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.

Laburnicolamine: A Rare Penillic Acid Congener from the Nematode Cyst-Associated Fungus Laburnicola nematophila.

A chemical investigation of a methanol extract derived from a solid-state rice culture of the nematode-cyst associated fungus Laburnicola nematophila K01 led to the isolation and characterization of a previously undescribed penillic acid analogue named laburnicolamine (1). The chemical structure was elucidated through comprehensive 1D and 2D NMR spectroscopic analyses in methanol-d4 and DMSO-d6, alongside with HR-ESI-MS spectrometry. The absolute configuration of 1 was concluded through the electronic circular dichroism (ECD) and time-dependent density functional theory-ECD (TDDFT-ECD) computations compared to its acquired spectrum. Biological assays revealed that compound 1 exhibited no significant cytotoxic, antimicrobial, or nematicidal activity.

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.

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.

Dentifragilones A-B and Other Benzoic Acid Derivatives from the European Basidiomycete Dentipellis fragilis.

A chemical and biological exploration of the European polypore Dentipellis fragilis afforded two previously undescribed natural products (1 and 2), together with three known derivatives (3-5). Chemical structures of the isolated compounds were confirmed through 1D/2D NMR spectroscopic analyses, mass spectrometry, and by comparison with the reported literature. The relative and absolute configurations of 1 were determined according to the ROESY spectrum and time-dependent density functional theory electronic circular dichroism (TDDFT-ECD), respectively. Furthermore, the absolute configuration of dentipellinol (3) was revisited and revealed to be of (R) configuration. All the isolated compounds were assessed for their cytotoxic and antimicrobial activities, with some being revealed to have weak to moderate antimicrobial activity, particularly against Gram-positive bacteria.

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.

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.

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.

Calamene-Type Sesqui-, Mero-, and Bis-sesquiterpenoids from Cultures of Heimiomyces sp., a Basidiomycete Collected in Africa.

New meroterpenoids bis-heimiomycins A-D (1-4) and heimiomycins D and E (5 and 6) were isolated from solid rice cultures of Heimiomyces sp., while new calamene-type sesquiterpenoids heimiocalamene A (7) and B (8) were isolated from shake cultures, respectively. Structures of the metabolites were elucidated by 1D and 2D NMR in addition to HRESIMS data. While relative configurations were assigned by ROESY data, absolute configurations were derived from the structurally related, previously described calamenes, which we herein name heimiocalamenes C-E (9-11). A plausible biosynthetic pathway was proposed for 1-6, with a radical reaction connecting their central para-benzoquinone building block to calamene-sesquiterpenoids. Based on the assumption of a common biosynthesis, we reviewed the structure of the known nitrogen-containing derivative 11, calling the validity of the originally proposed structure into question. Subsequently, the structure of 11 was revised by analysis of HMBC and ROESY NMR data. Only heimiomycin D (5) displayed cytotoxic effects against cell line KB3.1.

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.

Heimionones A-E, New Sesquiterpenoids Produced by Heimiomyces sp., a Basidiomycete Collected in Africa.

With heimionones A-E (1-5), five new terpenoids were isolated from submerged cultures of Heimiomyces sp. in addition to the previously described compounds hispidin, hypholomin B, and heimiomycins A and B. Planar structures of the metabolites were elucidated by 1D and 2D NMR in addition to HRESIMS data. While ROESY data assigned relative configurations, absolute configurations were determined by the synthesis of MTPA esters of 1, 3, and 5. The [6.3.0] undecane core structure of compounds 3-5 is of the asteriscane-type, however, the scaffold of 1 and 2 with their bicyclo [5.3.0] decane core and germinal methyl substitution is, to our knowledge, unprecedented. Together with several new compounds that were previously isolated from solid cultures of this strain, Heimiomyces sp. showed an exceptionally high chemical diversity of its secondary metabolite profile.

Bioactive Secondary Metabolites from Fungi of the Genus Cytospora Ehrenb. (Ascomycota).

Cytospora is a genus of fungi belonging to the Cytosporaceae family (Sordariomycetes, Ascomycota) considered as a prolific source of specialized metabolites due to their ability to produce diverse secondary metabolites with a broad range of biological activities. Since the first chemical investigation of this genus in the 1980s, further studies have led to the isolation and structural elucidation of several bioactive compounds including cytosporones, nonanolides, macrocyclic dilactones, and terpenoids. This review summarizes, for the first time, the chemical diversity of bioactive secondary metabolites from the genus Cytospora and highlights its potential as an alternative source of secondary metabolites for pharmacological studies. Moreover, this review will serve as a basis for future investigations of compounds of this genus.

