The Light-activated Effect of Natural Anthraquinone Parietin against Candida auris and Other Fungal Priority Pathogens.

Antimicrobial photodynamic therapy (aPDT) is an evolving treatment strategy against human pathogenic microbes such as the Candida species, including the emerging pathogen C. auris. Using a modified EUCAST protocol, the light-enhanced antifungal activity of the natural compound parietin was explored. The photoactivity was evaluated against three separate strains of five yeasts, and its molecular mode of action was analysed via several techniques, i.e., cellular uptake, reactive electrophilic species (RES), and singlet oxygen yield. Under experimental conditions (λ = 428 nm, H = 30 J/cm2, PI = 30 min), microbial growth was inhibited by more than 90% at parietin concentrations as low as c = 0.156 mg/L (0.55 µM) for C. tropicalis and Cryptococcus neoformans, c = 0.313 mg/L (1.10 µM) for C. auris, c = 0.625 mg/L (2.20 µM) for C. glabrata, and c = 1.250 mg/L (4.40 µM) for C. albicans. Mode-of-action analysis demonstrated fungicidal activity. Parietin targets the cell membrane and induces cell death via ROS-mediated lipid peroxidation after light irradiation. In summary, parietin exhibits light-enhanced fungicidal activity against all Candida species tested (including C. auris) and Cryptococcus neoformans, covering three of the four critical threats on the WHO's most recent fungal priority list.

Revised taxon definition in European Cortinarius subgenus Dermocybe based on phylogeny, chemotaxonomy, and morphology.

Cortinarius (Fr.) Fr. is one of the most species-rich genera in the Agaricales (Basidiomycota). Cortinarius subgen. Dermocybe (Fr.) Trog includes brightly coloured Cortinarii with anthraquinone pigments. The chemotaxonomic approach has always been as important as classical methods for species definition of Dermocybe and helped to improve overall species concepts. However, some species concepts within this group remain unclear. We therefore address this topic based on a combined phylogenetic, morphological, and pigment-chemical approach. For this, sequence data, HPLC-MS pigment profiles and spore sizes were included were included to obtain a better resolution of taxa. The study was based on 173 recent collections and 12 type specimens. A total of 117 rDNA ITS sequences were produced from the collections in this study, 102 sequences were retrieved from databases. We could detect and clearly delimit 19 Dermocybe species occurring in central European habitats, from which 16 are discussed in detail. Additionally, we grouped the detected anthraquinone pigments into four groups. This detailed analysis of dermocyboid Cortinarius species occurring in a restricted number of habitat types confirmed our hypothesis that species diversity is much higher than currently assumed. This high diversity is blurred by too wide and incorrect species concepts of several classical species like C. croceus and C. cinnamomeus. Molecular and chemotaxonomical studies carried out together with careful phenotypical analyses resulted in a good differentiation of species. A key is presented for these taxa to allow a better identification of Cortinarius subgenus Dermocybe spp. occurring in Central Europe mainly in the alpine range. Supplementary Information: The online version contains supplementary material available at 10.1007/s11557-024-01959-z.

Assessment of the chemical and genetic variability among accessions of Cicerbita alpina (L.) Wallr., an alpine plant with anthelmintic properties.

Cicerbita alpina (L.) Wallr, is a perennial alpine plant and a member of the Asteraceae family, typically found at altitudes above 1000 meters in the Italian Alps. Although previously utilized primarily as a local delicacy, recent studies have revealed strong antiparasitic activity through in vitro experiments. In Europe, numerous chemical drugs employed to combat nematodes - helminths that infest the digestive tract of livestock - are banned due to their environmental harm or show only reduced efficiency because of the development of resistance. Consequently, there is a growing demand for new alternative anthelmintic treatments in agricultural practices. Specialized metabolites found in the extracts of C. alpina could offer a sustainable and biological alternative to chemical drugs, specifically for nematode control. For this purpose, a unique germplasm collection originating from eight distinct natural populations in the Italian Alps was analyzed for its chemical diversity using state-of-the-art targeted LC-MS/MS spectrometry, including quantification based on multiple reaction monitoring. The predominant metabolites identified from the species were the caffeic acid derivatives chicoric acid, chlorogenic acid, and 3. 5-dicaffeoylquinic acid, the sesquiterpene lactone derivative 8-O-acetyl-15-ß-D-glucopyranosyl lactucin and the flavone glycosides, apigenin-7-O-glucoside and luteolin-7-O-glucoside, alongside their precursors apigenin and luteolin, respectively. Additionally, the genetic diversity of eighty individual plants within the germplasm collection was evaluated using ten DNA molecular markers (Simple Sequence Repeats), successfully transferred from two closely related species (Cichorium intybus and Tanacetum parthenium). This investigation unveiled a significant range of genetic diversity within the examined populations, resulting in the establishment of three distinct genetic groups. The findings were further correlated with the original ecological environment and local climate conditions spanning a biennial period, indicating substantial variations among the different accessions and the intricate interplay between genetic background and environmental factors. These results could serve as a basis for future domestication of the species through plant breeding programs ensuring product quality, but also facilitating the cultivation of C. alpina in more diverse geographic regions.

