Mushrooms such as the dermocyboid Cortinarius rubrophyllus are characterized by strikingly colorful fruiting bodies. The molecular dyes responsible for such colors recently experienced a comeback as photoactive compounds with remarkable photophysical and photobiological properties. One of them-7,7'-biphyscion-is a dimeric anthraquinone that showed promising anticancer effects in the low nanomolar range under blue-light irradiation. Compared to acidic anthraquinones, 7,7'-biphyscion was more efficiently taken up by cells and induced apoptosis after photoactivation. However, seasonal collection of mushrooms producing this compound, low extraction yields, and tricky fungal identification hamper further developments to the clinics. To bypass these limitations, we demonstrate here an alternative approach utilizing a precursor of 7,7'-biphyscion, i.e., the pre-anthraquinone flavomannin-6,6'-dimethyl ether, which is abundant in many species of the subgenus Dermocybe. Controlled oxidation of the crude extract significantly increased the yield of 7,7'-biphyscion by 100%, which eased the isolation process. We also present the mycochemical and photobiological characterization of the yet chemically undescribed species, i.e. C. rubrophyllus. In total, eight pigments (1-8) were isolated, including two new glycosylated anthraquinones (1 and 2). Light-dependent generation of singlet oxygen was detected for the first time for emodin-1-O-β-D-glucopyranoside (3) [photophysical measurement: Φ∆ = 0.11 (CD3OD)]. Furthermore, emodin (7) was characterized as promising compound in the photocytotoxicity assay with EC50-values in the low micromolar range under irradiation against cells of the cancer cell lines AGS, A549, and T24.

Reaction of 3-O-acetyl-oleanolic acid (3) with formic acid/hydrogen peroxide at 100 °C for several hours provides an extraordinary but simple pathway to a taraxeran-28,14 β -olide type triterpenoid while the same reaction at 0 °C occurred without re-arrangement of the carbon skeleton, and an oleanane-28,13 β -olide was obtained instead. The products from these reactions were subjected to a cytotoxicity screening employing several human tumor cell lines showing the latter compound not cytotoxic while the former was cytotoxic especially for MCF-7 (breast adenocarcinoma), and FaDu (hypopharyngeal carcinoma) cells. The highest cytotoxicity, however, was observed for 3 β, 12α, 13 β -trihydroxy-oleanan-28-oic acid (6) holding with EC50 = 4.2 μM for MCF-7 tumor cells.

The demand for natural pigments in general, and for fungi-derived pigments in particular, is constantly rising. Wood-decomposing fungi represent a promising source for natural pigments and they are usually easy to cultivate in pure culture. One of them, i.e., Pycnoporus cinnabarinus, offers a highly interesting spectrum of bioactivity, partly due to the formation of the orange-red pigment cinnabarin. However, apart from a few studies addressing its diverse potential biotechnological applications, there is still a large gap of knowledge concerning the influence of light on the formation of cinnabarin. The aim of this work was to investigate the effect of different irradiations on the cinnabarin content, the growth, and the morphology of three different P. cinnabarinus strains. We used highly standardized irradiation conditions and cultivation techniques in combination with newly developed methods for the extraction and direct quantification of cinnabarin.

