Formaldehyde as a Chemical Defence Agent of Fruiting Bodies of Mycena rosea and its Role in the Generation of the Alkaloid Mycenarubin†C.

Mycenarubin C, a previously unknown red pyrroloquinoline alkaloid, was isolated from fruiting bodies of the mushroom Mycena rosea and its structure was elucidated mainly by NMR spectroscopy and mass spectrometry. Unlike mycenarubin A, the major pyrroloquinoline alkaloid in fruiting bodies of M. rosea, mycenarubin C, contains an eight-membered ring with an additional C1 unit that is hitherto unprecedented for pyrroloquinoline alkaloids known in nature. Incubation of mycenarubin A with an excess of formaldehyde revealed that mycenarubin C was generated nearly quantitatively from mycenarubin A. An investigation into the formaldehyde content of fresh fruiting bodies of M. rosea showed the presence of considerable amounts of formaldehyde, with values of 5 µg per gram of fresh weight in fresh fruiting bodies. Although mycenarubin C did not show bioactivity against selected bacteria and fungi, formaldehyde inhibits the growth of the mycoparasite Spinellus fusiger at concentrations present in fruiting bodies of M. rosea. Therefore, formaldehyde might play an ecological role in the chemical defence of M. rosea against S. fusiger. In turn, S. fusiger produces gallic acid-presumably to detoxify formaldehyde by reaction of this aldehyde with amino acids and gallic acid to Mannich adducts.

Two New Cyathane Diterpenoids from Mycelial Cultures of the Medicinal Mushroom Hericium erinaceus and the Rare Species, Hericium flagellum.

Basidiomycetes of the genus Hericium are among the most praised medicinal and edible mushrooms, which are known to produce secondary metabolites with the potential to treat neurodegenerative diseases. This activity has been attributed to the discovery of various terpenoids that can stimulate the production of nerve growth factor (NGF) or (as established more recently) brain-derived neurotrophic factor (BDNF) in cell-based bioassays. The present study reports on the metabolite profiles of a Lion's Mane mushroom (Hericium erinaceus) strain and a strain of the rare species, Hericium flagellum (synonym H. alpestre). While we observed highly similar metabolite profiles between the two strains that were examined, we isolated two previously undescribed metabolites, given the trivial names erinacines Z1 and Z2. Their chemical structures were elucidated by means of nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry. Along with six further, previously identified cyathane diterpenes, the novel erinacines were tested for neurotrophin inducing effects. We found that erinacines act on BDNF, which is a neurotrophic factor that has been reported recently by us to be induced by the corallocins, but as well on NGF expression, which is consistent with the literature.

Corallocins A-C, Nerve Growth and Brain-Derived Neurotrophic Factor Inducing Metabolites from the Mushroom Hericium coralloides.

Three new natural products, corallocins A-C (1-3), along with two known compounds were isolated from the mushroom Hericium coralloides. Their benzofuranone and isoindolinone structures were elucidated by spectral methods. All corallocins induced nerve growth factor and/or brain-derived neurotrophic factor expression in human 1321N1 astrocytes. Furthermore, corallocin B showed antiproliferative activity against HUVEC and human cancer cell lines MCF-7 and KB-3-1.

Erinacine C Activates Transcription from a Consensus ETS DNA Binding Site in Astrocytic Cells in Addition to NGF Induction.

Medicinal mushrooms of the genus Hericium are known to produce secondary metabolites with homeostatic properties for the central nervous system. We and others have recently demonstrated that among these metabolites cyathane diterpenoids and in particular erinacine C possess potent neurotrophin inducing properties in astrocytic cells. Yet, the signaling events downstream of erinacine C induced neurotrophin acitivity in neural-like adrenal phaeochromocytoma cells (PC12) cells have remained elusive. Similar, signaling events activated by erinacine C in astrocytic cells are unknown. Using a combination of genetic and pharmacological inhibitors we show that erinacine C induced neurotrophic activity mediates PC12 cell differentiation via the TrkA receptor and likely its associated PLCγ-, PI3K-, and MAPK/ERK pathways. Furthermore, a small library of transcriptional activation reporters revealed that erinacine C induces transcriptional activation mediated by DNA consensus binding sites of selected conserved transcription factor families. Among these, transcription is activated from an ETS consensus in a concentration dependent manner. Interestingly, induced ETS-consensus transcription occurs in parallel and independent of neurotrophin induction. This finding helps to explain the many pleiotropic functions of cyathane diterpenoids. Moreover, our studies provide genetic access to cyathane diterpenoid functions in astrocytic cells and help to mechanistically understand the action of cyathanes in glial cells.

