Comparison of the Secondary Metabolism of the Basidiomycetes Armillaria mellea and Desarmillaria ectypa.

During the course of our ongoing studies on the secondary metabolism of cultures of Basidiomycota, a new meroterpenoid named 10, 15-dihydroxydihydromelleolide (1) was isolated along with the known armillaridin (2) and arnamiol (3) from cultures of the rare saprotrophic species, Desarmillaria ectypa. These are the first secondary metabolites that were ever isolated from the latter species. A concurrently studied strain of the common pathogenic A. mellea yielded other melleolides, with 5'-O-methylmelledonal (4), melledonal C (5), 10 α-hydroxydihydromelleolide (6) and melledonal (7). The chemical structures were elucidated using 1D and 2D NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). All compounds were studied for their antimicrobial and cytotoxic effects against a panel of microbes and mammalian cell lines, and the results are also reported.

Synthesis of a Non-Symmetrical Disorazole C1-Analogue and Its Biological Activity.

The synthesis of a novel disorazole C1 analogue is described, and its biological activity as a cytotoxic compound is reported. Based on our convergent and flexible route to the disorazole core, we wish to report a robust strategy to synthesize a non-symmetrical disorazole in which we couple one half of the molecule containing the naturally occurring oxazole heterocycle and the second half of the disorazole macrocycle containing a thiazole heterocycle. This resulted in a very unusual non-symmetrical disorazole C1 analogue containing two different heterocycles, and its biological activity was studied. This provided exciting information about SAR (structure-activity-relationship) for this highly potent class of antitumor compounds.

Mesoporous Silica as an Alternative Vehicle to Overcome Solubility Limitations.

Toxicological studies are a part of the drug development process and the preclinical stages, for which suitable vehicles ensuring easy and safe administration are crucial. However, poor aqueous solubility of drugs complicates vehicle screening for oral administration since non-aqueous solvents are often not tolerable. In the case of the anti-infective corallopyronin A, currently undergoing preclinical investigation for filarial nematode and bacterial infections, commonly used vehicles such as polyethylene glycol 200, aqueous solutions combined with cosolvents or solubilizers, or aqueous suspension have failed due to insufficient tolerability, solubility, or the generation of a non-homogeneous suspension. To this end, the aim of the study was to establish an alternative approach which offers suitable tolerability, dissolution, and ease of handling. Thus, a corallopyronin A-mesoporous silica formulation was successfully processed and tested in a seven-day toxicology study focused on Beagle dogs, including a toxicokinetic investigation on day one. Sufficient tolerability was confirmed by the vehicle control group. The vehicle enabled high-dose levels resulting in a low-, middle-, and high-dose of 150, 450, and 750 mg/kg. Overall, it was possible to achieve high plasma concentrations and exposures, leading to a valuable outcome of the toxicology study and establishing mesoporous silica as a valuable contender for challenging drug candidates.

Functionalization of Chlorotonils: Dehalogenil as Promising Lead Compound for In Vivo Application.

The natural product chlorotonil displays high potency against multidrug-resistant Gram-positive bacteria and Plasmodium falciparum. Yet, its scaffold is characterized by low solubility and oral bioavailability, but progress was recently made to enhance these properties. Applying late-stage functionalization, we aimed to further optimize the molecule. Previously unknown reactions including a sulfur-mediated dehalogenation were revealed. Dehalogenil, the product of this reaction, was identified as the most promising compound so far, as this new derivative displayed improved solubility and in vivo efficacy while retaining excellent antimicrobial activity. We confirmed superb activity against multidrug-resistant clinical isolates of Staphylococcus aureus and Enterococcus spp. and mature transmission stages of Plasmodium falciparum. We also demonstrated favorable in vivo toxicity, pharmacokinetics and efficacy in infection models with S. aureus. Taken together, these results identify dehalogenil as an advanced lead molecule.

Metagenomic insight to apprehend the fungal communities associated with leaf blight of Welsh onion in Taiwan.