The crystal structure and in vitro cytotoxicity of the amphiphilic ruthenium complex [3](PF6 )2 are reported. Complex [3](PF6 )2 contains a Ru-S bond that is stable in the dark in cell-growing medium, but is photosensitive. Upon blue-light irradiation, complex [3](PF6 )2 releases the cholesterol-thioether ligand 2 and an aqua ruthenium complex [1](PF6 )2 . Although ligand 2 and complex [1](PF6 )2 are by themselves not cytotoxic, complex [3](PF6 )2 was unexpectedly found to be as cytotoxic as cisplatin in the dark, that is, with micromolar effective concentrations (EC50 ), against six human cancer cell lines (A375, A431, A549, MCF-7, MDA-MB-231, and U87MG). Blue-light irradiation (λ=450 nm, 6.3 J cm(-2) ) had little influence on the cytotoxicity of [3](PF6 )2 after 6 h of incubation time, but it increased the cytotoxicity of the complex by a factor 2 after longer (24 h) incubation. Exploring the unexpected biological activity of [3](PF6 )2 in the dark elucidated an as-yet unknown bifaceted mode of action that depended on concentration, and thus, on the aggregation state of the compound. At low concentration, it acts as a monomer, inserts into the membrane, and can deliver [1](2+) inside the cell upon blue-light activation. At higher concentrations (>3-5 μm), complex [3](PF6 )2 forms supramolecular aggregates that induce non-apoptotic cell death by permeabilizing cell membranes and extracting lipids and membrane proteins.

With the steady increase of antibiotic resistance, several strategies have been proposed in the scientific community to overcome the crisis. One of many successful strategies is the re-evaluation of known compounds, which have been early discarded out of the pipeline, with state-of-the-art know-how. Xanthoepocin, a polyketide widespread among the genus Penicillium with an interesting bioactivity spectrum against gram-positive bacteria, is such a discarded antibiotic. The purpose of this work was to (i) isolate larger quantities of this metabolite and chemically re-evaluate it with modern technology, (ii) to explore which factors lead to xanthoepocin biosynthesis in P. ochrochloron, and (iii) to test if it is beside its known activity against methicillin-resistant Staphylococcus aureus (MRSA), also active against linezolid and vancomycin-resistant Enterococcus faecium (LVRE)-a very problematic resistant bacterium which is currently on the rise.

Triterpenoic acids (oleanolic, ursolic, betulinic, platanic and glycyrrhetinic acid) were acetylated and coupled with 1,3- or 1,4-diazabicyclo[3.2.2]nonanes to yield amides. Reaction of these amides with methyl iodide at the distal nitrogen of the bicyclic system gave the corresponding quaternary ammonium salts. These compounds were shown to act as excellent inhibitors of the enzyme butyrylcholinesterase (BChE) while being only weak inhibitors for acetylcholinesterase (AChE). Evaluation of the enzyme kinetics revealed these compounds to act as hyperbolic inhibitors for BChE while the results from molecular modeling gave an explanation for their selectivity between AChE and BChE.

Fungi have developed a wide array of defense strategies to overcome mechanical injuries and pathogen infections. Recently, photoactivity has been discovered by showing that pigments isolated from Cortinarius uliginosus produce singlet oxygen under irradiation. To test if this phenomenon is limited to dermocyboid Cortinarii, six colourful Cortinarius species belonging to different classical subgenera (i.e., Dermocybe, Leprocybe, Myxacium, Phlegmacium, and Telamonia) were investigated. Fungal extracts were explored by the combination of in vitro photobiological methods, UHPLC coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS2), feature-based molecular networking (FBMN), and metabolite dereplication techniques. The fungi C. rubrophyllus (Dermocybe) and C. xanthophyllus (Phlegmacium) exhibited promising photobiological activity in a low concentration range (1-7 µg/mL). Using UHPLC-HRMS2-based metabolomic tools, the underlying photoactive principle was investigated. Several monomeric and dimeric anthraquinones were annotated as compounds responsible for the photoactivity. Furthermore, the results showed that light-induced activity is not restricted to a single subgenus, but rather is a trait of Cortinarius species of different phylogenetic lineages and is linked to the presence of fungal anthraquinones. This study highlights the genus Cortinarius as a promising source for novel photopharmaceuticals. Additionally, we showed that putative dereplication of natural photosensitizers can be done by FBMN.

