Fungal natural products in research and development.

To date approximately 100 000 fungal species are known although far more than one million are expected. The variety of species and the diversity of their habitats, some of them less exploited, allow the conclusion that fungi continue to be a rich source of new metabolites. Besides the conventional fungal isolates, an increasing interest in endophytic and in marine-derived fungi has been noticed. In addition new screening strategies based on innovative chemical, biological, and genetic approaches have led to novel fungal metabolites in recent years. The present review focuses on new fungal natural products published from 2009 to 2013 highlighting the originality of the structures and their biological potential. Furthermore synthetic products based on fungal metabolites as well as new developments in the uses or the biological activity of known compounds or new derivatives are discussed.

Inhibition of TGF-ß signaling, vasculogenic mimicry and proinflammatory gene expression by isoxanthohumol.

TGF-ß is a multifunctional cytokine that regulates cell proliferation, differentiation, apoptosis and extracellular matrix production. Deregulation of TGF-ß production or signaling has been associated with a variety of pathological processes such as cancer, metastasis, angiogenesis and fibrosis. Therefore, TGF-ß signaling has emerged as an attractive target for the development of new cancer therapeutics. In a screening program of natural compounds from fungi inhibiting the TGF-ß dependent expression of a reporter gene in HepG2 cells, we found that the flavone isoxanthohumol inhibited the binding of the activated Smad2/3 transcription factors to the DNA and antagonized the cellular effects of TGF-ß including reporter gene activation and expression of TGF-ß induced genes in HepG2 and MDA-MB-231 cells. In an in vitro angiogenesis assay, isoxanthohumol (56 µM) strongly decreased the formation of capillary-like tubules of MDA-MB-231 cells on Matrigel. In addition, we found that isoxanthohumol blocked IFN-γ, IL-4 and IL-6 dependent Jak/Stat signaling and strongly inhibited the induction of pro-inflammatory genes in MonoMac6 cells at the transcriptional level after LPS/TPA treatment.

Induction of tolerogenic lung CD4+ T cells by local treatment with a pSTAT-3 and pSTAT-5 inhibitor ameliorated experimental allergic asthma.

Signal transducer and activator of transcription (STAT)-3 inhibitors play an important role in regulating immune responses. Galiellalactone (GL) is a fungal secondary metabolite known to interfere with the binding of phosphorylated signal transducer and activator of transcription (pSTAT)-3 as well of pSTAT-6 dimers to their target DNA in vitro. Intra nasal delivery of 50 µg GL into the lung of naive Balb/c mice induced FoxP3 expression locally and IL-10 production and IL-12p40 in RNA expression in the airways in vivo. In a murine model of allergic asthma, GL significantly suppressed the cardinal features of asthma, such as airway hyperresponsiveness, eosinophilia and mucus production, after sensitization and subsequent challenge with ovalbumin (OVA). These changes resulted in induction of IL-12p70 and IL-10 production by lung CD11c(+) dendritic cells (DCs) accompanied by an increase of IL-3 receptor α chain and indoleamine-2,3-dioxygenase expression in these cells. Furthermore, GL inhibited IL-4 production in T-bet-deficient CD4(+) T cells and down-regulated the suppressor of cytokine signaling-3 (SOCS-3), also in the absence of STAT-3 in T cells, in the lung in a murine model of asthma. In addition, we found reduced amounts of pSTAT-5 in the lung of GL-treated mice that correlated with decreased release of IL-2 by lung OVA-specific CD4(+) T cells after treatment with GL in vitro also in the absence of T-bet. Thus, GL treatment in vivo and in vitro emerges as a novel therapeutic approach for allergic asthma by modulating lung DC phenotype and function resulting in a protective response via CD4(+)FoxP3(+) regulatory T cells locally.

Isolactarane and sterpurane sesquiterpenoids from the basidiomycete Phlebia uda.

Three new sesquiterpenoids, named udasterpurenol A, udalactarane A, and udalactarane B, as well as the known compounds hyphodontal and sterpuric acid have been isolated from the basidiomycete Phlebia uda. These compounds represent the first natural products described from this species. The structures were elucidated by NMR spectroscopy and mass spectrometry. Udalactaranes A and B were isolated as mixtures with their respective epimeric acetals. These mixtures inhibited the spore germination of the plant pathogenic fungus Fusarium graminearum at 10 and 5 µg/mL, respectively, and were active against Jurkat cells with IC(50) values of 101 and 42 µM, respectively.

Elucidation of the biosynthesis and degradation of allantofuranone by isotopic labelling and fermentation of modified precursors.

Feeding experiments with the ascomycete Allantophomopsis lycopodina indicated that the potent fungistatic allantofuranone is biosynthesized from phenylalanine. Further experiments with synthetic precursors gave evidence that the naturally occurring polyporic acid serves as a key intermediate in the biosynthesis. In addition to the formation of allantofuranone, its abiotic and metabolic degradation were investigated.

Hirsutane-type sesquiterpenes with uncommon modifications from three basidiomycetes.