Growth, morphology, and formation of cinnabarin in Pycnoporus cinnabarinus in relation to different irradiation spectra.

BACKGROUND: 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. RESULTS: Red, green, blue, and UV-A irradiation (mean irradiance Ee = 1.5 ± 0.18 W m-2) had considerable effects on the growth and colony appearance of all three P. cinnabarinus strains tested. The cinnabarin content determined was, thus, dependent on the irradiation wavelength applied, allowing strain-specific thresholds to be defined. Irradiation with wavelengths below this strain-specific threshold corresponded to a lower cinnabarin content, at least at the intensity applied. The orange-red pigment appeared by light microscopy as incrusted extracellular plaques present on the hyphal walls. Highly efficient vegetative propagation occurred by arthroconidia, and we observed the tendency that this asexual reproduction was (i) most frequent in the dark but (ii) never occurred under UV-A exposure. CONCLUSION:  This study highlights a differential photo-dependence of growth, morphology, and cinnabarin formation in P. cinnabarinus. This confirms that it is advisable to consider the wavelength of the light used in future biotechnological productions of natural pigments.

Fungal Anthraquinone Photoantimicrobials Challenge the Dogma of Cationic Photosensitizers.

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.

Identification of Novel ß-Tubulin Inhibitors Using a Combined In Silico/In Vitro Approach.

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 ß-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.

Light in the box-photobiological examination chamber with light trap ventilation system for studying fungal surface cultures illustrated with Metarhizium brunneum and Beauveria brongniartii.

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.

Highlighting the Phototherapeutical Potential of Fungal Pigments in Various Fruiting Body Extracts with Informed Feature-Based Molecular Networking.

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.

Co-cultivation of Mortierellaceae with Pseudomonas helmanticensis affects both their growth and volatilome.

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.

In vitro evaluation of the effects of methanolic plant extracts on the embryonation rate of Ascaridia galli eggs.

The present study aims to find efficient alternatives to synthetic anthelmintics among ethno-veterinary herbs. Ascaridia galli eggs isolated from the worm uterus were exposed in vitro to methanolic extracts (ME) of nine plant species such as Achillea millefolium (AM), Artemisia absinthium (AA), Artemisia vulgaris (AV), Cicerbita alpina (CA), Cichorium intybus (CI), Inula helenium (IH), Origanum vulgare (OV), Tanacetum vulgare (TV), Tanacetum parthenium (TP). Flubendazole (FL), 0.5% formalin with dimethylsulfoxide and Petri dishes without the addition of reagents were used as positive, negative and untreated control respectively. The effects of the different ME at concentrations 0.500, 0.325, 0.200 mg/ml were assessed on the embryonic development (ED) of the eggs in duplicate. Logit analysis was used to calculate EC50 values. A generalized linear mixed model, having plant species and concentration as fixed effect and day as repeated measure, was used to determine differences in ED. Estimated EC50 was the lowest for FL at 0.11 mg/ml. CA and TV followed with 0.27 mg/ml and 0.32 mg/ml. ED for FL was significantly lower (25%) than that of CA (47%). The analysis showed 0.5 mg/ml of the ME of CA and TV significantly affected the ED at 35% and 42% inhibitions respectively. The ED for all ME showed similar pattern i.e., relatively higher efficacy in the first experimental week compared to the rest of the experimental period. The effect from all multicomponent extracts is time and dose dependent. The plants have promising results in inhibiting ED, contributing to the identification of alternative anthelmintic treatments.

Photochemical defense as trait of fungi from Cortinarius subgenus Dermocybe.