Two new lanostanoid glycosides isolated from a Kenyan polypore Fomitopsis carnea.

Chemical exploration of solid-state cultures of the polypore Fomitopsis carnea afforded two new C31 lanostane-type triterpenoid glycosides, forpiniosides B (1) and C (2) together with two known derivatives, namely 3-epipachymic acid (3) and (3α,25S)-3-O-malonyl-23-oxolanost-8,24(31)-dien-26-oic acid (4). The structures of the isolated compounds were established based on HRESIMS and extensive 1D and 2D NMR experiments. All the isolated compounds were assessed for their antimicrobial and cytotoxic activities. Among the tested compounds, forpinioside B (1) exhibited significant antimicrobial activity against Staphylococcus aureus and Bacillus subtilis at MIC values comparable to gentamycin and oxytetracycline (positive controls), respectively.

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.

Corallopyronin A: antimicrobial discovery to preclinical development.

Covering: August 1984 up to January 2022Worldwide, increasing morbidity and mortality due to antibiotic-resistant microbial infections has been observed. Therefore, better prevention and control of infectious diseases, as well as appropriate use of approved antibacterial drugs are crucial. There is also an urgent need for the continuous development and supply of novel antibiotics. Thus, identifying new antibiotics and their further development is once again a priority of natural product research. The antibiotic corallopyronin A was discovered in the 1980s in the culture broth of the Myxobacterium Corallococcus coralloides and serves, in the context of this review, as a show case for the development of a naturally occurring antibiotic compound. The review demonstrates how a hard to obtain, barely water soluble and unstable compound such as corallopyronin A can be developed making use of sophisticated production and formulation approaches. Corallopyronin A is a bacterial DNA-dependent RNA polymerase inhibitor with a new target site and one of the few representatives of this class currently in preclinical development. Efficacy against Gram-positive and Gram-negative pathogens, e.g., Chlamydia trachomatis, Orientia tsutsugamushi, Staphylococcus aureus, and Wolbachia has been demonstrated. Due to its highly effective in vivo depletion of Wolbachia, which are essential endobacteria of most filarial nematode species, and its robust macrofilaricidal efficacy, corallopyronin A was selected as a preclinical candidate for the treatment of human filarial infections. This review highlights the discovery and production optimization approaches for corallopyronin A, as well as, recent preclinical efficacy results demonstrating a robust macrofilaricidal effect of the anti-Wolbachia candidate, and the solid formulation strategy which enhances the stability as well as the bioavailability of corallopyronin A.

Prognosis of patients with hepatocellular carcinoma treated with immunotherapy - development and validation of the CRAFITY score.

BACKGROUND & AIMS: Immunotherapy with atezolizumab plus bevacizumab represents the new standard of care in systemic front-line treatment of hepatocellular carcinoma (HCC). However, biomarkers that predict treatment success and survival remain an unmet need. METHODS: Patients with HCC put on PD-(L)1-based immunotherapy were included in a training set (n = 190; 6 European centers) and a validation set (n = 102; 8 European centers). We investigated the prognostic value of baseline variables on overall survival using a Cox model in the training set and developed the easily applicable CRAFITY (CRP and AFP in ImmunoTherapY) score. The score was validated in the independent, external cohort, and evaluated in a cohort of patients treated with sorafenib (n = 204). RESULTS: Baseline serum alpha-fetoprotein ≥100 ng/ml (hazard ratio [HR] 1.7; p = 0.007) and C-reactive protein ≥1 mg/dl (HR, 1.7; p = 0.007) were identified as independent prognostic factors in multivariable analysis and were used to develop the CRAFITY score. Patients who fulfilled no criterion (0 points; CRAFITY-low) had the longest median overall survival (27.6 (95% CI 19.5-35.8) months), followed by those fulfilling 1 criterion (1 point; CRAFITY-intermediate; 11.3 (95% CI 8.0-14.6) months), and patients meeting both criteria (2 points; CRAFITY-high; 6.4 (95% CI 4.8-8.1) months; p <0.001). Additionally, best radiological response (complete response/partial response/stable disease/progressive disease) was significantly better in patients with lower CRAFITY score (CRAFITY-low: 9%/20%/52%/20% vs. CRAFITY-intermediate: 3%/25%/36%/36% vs. CRAFITY-high: 2%/15%/22%/61%; p = 0.003). These results were confirmed in the independent validation set and in different subgroups, including Child-Pugh A and B, performance status 0 and ≥1, and first-line and later lines. In the sorafenib cohort, CRAFITY was associated with survival, but not radiological response. CONCLUSIONS: The CRAFITY score is associated with survival and radiological response in patients receiving PD-(L)1 immunotherapy. The score may help with patient counseling but requires prospective validation. LAY SUMMARY: The immunotherapy-based regimen of atezolizumab plus bevacizumab represents the new standard of care in systemic first-line therapy of hepatocellular carcinoma (HCC). Biomarkers to predict treatment outcome are an unmet need in patients undergoing immunotherapy for HCC. We developed and externally validated a score that predicts outcome in patients with HCC undergoing immunotherapy with immune checkpoint blockers.