As recently shown, some fungal pigments exhibit significant photoactivity turning them into promising agents for the photodynamic treatment of microbial infections or malignant diseases. In the present study, a separation strategy for fungal anthraquinones was developed based on centrifugal partition chromatography. A suitable method was explored employing a methanolic extract of the fruiting bodies of Cortinarius sanguineus (Agaricales, Basidiomycota). An excellent fractionation was achieved using a biphasic solvent system comprising chloroform/ethyl acetate/methanol/water/acetic acid (3:1:3:2:1, v/v/v/v/v) operating in ascending mode. Experiments on an analytical scale with extracts of closely related Cortinarius species exhibited broad applicability of the devised system. Up to six pigments could be purified directly from the crude extract. Preparative-scale fractionation of the methanol extracts of C. malicorius and C. sanguineus demonstrated that up-scaling was possible without compromising selectivity.

Fungal pigments are characterized by a diverse set of chemical backbones, some of which present photosensitizer-like structures. From the genus Cortinarius, for example, several biologically active photosensitizers have been identified leading to the hypothesis that photoactivity might be a more general phenomenon in the kingdom Fungi. This paper aims at testing the hypothesis. Forty-eight fruiting body-forming species producing pigments from all four major biosynthetic pathways (i.e., shikimate-chorismate, acetate-malonate, mevalonate, and nitrogen heterocycles) were selected and submitted to a workflow combining in vitro chemical and biological experiments with state-of-the-art metabolomics. Fungal extracts were profiled by high-resolution mass spectrometry and subsequently explored by spectral organization through feature-based molecular networking (FBMN), including advanced metabolite dereplication techniques. Additionally, the photochemical properties (i.e., light-dependent production of singlet oxygen), the phenolic content, and the (photo)cytotoxic activity of the extracts were studied. Different levels of photoactivity were found in species from all four metabolic groups, indicating that light-dependent effects are common among fungal pigments. In particular, extracts containing pigments from the acetate-malonate pathway, e.g., extracts from Bulgaria inquinans, Daldinia concentrica, and Cortinarius spp., were not only efficient producers of singlet oxygen but also exhibited photocytotoxicity against three different cancer cell lines. This study explores the distribution of photobiological traits in fruiting body forming fungi and highlights new sources for phototherapeutics.

Due to their potential as leads for various therapeutic applications, including as antimitotic and antiparasitic agents, the development of tubulin inhibitors offers promise for drug discovery. In this study, an in silico pharmacophore-based virtual screening approach targeting the colchicine binding site of β-tubulin was employed. Several structure- and ligand-based models for known tubulin inhibitors were generated. Compound databases were virtually screened against the models, and prioritized hits from the SPECS compound library were tested in an in vitro tubulin polymerization inhibition assay for their experimental validation. Out of the 41 SPECS compounds tested, 11 were active tubulin polymerization inhibitors, leading to a prospective true positive hit rate of 26.8%. Two novel inhibitors displayed IC50 values in the range of colchicine. The most potent of which was a novel acetamide-bridged benzodiazepine/benzimidazole derivative with an IC50 = 2.9 μM. The screening workflow led to the identification of diverse inhibitors active at the tubulin colchicine binding site. Thus, the pharmacophore models show promise as valuable tools for the discovery of compounds and as potential leads for the development of cancer therapeutic agents.

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health.

Photodynamic therapy (PDT) is an alternative approach for the treatment of neoplastic diseases employing photosensitizers activated by light. In order to discover new natural photosensitizers, a convenient workflow was established. To validate the workflow, fungi were selected, because we hypothesized that fruiting bodies and mycelia are an overlooked source. The results proved the hypothesis, as exorbitant high photo-cytotoxicity values were detected. For example, the acetone extract of Cortinarius croceus was characterized by an EC50, 9.3 J cm-2 of 1 μg mL-1 against cells of a lung cancer cell-line (A549). In sum, a low-cost workflow for the detection and biological evaluation of photosensitizers is presented and discussed. Furthermore, this paper provides the first experimental evidence for phototoxic metabolites in basidiomycetes. This hints towards a new assignable function of fungal pigments, i.e. photochemical defense.