The photobiological activity of ten colorful species belonging to subgenus Dermocybe of the basidiomycete genus Cortinarius was investigated. Extracts of all species produced singlet oxygen and are thus photoactive. Pigment analysis was performed and showed similarities of the anthraquinone pigments across the species in dependency to their respective pigmentation types. Detailed content analysis of the pigments in the whole agaricoid fruiting body compared to the three different tissue types (pileus, stipe, and lamellae) revealed that the pigments emodin, dermocybin, and dermorubin, as well as their respective glycosides, are enhanced in the gills. In an independent experiment, the gills were shown to be the most photoactive tissues of the fruiting body. Photobiological experiments with invertebrates (i.e., glassworm Chaoborus crystallinus) proved a phototoxic effect of the methanolic extract of the red blood webcap (Cortinarius sanguineus var. aurantiovaginatus). This work adds further evidence to a common photobiological trait in Cortinarius subgenus Dermocybe and underpins the possibility of a photochemical defense mechanism in fungi.

The photosensitizer emodin is concentrated in the gills of the fungus Cortinarius rubrophyllus.

The colorful agaricoid fruiting bodies of dermocyboid Cortinarii owe their magnificent hue to a mixture of anthraquinone (AQ) pigments. Recently, it was discovered that some of these fungal anthraquinones have an impressive photopharmacological effect. The question, therefore, arises as to whether these pigments are also of ecological or functional significance. According to the optimal defense hypothesis, toxic molecules should be enriched in spore-producing structures, such as the gills of agarics. To test this hypothesis, we studied the distribution of fungal AQs in the fruiting body of Cortinarius rubrophyllus. The fungus belongs to the well-studied Cortinarius subgenus Dermocybe but has not been chemically characterized. Here, we report on the pigment profile of this beautiful fungus and focus on the distribution of anthraquinone pigments in the fruiting body for the first time. Here it is statistically confirmed that the potent photosensitizer emodin is significantly enriched in the gills. Furthermore, we show that the extract is photoactive against cancer cells and bacteria.

Targeted isolation of photoactive pigments from mushrooms yielded a highly potent new photosensitizer: 7,7'-biphyscion.

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.

Xanthoepocin, a photolabile antibiotic of Penicillium ochrochloron CBS 123823 with high activity against multiresistant gram-positive bacteria.

BACKGROUND: 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. RESULTS: In this work, we developed several new protocols to isolate, extract, and quantify xanthoepocin out of bioreactor batch and petri dish-grown mycelium of P. ochrochloron. The (photo)chemical re-evaluation with state-of-the-art techniques revealed that xanthoepocin is a photolabile molecule, which produces singlet oxygen under blue light irradiation. The intracellular xanthoepocin content, which was highest under ammonium-limited conditions, varied considerably with the applied irradiation conditions in petri dish and bioreactor batch cultures. Using light-protecting measures, we achieved MIC values against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), which were up to 5 times lower than previously published. In addition, xanthoepocin was highly active against a clinical isolate of linezolid and vancomycin-resistant Enterococcus faecium (LVRE). CONCLUSIONS: This interdisciplinary work underlines that the re-evaluation of known compounds with state-of-the-art techniques is an important strategy in the combat against multiresistant bacteria and that light is a crucial factor on many levels that needs to receive more attention. With appropriate light protecting measures in the susceptibility tests, xanthoepocin proved to be a powerful antibiotic against MRSA and LVRE. Exploring the light response of other polyketides may be pivotal for re-introducing previously discarded metabolites into the antibiotic pipeline and to identify photosensitizers which might be used for (antimicrobial) photodynamic therapies.

A convenient separation strategy for fungal anthraquinones by centrifugal partition chromatography.

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.

N-methylated diazabicyclo[3.2.2]nonane substituted triterpenoic acids are excellent, hyperbolic and selective inhibitors for butyrylcholinesterase.

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.

Optimized isolation of 7,7'-biphyscion starting from Cortinarius rubrophyllus, a chemically unexplored fungal species rich in photosensitizers.

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-ß-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.

Feature-Based Molecular Networking-An Exciting Tool to Spot Species of the Genus Cortinarius with Hidden Photosensitizers.

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.

A New High-Throughput-Screening-Assay for Photoantimicrobials Based on EUCAST Revealed Unknown Photoantimicrobials in Cortinariaceae.

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).

A simple but unusual rearrangement of an oleanane to a taraxerane-28,14 ß -olide.

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 ß -olide type triterpenoid while the same reaction at 0 °C occurred without re-arrangement of the carbon skeleton, and an oleanane-28,13 ß -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 ß, 12α, 13 ß -trihydroxy-oleanan-28-oic acid (6) holding with EC50 = 4.2 µM for MCF-7 tumor cells.