Extending the Structure-Activity Relationship of Disorazole C1 : Exchanging the Oxazole Ring by Thiazole and Influence of Chiral Centers within the Disorazole Core on Cytotoxicity.

The synthesis of novel disorazole C1 analogues is described and their biological activity as cytotoxic compounds is reported. Based on our convergent entry to the disorazole core we present a flexible and robust strategy to construct a variety of interesting new analogues. In particular, two regions of the molecules were examined for structural modification: 1. Replacement of the heterocyclic moiety by an exchange of the oxazole ring by a thiazole; and 2. Evaluation of the influence of the absolute configuration of the chiral centers of the molecule. Predicated on our flexible strategy we were able to construct all analogues in an efficient way and could perform an exciting SAR (structure-activity-relationship) study to obtain insight in the cytotoxic activity influenced by the chiral centers of the disorazole core.

Terpenoids and Meroterpenoids from Cultures of Two Grass-Associated Species of Amylosporus (Basidiomycota).

An investigation of the chemical components of the fermentation extract of two cultures of Amylosporus cf. graminicola and Amylosporus cf. campbelii from Cuba and Zimbabwe, respectively, led to the isolation of seven previously undescribed secondary metabolites for which we proposed the trivial names amylosporanes A-G (1-7) along with the known compounds orsellinic acid (11), colletorin D acid (12), colletorin B (13), colletochlorin B (14), and the ß-lactam cyclo-(S-Pro-R-Leu) (15). Three additional compounds (8-10) previously unknown from a fungal source were also characterized for the first time, and two of them were assigned the trivial names amylosporanes H-I (8-9) while the other was identified as cannabigerorcinic acid (10). The structures of the isolated compounds were determined based on their high-resolution electrospray ionization mass spectrometry (HR-ESIMS) spectra and an extensive analysis of their 1D and 2D NMR spectroscopic data. Based on literature searches, we hypothesized that a majority of the isolated metabolites have orsellinic acid (11) as a biosynthetic precursor following a combined route of mevalonate-associated and orsellinic acid-associated pathways. Colletochlorin B (14), the only compound possessing chlorine in its structure, exhibited significant activity against Bacillus subtilis (minimum inhibitory concentration, 2 µg/mL), stronger than that of oxytetracycline, and significant cytotoxicity against A431 cells with an IC50 value of 4.6 µM.

Alliacane-Type Secondary Metabolites from Submerged Cultures of the Basidiomycete Clitocybe nebularis.

Seven sesquiterpenoids, named nebucanes A-G (1-7), featuring a rare alliacane scaffold with unprecedented furan or pyrrole functions, were isolated from the fermentation broth of Clitocybe nebularis. Their structures were established on the basis of 1D/2D NMR spectroscopic analyses, HR-(+)-ESIMS spectra, and comparison of measured and calculated CD spectra for determination of the absolute configuration. Assessing the biological activities, nebucane D (4) exhibited antifungal effects against Rhodotorula glutinis, while nebucane G (7) displayed significant cytotoxicity against MCF-7 and A431 cell lines.

Ten decadal advances in fungal biology leading towards human well-being.

Fungi are an understudied resource possessing huge potential for developing products that can greatly improve human well-being. In the current paper, we highlight some important discoveries and developments in applied mycology and interdisciplinary Life Science research. These examples concern recently introduced drugs for the treatment of infections and neurological diseases; application of -OMICS techniques and genetic tools in medical mycology and the regulation of mycotoxin production; as well as some highlights of mushroom cultivaton in Asia. Examples for new diagnostic tools in medical mycology and the exploitation of new candidates for therapeutic drugs, are also given. In addition, two entries illustrating the latest developments in the use of fungi for biodegradation and fungal biomaterial production are provided. Some other areas where there have been and/or will be significant developments are also included. It is our hope that this paper will help realise the importance of fungi as a potential industrial resource and see the next two decades bring forward many new fungal and fungus-derived products.