Plants are associated with a large diversity of microbes, and these complex plant-associated microbial communities are critical for plant health. Welsh onion (Allium fistulosum L.) is one of the key and oldest vegetable crops cultivated in Taiwan. The leaf of the Welsh onion is one of the famous spices in Taiwanese cuisine, thus, it is crucial to control foliar diseases. In recent years, Welsh onion cultivation in Taiwan has been severely threatened by the occurrence of leaf blight disease, greatly affecting their yield and quality. However, the overall picture of microbiota associated with the Welsh onion plant is still not clear as most of the recent etiological investigations were heavily based on the isolation of microorganisms from diseased plants. Therefore, studying the diversity of fungal communities associated with the leaf blight symptoms of Welsh onion may provide information regarding key taxa possibly involved in the disease. Therefore, this investigation was mainly designed to understand the major fungal communities associated with leaf blight to identify key taxa potentially involved in the disease and further evaluate any shifts in both phyllosphere and rhizosphere mycobiome assembly due to foliar pathogen infection by amplicon sequencing targeting the Internal Transcribed Spacer (ITS) 1 region of the rRNA. The alpha and beta-diversity analyses were used to compare the fungal communities and significant fungal groups were recognized based on linear discriminant analyses. Based on the results of relative abundance data and co-occurrence networks in symptomatic plants we revealed that the leaf blight of Welsh onion in Sanxing, is a disease complex mainly involving Stemphylium and Colletotrichum taxa. In addition, genera such as Aspergillus, Athelia and Colletotrichum were abundantly found associated with the symptomatic rhizosphere. Alpha-diversity in some fields indicated a significant increase in species richness in the symptomatic phyllosphere compared to the asymptomatic phyllosphere. These results will broaden our knowledge of pathogens of Welsh onion associated with leaf blight symptoms and will assist in developing effective disease management strategies to control the progress of the disease.

Discovery of novel secondary metabolites from the basidiomycete Lentinus cf. sajor-caju and their inhibitory effects on Staphylococcus aureus biofilms.

Three novel derivatives of microporenic acid, microporenic acids H-J, were identified from submerged cultures of a Lentinus species obtained from a basidiome collected during a field trip in the tropical rainforest in Western Kenya. Their structures were elucidated via HR-ESIMS spectra and 1D/2D NMR spectroscopic analyses, as well as by comparison with known derivatives. Applying biofilm assays based on crystal violet staining and confocal microscopy, two of these compounds, microporenic acids H and I, demonstrated the ability to inhibit biofilm formation of the opportunistic pathogen Staphylococcus aureus. Thereby, they were effective in a concentration range that did not affect planktonic growth. Additionally, microporenic acid I enhanced the anti-biofilm activity of the antibiotics vancomycin and gentamicin when used in combination. This opens up possibilities for the use of these compounds in combination therapy to prevent the formation of S. aureus biofilms.

Omnipolyphilins A and B: Chlorinated Cyclotetrapeptides and Naphtho-α-pyranones from the Plant Nematode-Derived Fungus Polyphilus sieberi.

Chemical exploration for two isolates of the recently described ascomycete species Polyphilus sieberi, derived from the eggs of the plant parasitic nematode Heterodera filipjevi, afforded the identification of many compounds that belong to various metabolite families: two previously undescribed chlorinated cyclotetrapeptides, omnipolyphilins A (1) and B (2), one new pyranonaphthoquinone, ventiloquinone P (3), a 6,6'-binaphto-α-pyranone dimer, talaroderxine D (4) in addition to nine known metabolites (5-13) were isolated from this biocontrol candidate. All isolated compounds were characterized by comprehensive 1D, 2D NMR, and HR-ESI-MS analyses. The absolute configurations of the cyclotetrapeptides were determined by a combination of advanced Marfey's method, ROE correlation aided by conformational analysis, and TDDFT-ECD calculations, while ECD calculations, Mosher's method, and experimental ECD spectra were used for ventiloquinone P (3) and talaroderxine D (4). Among the isolated compounds, talaroderxine D (4) showed potent antimicrobial activities against Bacillus subtilis and Staphylococcus aureus with MIC values of 2.1 and 8.3 µg mL-1, respectively. Additionally, promising inhibitory effects on talaroderxine D (4) against the formation of S. aureus biofilms were observed up to a concentration of 0.25 µg mL-1. Moreover, ophiocordylongiiside A (10) showed activity against the free-living nematode Caenorhabditis elegans.

Hidden diversity of Pestalotiopsis and Neopestalotiopsis (Amphisphaeriales, Sporocadaceae) species allied with the stromata of entomopathogenic fungi in Taiwan.