From three basidiomycetes, Xeromphalina sp., Stereum sp., and Pleurocybella porrigens, six triquinane sesquiterpenes with unprecendented modifications and a rearranged sesquiterpene related to coriolin C have been isolated. Their isolation, structure elucidation, and biological evaluation are described.

Antimicrobial activity of submerged cultures of Chilean basidiomycetes.

This study is part of a screening program aimed at searching for bioactive metabolites from Chilean basidiomycetes. Submerged cultivation of fungal mycelia in liquid media was evaluated for antimicrobial activity. A total of 148 strains were obtained in vitro. The extracts produced from submerged cultures were evaluated against bacteria and fungi. In the primary antimicrobial assay, approximately 60% of the extracts presented positive biological activity. The highest frequencies of active strains were from the orders Agaricales (31.0%), Polyporales (20.6%), Sterales (18.3%), Boletales (11.4%), and Cortinariales (9.1%). Antifungal activity was more pronounced than antibacterial activity. Twelve extracts that exhibited strong antimicrobial activity showed minimum inhibitory concentration (MIC) values of 50 µL/mL against Bacillus brevis and 25∼50 µL/mL against Penicillium notatum and Paecilomyces variotii. The biological activity of some strains did not vary considerably, regardless of the substrate or collection site whereas, for others, it showed marked variations. Differences in antimicrobial activities observed in the different fungal genera suggested that the ability to produce bioactive compounds is not homogenously distributed among basidiomycetes. The information obtained from this study reveals that Chilean basidiomycetes are able to generate small and/or large variations in the normal pathway of compounds production. Thus, it is necessary to evaluate this biological and chemical wealth, which could be an unsuspected reservoir of new and potentially useful molecules.

The fungal secondary metabolite trichodimerol inhibits TGF-ß dependent cellular effects and tube formation of MDA-MB-231 cells.

The transforming growth factor-beta (TGF-beta) family of ligands has a pivotal role as regulators of cell growth, differentiation and migration. Overexpression of TGF-beta has been associated with breast, colon, hepatocellular, lung and pancreatic cancer. Importantly, overexpression of TGF-beta correlates with tumor progression, metastasis, angiogenesis and poor prognostic outcome. Therefore, TGF-beta signaling has emerged as an attractive target for the development of new cancer therapeutics. In a search for metabolites from fungi inhibiting the TGF-beta dependent expression of a reporter gene in HepG2 cells, we found that trichodimerol, a previously isolated bisorbicillinoid, inhibited serine phosphorylation of the TGF-beta activated Smad2/3 transcription factors and antagonized the cellular effects of TGF-beta including reporter gene activation and expression of TGF-beta inducible genes in HepG2 and MDA-MB-231 cells. In addition, trichodimerol blocked IFN-gamma, IL-6 and IL-4 induced activation of Stat1, Stat3 and Stat6 transcription factors by inhibiting serine and tyrosine phosphorylation. In an in vitro angiogenesis assay, 20 muM trichodimerol completely abrogated the capillary-like tube formation of MDA-MB-231 cells on Matrigel.

New caloporoside derivatives and their inhibition of fungal spore germination.

In our ongoing screening culture fluid extracts of Gloeoporus (Caloporus) dichrous strain 83065 inhibited the germination of Magnaporthe grisea and Fusarium graminearum spores. While isolating the active metabolites two new caloporosides, caloporoside G and caloporoside H, in addition to the known caloporoside derivatives F-16438G, caloporoside A, and 2-hydroxy-6-(16-hydroxyheptadecyl)benzoic acid were obtained.

Imidazo[1,2-a]pyridine derivatives as inhibitors of TNF-alpha expression in T cells.

Novel hexahydroimidazo[1,2-a]pyridines prepared by the addition of ethyl (1-benzylimidazolidin-2-ylidene)acetate (2) to the fungal metabolite podoscyphic acid (1a) and esters of 1a have been evaluated for their ability to inhibit the inducible TNF-alpha promoter activity in T cells. The methyl ester 3b is the most potent, inhibiting the TNF-alpha driven reporter gene expression in Jurkat T cells with an IC(50)-value of 2.0 microg/ml (3.6 microM). In addition, compound 3b inhibited the inducible TNF-alpha production in the myelomonocytic U937 cells with an IC(50)-value of 4.6 microM.

Oxacyclododecindione, a novel inhibitor of IL-4 signaling from Exserohilum rostratum.

In a screening program for new metabolites from fungi inhibiting the IL-4 mediated signal transduction, a novel chlorinated macrocyclic lactone, designated as oxacyclododecindione, was isolated from fermentations of the imperfect fungus Exserohilum rostratum. The structure was determined by a combination of spectroscopic techniques. Oxacyclododecindione inhibits the IL-4 induced expression of the reporter gene secreted alkaline phosphatase (SEAP) in transiently transfected HepG2 cells with IC50 values of 20-25 ng/ml (54-67.5 nM). Studies on the mode of action of the compound revealed that the inhibition of the IL-4 dependent signaling pathway is caused by blocking the binding of the activated STAT6 transcription factors to the DNA binding site without inhibiting tyrosine phosphorylation. The compound has no antibacterial or antifungal activity.

