigenic mechanism activity of freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 on the two aflatoxigenic fungi and identification of its active component.

AIMS: The study reports the antifungal and antiaflatoxigenic mechanism activity of freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 (DCF RL-1-178) against two aflatoxigenic strains (Aspergillus parasiticus and A. flavus) and identification of its active component. METHODS AND RESULTS: Significant inhibition in ergosterol biosynthesis by the DCF RL-1-178 appeared on the plasma membrane. Moreover, the DCF RL-1-178 showed dose-dependent inhibition of methylglyoxal (MG) (an aflatoxin inducer) biosynthesis and exhibited a novel antiaflatoxigenic action mechanism. Significant impairments in enzymatic [superoxide dismutase (SOD) and catalase (CAT)] and nonenzymatic [oxidized and reduced glutathione (GSH) and ratio of oxidized and reduced glutathione (GSSG)] anti-oxidative defense molecules were observed in the two aflatoxigenic cells. The active component of the DCF RL-1-178 was identified as natamycin. The natamycin exhibited against A. parasiticus and A. flavus with the minimum inhibitory concentration (MIC) values of 0.5 and 1.0 µg ml-1, respectively, while the minimum fungicidal concentration values were the same (4.0 µg ml-1). CONCLUSIONS: The DCF RL-1-178 containing natamycin exhibited the following effects: (1) inhibition of cellular ergosterol biosynthesis on plasma membrane, (2) reduction in MG (aflatoxin inducer) confirmed novel antiaflatoxigenic mechanism of action, and (3) caused remarkable debasement in antioxidant defense enzymes (SOD and CAT) and nonenzymatic defense molecules (GSH and GSSG) revealing biochemical mechanism of action.

Bulbiferamide, an Antitrypanosomal Hexapeptide Cyclized via an N-Acylindole Linkage from a Marine Obligate Microbulbifer.

UV absorption spectroscopy-guided fractionation of the culture extract of a marine obligate bacterium of the genus Microbulbifer yielded a novel cyclic hexapeptide, bulbiferamide (1). NMR spectroscopic and mass spectrometric analyses revealed the structure of 1 to be a cyclic tetrapeptide appending a ureido-bridged two amino acid unit. Notably, Trp is a junction residue, forming on one hand a very rare N-aminoacylated indole linkage for cyclization and on the other hand connecting the ureido-containing tail structure, which is an unprecedented way of configuring peptides. The component amino acids were determined to be l by the advanced Marfey's method. Compound 1 displayed growth inhibitory activity against Trypanosoma cruzi epimastigotes with an IC50 value of 4.1 µM, comparable to the currently approved drug benznidazole, while it was not cytotoxic to P388 murine leukemia cells at 100 µM.

Taxonomic Positions and Secondary Metabolite-Biosynthetic Gene Clusters of Akazaoxime- and Levantilide-Producers.

Micromonospora sp. AKA109 is a producer of akazaoxime and A-76356, whereas Micromonospora sp. AKA38 is that of levantilide C. We aimed to clarify their taxonomic positions and identify biosynthetic gene clusters (BGCs) of these compounds. In 16S rRNA gene and DNA gyrase subunit B gene (gyrB) sequence analyses, strains AKA109 and AKA38 were the most closely related to Micromonospora humidisoli MMS20-R2-29T and Micromonospora schwarzwaldensis HKI0641T, respectively. Although Micromonospora sp. AKA109 was identified as M. humidisoli by the gyrB sequence similarity and DNA-DNA relatedness based on whole genome sequences, Micromonospora sp. AKA38 was classified to a new genomospecies. M. humidisoli AKA109 harbored six type-I polyketide synthase (PKS), one type-II PKS, one type-III PKS, three non-ribosomal peptide synthetase (NRPS) and three hybrid PKS/NRPS gene clusters, among which the BGC of akazaoxime and A-76356 was identified. These gene clusters are conserved in M. humidisoli MMS20-R2-29T. Micromonospora sp. AKA38 harbored two type-I PKS, one of which was responsible for levantilide C, one type-II PKS, one type-III PKS, two NRPS and five hybrid PKS/NRPS gene clusters. We predicted products derived from these gene clusters through bioinformatic analyses. Consequently, these two strains are revealed to be promising sources for diverse non-ribosomal peptide and polyketide compounds.

Click-designed vanilloid-triazole conjugates as dual inhibitors of AChE and Aß aggregation.

Based on their reported neuroprotective properties, vanilloids provide a good starting point for the synthesis of anti-Alzheimer's disease (AD) agents. In this context, nine new 1,2,3-triazole conjugates of vanilloids were synthesized via click chemistry. The compounds were tested for their effect on acetylcholine esterase (AChE) and amyloid-beta peptide (Aß) aggregation. The triazole esters (E)-(1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl 3-(4-hydroxy-3 methoxyphenyl)acrylate 9 and (1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl-4-hydroxy-3-methoxybenzoate 8 displayed dual inhibitory activity for AChE and Aß aggregation with IC50 values of 0.47/0.31 µM and 1.2/0.95 µM, respectively, as compared to donepezil (0.27 µM) and tacrine (0.41 µM), respectively. The results showed that the triazole ester moiety is more favorable for the activity than the triazole ether moiety. This could be attributed to the longer length of the spacer between the two vanillyl moieties in the triazole esters. Furthermore, the binding affinities and modes of the triazole esters 9 and 8 were examined against AChE and Aß utilizing a combination of docking predictions and molecular dynamics (MD) simulations. Docking computations revealed promising binding affinity of triazole esters 9 and 8 as potential AChE, Aß40, and Aß42 inhibitors with docking scores of -10.4 and -9.4 kcal mol-1, -5.8 and -4.7 kcal mol-1, and -3.3 and -2.9 kcal mol-1, respectively. The stability and binding energies of triazole esters 9 and 8 complexed with AChE, Aß40, and Aß42 were measured and compared to donepezil and tacrine over 100 ns MD simulations. According to the estimated binding energies, compounds 9 and 8 displayed good binding affinities with AChE, Aß42, and Aß40 with average ΔG binding values of -32.9 and -31.8 kcal mol-1, -12.0 and -10.5 kcal mol-1, and -20.4 and -16.6 kcal mol-1, respectively. Post-MD analyses demonstrated high steadiness for compounds 9 and 8 with AChE and Aß during the 100 ns MD course. This work suggests the triazole conjugate of vanilloids as a promising skeleton for developing multi-target potential AD therapeutics.

1-(6-Methylsalicyloyl)glycerol from stony coral-derived Micromonospora sp.

A new natural product, 1-(6-methylsalicyloyl)glycerol (1) was isolated from the culture extract of the stony coral-derived Micromonospora sp. C029. The structure of 1 was determined by extensive analysis of 1D and 2D NMR spectroscopic data. The absolute configuration was determined to be S by comparison of specific rotation with synthetic (R)- and (S)-1. Compound 1 showed weak antimicrobial activity against Kocuria rizhophila. Structurally related benzoyl glycerol is not reported from actinomycetes, suggesting that isolation of actinomycetes from little studied environments should be important for the discovery of novel natural products.