The photoantimicrobial potential of four mushroom species (i.e., Cortinarius cinnabarinus, C. holoxanthus, C. malicorius, and C. sanguineus) was explored by studying the minimal inhibitory concentrations (MIC) via a light-modified broth microdilution assay based on the recommended protocols of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The extracts were tested against Candida albicans, Escherichia coli, and Staphylococcus aureus under blue (λ = 428 and 478 nm, H = 30 J/cm2) and green light (λ = 528 nm, H = 30 J/cm2) irradiation. Three extracts showed significant photoantimicrobial effects at concentrations below 25 μg/mL. Targeted isolation of the major pigments from C. sanguineus led to the identification of two new potent photoantimicrobials, one of them (i.e., dermocybin) being active against S. aureus and C. albicans under green light irradiation [PhotoMIC530 = 39.5 μM (12.5 μg/mL) and 2.4 μM (0.75 μg/mL), respectively] and the other one (i.e., emodin) being in addition active against E. coli in a low micromolar range [PhotoMIC428 = 11.1 μM (3 μg/mL)]. Intriguingly, dermocybin was not (photo)cytotoxic against the three tested cell lines, adding an additional level of selectivity. Since both photoantimicrobials are not charged, this discovery shifts the paradigm of cationic photosensitizers.

Antimicrobial resistance is one of the biggest health and subsequent economic threat humanity faces. Next to massive global awareness campaigns, governments and NGOs alike stress the need for new innovative strategies to treat microbial infections. One of such innovative strategies is the photodynamic antimicrobial chemotherapy (PACT) in which the synergistic effects of photons and drugs are exploited. While many promising reports are available, PACT - and especially the drug-design part behind - is still in its infancy. Common best-practice rules, such as the EUCAST or CLSI protocols for classic antibiotics as well as high-throughput screenings, are missing, and this, in turn, hampers the identification of hit structures. Hit-like structures might come from synthetic approaches or from natural sources. They are identified via activity-guided synthesis or isolation strategies. As source for new antimicrobials, fungi are highly ranked. They share the same ecological niche with many other microbes and consequently established chemical strategies to combat with the others. Recently, in members of the Cortinariaceae, especially of the subgenus Dermocybe, photoactive metabolites were detected. To study their putative photoantimicrobial effect, a photoantimicrobial high-throughput screening (HTS) based on The European Committee on Antimicrobial Susceptibility Testing (EUCAST) was established. After validation, the established HTS was used to evaluate a sample set containing six colorful representatives from the genus Cortinarius (i.e., Cortinarius callisteus, C. rufo-olivaceus, C. traganus, C. trivialis, C. venetus, and C. xanthophyllus). The assay is built on a uniform, light-emitting diode (LED)-based light irradiation across a 96-well microtiter plate, which was achieved by a pioneering arrangement of the LEDs. The validation of the assay was accomplished with well-known photoactive drugs, so-called photosensitizers, utilizing six distinct emission wavelengths (λexc = 428, 478, 523, 598, or 640 nm) and three microbial strains (Candida albicans, Staphylococcus aureus, and Escherichia coli). Evaluating the extracts of six Cortinarius species revealed two highly promising species, i.e., C. rufo-olivaceus and C. xanthophyllus. Extracts from the latter were photoactive against the Gram-positive S. aureus (c = 7.5 μg/ml, H = 30 J/cm2, λ = 478 nm) and the fungus C. albicans (c = 75 μg/ml, H = 30 J/cm2, λ = 478 nm).