Pestalotiopsissensu lato, commonly referred to as pestalotiopsis-like fungi, exhibit a broad distribution and are frequently found as endophytes, saprobes and pathogens across various plant hosts. The taxa within pestalotiopsis-like fungi are classified into three genera viz. Pestalotiopsis, Pseudopestalotiopsis and Neopestalotiopsis, based on the conidial colour of their median cells and multi-locus molecular phylogenies. In the course of a biodiversity investigation focusing on pestalotiopsis-like fungi, a total of 12 fungal strains were identified. These strains were found to be associated with stromata of Beauveria, Ophiocordyceps and Tolypocladium in various regions of Taiwan from 2018 to 2021. These strains were evaluated morphologically and multi-locus phylogenetic analyses of the ITS (internal transcribed spacer), tef1-α (translation elongation factor 1-α) and tub2 (beta-tubulin) gene regions were conducted for genotyping. The results revealed seven well-classified taxa and one tentative clade in Pestalotiopsis and Neopestalotiopsis. One novel species, Pestalotiopsismanyueyuanani and four new records, N.camelliae-oleiferae, N.haikouensis, P.chamaeropis and P.hispanica, were reported for the first time in Taiwan. In addition, P.formosana and an unclassified strain of Neopestalotiopsis were identified, based on similarities of phylogeny and morphology. However, the data obtained in the present study suggest that the currently recommended loci for species delimitation of pestalotiopsis-like fungi do not deliver reliable or adequate resolution of tree topologies. The in-vitro mycelial growth rates of selected strains from these taxa had an optimum temperature of 25 °C, but growth ceased at 5 °C and 35 °C, while all the strains grew faster under alkaline than acidic or neutral pH conditions. This study provides the first assessment of pestalotiopsis-like fungi, associated with entomopathogenic taxa.

Lachnuoic Acids A-F: Ambuic Acid Congeners from a Saprotrophic Lachnum Species.

Chemical prospection of an extract derived from a saprotrophic fungus Lachnum sp. IW157 resulted in the isolation and characterization of six unprecedentedly reported ambuic acid analogues named lachnuoic acids A-F (1-6). Chemical structures of 1-6 were determined based on comprehensive 1D and 2D NMR spectroscopic analyses together with HR-ESI-MS spectrometry. The relative configurations of 1-3 were defined by ROESY spectroscopic analyses while their absolute configurations were unambiguously determined by Mosher's esters method. All isolated compounds were subjected to cytotoxic, antimicrobial, antibiofilm and nematicidal activity assays where only lachnuoic acid A (1) revealed potent antimicrobial activity against Staphylococcus aureus and Bacillus subtilis at MIC values of 16.6 and 8.3 µg/mL, respectively.

Depicting the Chemical Diversity of Bioactive Meroterpenoids Produced by the Largest Organism on Earth.

In this investigation, we explored the diversity of melleolide-type meroterpenoids produced by Armillaria ostoyae, one of the largest and oldest organisms on Earth, using extracts from liquid and solid fermentation media. The study unveiled three unprecedented dimeric bismelleolides and three novel fatty-acid-substituted congeners, along with 11 new and 21 known derivatives. The structures were elucidated by 1D and 2D NMR spectroscopy and HRESI-MS, and ROESY spectral analysis for relative configurations. Absolute configurations were determined from crystal structures and through ECD spectra comparison. A compound library of melleolide-type meroterpenoids facilitated metabolomics-wide associations, revealing production patterns under different culture conditions. The library enabled assessments of antimicrobial and cytotoxic activities, revealing that the Δ2,4 double bond is not crucial for antifungal activity. Cytotoxicity was linked to the presence of an aldehyde at C1, but lost with hydroxylation at C13. Chemoinformatic analyses demonstrated the intricate interplay of chemical modifications on biological properties. This study marks the first systematic exploration of Armillaria spp. meroterpenoid diversity by MS-based untargeted metabolomics, offering insight into structure-activity relationships through innovative chemoinformatics.

Bioprospection of Tenellins Produced by the Entomopathogenic Fungus Beauveria neobassiana.