Labyrinthopeptins as virolytic inhibitors of respiratory syncytial virus cell entry.

Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 µM and 4.97 µM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.

Synthesis and biological evaluation of (±)-hippolachnin and analogs.

Due its unique structure and its reported potent antifungal activity, the marine polyketide hippolachnin A (1) has attracted much attention in the synthetic community. Herein, we describe the development of a concise, diversifiable and scalable synthesis of the racemic natural product, which serves as a platform for the generation of bioactive analogues. Biological testing of our synthetic material did not confirm the reported antifungal activity of hippolachnin A but unraveled moderate activity against nematodes and microbes.

Nematicidal anthranilic acid derivatives from Laccaria species.

Three undescribed natural products, the anthranilic acid derivatives laccanthrilic acids A, B, and C, as well as the known (3S)-1,2,3,4-tetrahydro-3-ß-carboline-3-carboxylic acid were isolated from fruiting bodies of Laccaria laccata. The structures were established by 1D and 2D NMR spectroscopy, HR-(+)-ESIMS and chemical synthesis. The absolute configuration of laccanthrilic acids A and B was determined by GC-MS after hydrolytic cleavage and derivatisation of the resulting glutamic acid with methanol and Mosher's reagent and subsequent comparison with authentic synthetic samples of known absolute configuration. The absolute configuration of laccanthrilic acid C was determined by comparison of the CD spectra of laccanthrilic acids B and C with each other. Metabolic profiling of related species showed that the compounds are common in the genus Laccaria. Laccanthrilic acid B exhibited moderate nematicidal effects against Caenorhabditis elegans, which might explain to some degree the beneficial role of these fungi for the growth and survival of their host plants.

Large Scale Production and Downstream Processing of Labyrinthopeptins from the Actinobacterium Actinomadura namibiensis.

A method was established for the production of 1.2-fold and 4.2-fold increased amounts of the antiviral and central nervous system-active lantipeptides, labyrinthopeptins A1 and A2, respectively, isolated from the actinobacterium Actinomadura namibiensis, to enable production in gram scale. We then performed in vivo characterization of this promising compound class. The labyrinthopeptins A1 and A2 have similar chemical structures and physical properties but differ drastically in their bioactivities. Therefore, large quantities of highly pure material are required for pharmacological studies. An effective methodology was established for the first time for their production in bioreactors, their separation involving gel permeation chromatography on LH20 material, followed by reversed phase-high performance liquid chromatography. With an optimized methodology, 580 mg of labyrinthopeptin A1 and 510 mg of labyrinthopeptin A2 were quantitatively isolated with recovery rates of 72.5% and 42.3% from 7.5 L of culture broth, respectively. However, the fermentation that had already resulted in maximum yields of over 100 mg/L of both target molecules after 300 h in a 10-L scale bioreactor, still requires further optimisation.

New nematicidal and antimicrobial secondary metabolites from a new species in the new genus, Pseudobambusicola thailandica.

During the course of a study on the functional biodiversity of the mycobiota inhabiting rainforests in Thailand, a fungal strain was isolated from a plant sample and shown to represent an undescribed species, as inferred from a combination of morphological and molecular phylogenetic methods. Molecular phylogenetic analyses, based on four DNA loci, revealed a phylogenetic tree with the newly generated sequences clustering in a separate branch, together with members of the Sulcatisporaceae (Pleosporales, Ascomycota). The Thai specimen morphologically resembled Neobambusicola strelitziae in having pycnidial conidiomata with phialidic conidiogenous cells that produce both fusoid-ellipsoid macroconidia and subcylindrical microconidia. However, the new fungus, for which the name Pseudobambusicola thailandica is proposed, differs from N. strelitziae in having conidiomata with well-defined necks, the presence of globose to subglobose thick-walled cells adjacent to conidiomata and the production of chlamydospores in culture. When cultures of P. thailandica, growing on water agar, were confronted with Caenorhabditis elegans nematodes, worms approaching the fungal mycelia were killed. This observation gave rise to a study of its secondary metabolites and six novel and two known compounds were isolated from submerged cultures of P. thailandica. The structures of metabolites 1-6, for which the trivial names thailanones A-F are proposed, were elucidated using a combination of spectral methods, including extensive 1 and 2D NMR analysis and high resolution mass spectrometry. Compounds 4 and 8 showed strong nematicidal and weak antifungal activity, whereas all other tested compounds showed moderate to weak nematicidal activity but no significant effects in the serial dilution assay against various fungi and bacteria. Compounds 1 and 8 also inhibited growth of the pathogenic basidiomycete Phellinus tremulae in a plate diffusion assay.