Pigments of fungi are a fertile ground of inspiration: they spread across various chemical backbones, absorption ranges, and bioactivities. However, basidiomycetes with strikingly colored fruiting bodies have never been explored as agents for photodynamic therapy (PDT), even though known photoactive compound classes (e.g., anthraquinones or alkaloids) are used as chemotaxonomic markers. In this study, we tested the hypothesis that the dyes of skin-heads (dermocyboid Cortinarii) can produce singlet oxygen under irradiation and thus are natural photosensitizers. Three photosensitizers based on anthraquinone structures were isolated and photopharmaceutical tests were conducted. For one of the three, i.e., (-)-7,7'-biphyscion (1), a promising photoyield and photocytotoxicity of EC50 = 0.064 µM against cancer cells (A549) was found under blue light irradiation (λexc = 468 nm, 9.3 J/cm2). The results of molecular biological methods, e.g., a viability assay and a cell cycle analysis, demonstrated the harmlessness of 1 in the dark and highlighted the apoptosis-inducing PDT potential under blue light irradiation. These results demonstrate for the first time that pigments of dermocyboid Cortinarii possess a so far undescribed activity, i.e., photoactivity, with significant potential for the field of PDT. The dimeric anthraquinone (-)-7,7'-biphyscion (1) was identified as a promising natural photosensitizer.

Volatile organic compounds (VOCs) might mediate microbial interactions, especially in spatially structured environments, such as soil. However, the variety and specificity of VOC production are poorly understood. Here, we studied 25 Mortierellaceae strains belonging to the genera Linnemannia and Entomortierella in both pure and co-culture with Pseudomonas helmanticensis under laboratory conditions. We analysed both the fungal growth depending on co-cultivation and the cultures' volatilomes applying proton-transfer-reaction time-of-flight and gas chromatography-mass spectrometry (PTR-ToF-MS and GC-MS). In a strain-specific manner, we found the fungi's radial growth rate and colony morphology affected by the presence of P. helmanticensis. The fungus seemed to generally reduce the bacterial growth. The volatilomes of the fungal and bacterial pure and co-cultures were diverse. While the fungi frequently consumed VOCs, P. helmanticensis produced a higher diversity and amount of VOCs than any fungal strain. Our results support that both the pure and co-culture volatilomes are taxonomically conserved. Taken together, our data supports the relevance of VOCs in Mortierellaceae-P. helmanticensis interaction. We also discuss individual VOCs that appear relevant in the interaction.

Due to their versatile way of life as saprophytes, endophytes, and entomopathogens, fungi of the genera Metarhizium and Beauveria are exposed to varying illumination conditions in their natural habitats, which makes a thorough adaptation to light very likely. While the few available studies for these genera support this assumption, research in this field is still in its infancy and the data material restricted to only a few fungal species. Thus, the aim of this work was to explore how light influences growth, conidial production and secondary metabolite formation of two industrial relevant strains of M. brunneum (MA 43, formerly M. anisopliae var. anisopliae BIPESCO 5/F52) and B. brongniartii (BIPESCO 2). To achieve this, we constructed an easily adjustable illumination device for highly standardized photophysiological studies of fungi on Petri dishes, the so-called LIGHT BOX. With the aid of this device, M. brunneum and B. brongniartii were grown on S4G or S2G agar at 25 °C for 14 days either in complete darkness or under constant illumination with red light (λpeak = 635 nm), green light (λpeak = 519 nm) or blue light (λpeak = 452 nm). In addition, for each wavelength the effect of different illumination intensities was tested, i.e., intensities of red light ranging from 22.1 ± 0.1 to 136.5 ± 0.3 µW cm-2, green light from 16.5 ± 0.1 to 96.2 ± 0.1 µW cm-2, and blue light from 56.1 ± 0.2 to 188.9 ± 0.6 µW cm-2. Both fungi strongly responded in terms of growth, conidial production, pigmentation and morphology to changes in the wavelength and irradiation intensity. The wavelength-dependent production of the well-known secondary metabolite oosporein which is secreted by the genus Beauveria in particular, was also increased under green and blue light exposure. The established LIGHT BOX system allows not only to optimize conidial production yields with these biotechnologically relevant fungi, but also allows the photobiological exploration of other fungi.