Isolation and biological activity of new norhirsutanes from Creolophus cirrhatus.

Five new norhirsutanes, named creolophins A-E, and complicatic acid were isolated from the culture broth of the rare tooth fungus Creolophus cirrhatus by solvent extraction, silica gel column chromatography and HPLC. In addition, neocreolophin, a complex dimerization product, was formed as an artefact during purification. The structures were elucidated by spectroscopic methods and are published in a separate paper. Two of the metabolites showed moderate antibacterial, antifungal and cytotoxic activities.

Mollisianitrile, a new antibiotic from Mollisia sp. A59-96.

Mollisianitrile (1), a new antibiotic was isolated from the fermentation broth of Mollisa sp. A59-96 together with the two known isocoumarins 2 and 3. 1 exhibited antimicrobial, cytotoxic, and phytotoxic activities. 1 contains a reactive propiolonitrile moiety which is believed to be responsible for its antibiotic activities. Upon incubation with L-cysteine the biological activity was lost.

Biologically active metabolites from the basidiomycete Limacella illinita (Fr.) Murr.

In the course of our search for new bioactive compounds from basidiomycetes, four new compounds were isolated from fermentations of Limacella illinita. Illinitone A (1) exhibited weak phytotoxic and moderate nematicidal activities against Caenorhabditis elegans, illinitone B (2) was moderately cytotoxic, while limacellone (3) exhibited weak cytotoxic and phytotoxic activities. The muurolane sesquiterpene 4a was found to be inactive in the assays performed here. Limacellone (3), which appeared to be related with the illinitones 1 and 2, has a new C15 carbon skeleton. It is possible that compounds 1, 2 and 3 are terpenoids/ secoterpenoids, but their biosyntheses were not investigated.

Phomopsidone, a novel depsidone from an endophyte of the medicinal plant Eupatorium arnottianum.

The medicinal plant Eupatorium arnottianum can be found in the Northeast and center of Argentina and the South of Bolivia. From plant material collected in Argentina an endophytic Phomopsis was isolated. The fungus was identified by microscopic features and analysis of its ITS sequence. Cultures yielded, besides mellein and nectriapyrone, a novel depsidone derivative for which we propose the name phomopsidone (1). The structure of 1 was determined from its spectroscopic data.

Antimicrobial isoflavonoids from Erythrina crista galli infected with Phomopsis sp.

The isoflavonoids coumestrol, genistein and daidzein have been isolated and identified by bioassay-guided fractionation from the acetone extract of Erythrina crista galli young twigs infected with Phomopsis sp. These compounds showed antimicrobial activity against Bacillus brevis (MIC values 16.3, 64.8 and 137.8 microM, respectively). This is the first time that coumestrol, besides lutein and n-nonacosane, are reported in this species.

Cylindrocyclin A, a new cytotoxic cyclopeptide from Cylindrocarpon sp.

In the course of a screening of fungal extracts for new metabolites with cytotoxic activities cylindrocyclin A (1) was isolated. The producing strain was identified as Cylindrocarpon sp. by microscopy and ITS rDNA sequence analysis. 1 is a novel compound that exhibits cytotoxic acticity against six different cell lines with IC50 values ranging from 11 to 53 microM. 1 has no antibacterial or antifungal activity. The compound is a cyclic nonapeptide comprising three alanines, five leucines and one isoleucine. Four amino acids are N-methylated. Its structure was elucidated by spectroscopic methods.

The dasyscyphins A-C and niveulone, new biologically active compounds from the ascomycete Dasyscyphus niveus.

In the course of a screening for cytotoxic compounds from fungi four new terpenoid metabolites, named dasyscyphins A, B, C, and niveulone were isolated from fermentations of Dasyscyphus niveus strain A0101. While dasyscyphin A (1) exhibited no significant biological activities in our test systems dasyscyphin B (2) and dasyscyphin C (3) showed cytotoxic activities against human (HepG2, Hela S3, U937, Colo-320, Jurkat) cell lines with IC50-values of 0.5-3 microg/ml while the activity of niveulone (4) was less pronounced. Only modest or weak antibiotic properties were observed for (2) and (3).

Niveulone, a heterocyclic spiro terpenoid from the ascomycete Dasyscyphus niveus.

The new heterocyclic spiro terpenoid niveulone (1) was isolated from the cultural fluid of the ascomycete Dasyscyphus niveus, and its chemical structure and relative configuration were determined by spectroscopic techniques.

Isolation and characterization of two verrucarins from Myrothecium roridum.

In the course of a screening for compounds inhibiting the growth of two human breast cancer cells lines, two highly cytotoxic compounds were isolated from fermentations of Myrothecium roridum. The elucidation of their structures revealed that they are macrocyclic trichothecenes of the verrucarin type, 16-hydroxyverrucarin A (1), reported here as a natural product for the first time, and verrucarin X (2), a new compound. Both 1 and 2 exhibit moderate antifungal activity and pronounced cytotoxic activity, with IC50 values in the nanomolar (1) and micromolar (2) range. Both compounds preferentially inhibit in vivo protein biosynthesis.