Fungi are known as prolific producers of bioactive secondary metabolites with applications across various fields, including infectious diseases, as well as in biological control. However, some of the well-known species are still underexplored. Our current study evaluated the production of secondary metabolites by the entomopathogenic fungus Beauveria neobassiana from Thailand. The fermentation of this fungus in a liquid medium, followed by preparative high-performance liquid chromatography (HPLC) purification, resulted in the isolation of a new tenellin congener, namely pretenellin C (1), together with five known derivatives (2-6). Their chemical structures were elucidated by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). We evaluated the antimicrobial and cytotoxic activities from all isolated compounds, as well as their inhibitory properties on biofilm formation by Staphylococcus aureus. Generally, tenellins displayed varying antibiofilm and cytotoxic effects, allowing us to propose preliminary structure-activity relationships (SARs). Among the tested compounds, prototenellin D (2) exhibited the most prominent antibiofilm activity, while its 2-pyridone congener, tenellin (4), demonstrated potent cytotoxic activity against all tested cell lines. Given the fact that the biological activities of the tenellins have so far been neglected in the past, our study could provide a good starting point to establish more concise structure-activity relationships in the near future.

Pharmacokinetic and pharmacodynamic evaluation of the atypical tetracyclines chelocardin and amidochelocardin in murine infection models.

IMPORTANCE: There is a strong need to find novel treatment options against urinary tract infections associated with antimicrobial resistance. This study evaluates two atypical tetracyclines, namely chelocardin (CHD) and amidochelocardin (CDCHD), with respect to their pharmacokinetics and pharmacodynamics. We show CHD and CDCHD are cleared at high concentrations in mouse urine. Especially, CDCHD is highly effective in an ascending urinary tract infection model, suggesting further preclinical evaluation.

Neurite Outgrowth-Inducing Drimane-Type Sesquiterpenoids Isolated from Cultures of the Polypore Abundisporus violaceus MUCL 56355.

Abundisporin A (1), together with seven previously undescribed drimane sesquiterpenes named abundisporins B-H (2-8), were isolated from a polypore, Abundisporus violaceus MUCL 56355 (Polyporaceae), collected in Kenya. Chemical structures of the isolated compounds were elucidated based on exhaustive 1D and 2D NMR spectroscopic measurements and supported by HRESIMS data. The absolute configurations of the isolated compounds were determined by using Mosher's method for 1-4 and TDDFT-ECD calculations for 4 and 5-8. None of the isolated compounds exhibited significant activities in either antimicrobial or cytotoxicity assays. Notably, all of the tested compounds demonstrated neurotrophic effects, with 1 and 6 significantly increasing outgrowth of neurites when treated with 5 ng/mL NGF.

Secondary metabolites of Diaporthe cameroonensis, isolated from the Cameroonian medicinal plant Trema guineensis.

From a fresh root of Trema guineensis (Ulmaceae), endophytic fungi were isolated, among which a taxon belonging to the new species Diaporthe cameroonensis. This strain was fermented in shake flask batch cultures and the broth was extracted with ethyl acetate. From the crude extract, a hemiketal polyketide 1, and an acetylated alternariol 2 were isolated, along with fifteen known secondary metabolites. Their structures were established by extensive NMR spectroscopy and mass spectrometry analyses, as well as by comparison with literature data of their analogs.

Diaporphasines E and F: New Polyketides from the Saprotrophic Fungus Lachnum sp. IW157 Growing on the Reed Grass Phragmites communis.

Chemical investigation for the mycelial extract of a saprotrophic fungus Lachnum sp. IW157 growing on the common reed grass Phragmites communis afforded the identification of two polyketide metabolites diaporphasines E (1) and F (2). Chemical structures of isolated compounds were unambiguously elucidated based on extensive 1D and 2D NMR spectral analyses in addition to their high-resolution mass spectrometry. The isolated compounds were assessed for their cytotoxicity and antimicrobial and biofilm inhibitory activities. While compound 1 revealed potent cytotoxicity against the tested cell lines L929 and KB3.1 with IC50 values of 0.9 and 3.7 µM, respectively, compound 2 exhibited moderate effects on the formation of S. aureus biofilms at 31.25 µg/mL.

Bioactive Compounds from an Endophytic Pezicula sp. Showing Antagonistic Effects against the Ash Dieback Pathogen.

A fungal endophyte originating from the Canary Islands was identified as a potent antagonist against the fungal phytopathogen Hymenoscyphus fraxineus, which causes the devastating ash dieback disease. This endophyte was tentatively identified as Pezicula cf. ericae, using molecular barcoding. Isolation of secondary metabolites by preparative high-performance liquid chromatography (HPLC) yielded the known compounds CJ-17,572 (1), mycorrhizin A (3) and cryptosporioptides A-C (4-6), besides a new N-acetylated dihydroxyphenylalanin derivative 2, named peziculastatin. Planar structures were elucidated by NMR and HRMS data, while the relative stereochemistry of 2 was assigned by H,H and C,H coupling constants. The assignment of the unknown stereochemistry of CJ-17,572 (1) was hampered by the broadening of NMR signals. Nevertheless, after semisynthetic conversion of 1 into its methyl derivatives 7 and 8, presumably preventing tautomeric effects, the relative configuration could be assigned, whereas comparison of ECD data to those of related compounds determined the absolute configuration. Metabolites 1 and 3 showed significant antifungal effects in vitro against H. fraxineus. Furthermore, 4-6 exhibited significant dispersive effects on preformed biofilms of S. aureus at concentrations up to 2 µg/mL, while the biofilm formation of C. albicans was also inhibited. Thus, cryptosporioptides might constitute a potential source for the development of novel antibiofilm agents.

Discovery and characterisation of a broderol-like illudin, omphaderol in the mycelial extracts of Omphalotus mexicanus (Omphalotaceae) using UPLC-QTOF-MS and NMR spectroscopy.

INTRODUCTION: The genus Omphalotus, in particular the "Jack-O'Lantern mushrooms" Omphalotus illudens and Omphalotus olearius, are famous for the production of the DNA-alkylating illudins. A lesser-known species, Omphalotus mexicanus, native to Central America, also produces cytotoxic illudins S and M, but its minor secondary metabolites are yet to be investigated. OBJECTIVE: To identify, isolate, and elucidate the structure of novel secondary metabolites of the illudin family in mycelial extracts of O. mexicanus from submerse cultivation. METHODOLOGY: A fermentation of the fungus in 15 L stirred tank bioreactors is described. Mycelial extracts were separated using a combination of flash chromatography with preparative RP-C18 high-performance liquid chromatography (HPLC). Analysis of metabolites was done using an ultrahigh-performance liquid chromatography ultraviolet diode array detector (UPLC-UV-DAD) system coupled to an electrospray ionisation quadrupole time-of-flight (ESI-QTOF) mass spectrometer. Structures were elucidated using one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance spectroscopy (NMR) techniques followed by comparison of experimental and simulated electronic circular dichroism (ECD) spectra to determine absolute configurations. RESULTS: Two novel illudin derivatives, for which we propose the names omphaderol (1) and illudaneol B (2), as well as illudaneol (3) and the unusual cyclobutylcyclopentane illudosin (4), were isolated from the mycelia and characterised. CONCLUSION: Particularly the illudaneol derivatives with their high titers may be potential building blocks for an alternative semisynthetic route to new illudin derivatives with improved medical properties. Additionally, the findings improve the knowledge of minor illudin compounds in the mycelial extract of this fungus and may be of significance for future biosynthetic studies of the illudins.

Rapid discovery of terpene tailoring enzymes for total biosynthesis.

Twenty oxygenated aristolochene congeners were rapidly synthesised by combining genes from four different fungal pathways in the fungal host organism Aspergillus oryzae. Compounds produced in a single step include the natural product hypoxylan A and an epimer of guignaderemophilane C. A new fungal aromatase was discovered that produces phenols by oxidative demethylation.

Analysis of SARS-CoV-2 spike RBD binding to ACE2 and its inhibition by fungal cohaerin C using surface enhanced Raman spectroscopy.

The structure of the SARS-CoV-2 spike RBD and human ACE2 as well as changes in the structure due to binding activities were analysed using surface enhanced Raman spectroscopy. The inhibitor cohaerin C was applied to inhibit the binding between spike RBD and ACE2. Differences and changes in the Raman spectra were determined using deconvolution of the amide bands and principal component analysis. We thus demonstrate a fast and label-free analysis of the protein structures and the differentiation between bound and unbound states. The approach is suitable for sensing and screening and might be relevant to investigate other protein systems as well.