Trachymyrmex septentrionalis ants promote fungus garden hygiene using Trichoderma-derived metabolite cues.

Fungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that Trichoderma spp. can act as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant noncultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens.

Exploring the in vivo anti-cancer potential of Neosetophomone B in leukemic cells using a zebrafish xenograft model.

Neosetophomone B (NSP-B) is a unique meroterpenoid fungal secondary metabolite that has previously demonstrated promising anti-cancer properties against various cancer cell lines in vitro. However, its in vivo anti-cancer potential remaines unexplored. To fill this gap in our knowledge, we tested NSP-B's in vivo anti-cancer activity using a zebrafish model, an organism that has gained significant traction in biomedical research due to its genetic similarities with humans and its transparent nature, allowing real-time tumor growth observation. For our experiments, we employed the K562-injected zebrafish xenograft model. Upon treating these zebrafish with NSP-B, we observed a marked reduction in the size and number of tumor xenografts. Delving deeper, our analyses indicated that NSP-B curtailed tumor growth and proliferation of leukemic grafted xenograft within the zebrafish. These results show that NSP-B possesses potent in vivo anti-cancer properties, making it a potential novel therapeutic agent for addressing hematological malignancies.

Verticillins: fungal epipolythiodioxopiperazine alkaloids with chemotherapeutic potential.

Covering: 1970 through June of 2023Verticillins are epipolythiodioxopiperazine (ETP) alkaloids, many of which possess potent, nanomolar-level cytotoxicity against a variety of cancer cell lines. Over the last decade, their in vivo activity and mode of action have been explored in detail. Notably, recent studies have indicated that these compounds may be selective inhibitors of histone methyltransferases (HMTases) that alter the epigenome and modify targets that play a crucial role in apoptosis, altering immune cell recognition, and generating reactive oxygen species. Verticillin A (1) was the first of 27 analogues reported from fungal cultures since 1970. Subsequent genome sequencing identified the biosynthetic gene cluster responsible for producing verticillins, allowing a putative pathway to be proposed. Further, molecular sequencing played a pivotal role in clarifying the taxonomic characterization of verticillin-producing fungi, suggesting that most producing strains belong to the genus Clonostachys (i.e., Bionectria), Bionectriaceae. Recent studies have explored the total synthesis of these molecules and the generation of analogues via both semisynthetic and precursor-directed biosynthetic approaches. In addition, nanoparticles have been used to deliver these molecules, which, like many natural products, possess challenging solubility profiles. This review summarizes over 50 years of chemical and biological research on this class of fungal metabolites and offers insights and suggestions on future opportunities to push these compounds into pre-clinical and clinical development.

Neosetophomone B induces apoptosis in multiple myeloma cells via targeting of AKT/SKP2 signaling pathway.

Multiple myeloma (MM) is a hematologic malignancy associated with malignant plasma cell proliferation in the bone marrow. Despite the available treatments, drug resistance and adverse side effects pose significant challenges, underscoring the need for alternative therapeutic strategies. Natural products, like the fungal metabolite neosetophomone B (NSP-B), have emerged as potential therapeutic agents due to their bioactive properties. Our study investigated NSP-B's antitumor effects on MM cell lines (U266 and RPMI8226) and the involved molecular mechanisms. NSP-B demonstrated significant growth inhibition and apoptotic induction, triggered by reduced AKT activation and downregulation of the inhibitors of apoptotic proteins and S-phase kinase protein. This was accompanied by an upregulation of p21Kip1 and p27Cip1 and an elevated Bax/BCL2 ratio, culminating in caspase-dependent apoptosis. Interestingly, NSP-B also enhanced the cytotoxicity of bortezomib (BTZ), an existing MM treatment. Overall, our findings demonstrated that NSP-B induces caspase-dependent apoptosis, increases cell damage, and suppresses MM cell proliferation while improving the cytotoxic impact of BTZ. These findings suggest that NSP-B can be used alone or in combination with other medicines to treat MM, highlighting its importance as a promising phytoconstituent in cancer therapy.

Semisynthetic derivatives of the fungal metabolite eupenifeldin via targeting the tropolone hydroxy groups.

Eupenifeldin (1) is a fungal secondary metabolite possessing bis-tropolone moieties that demonstrates nanomolar cytotoxic activity against a number of cancer cell types. As a potential anticancer lead, this meroterpenoid was used to access 29 semisynthetic analogues via functionalization of the reactive hydroxy groups of the bis-tropolones. A series of ester (2-6), carbonate (7-8), sulfonate (9-16), carbamate (17-20), and ether (21-30) analogues of 1 were generated via 22 reactions. Most of these compounds were disubstituted, produced via functionalization of both of the tropolonic hydroxy moieties, although three mono-functionalized analogues (6, 8, and 24) and one tri-functionalized analogue (3) were also obtained. The cytotoxic activities of 1-30 were evaluated against human melanoma and ovarian cancer cell lines (i.e., MDA-MB-435 and OVCAR3, respectively). Ester and carbonate analogues of 1 (i.e., 2-8) maintained cytotoxicity at the nanomolar level, and the greatest improvement in aqueous solubility came from the monosuccinate analogue, which was acylated on the secondary hydroxy at the 11 position (6).

Semisynthesis, Characterization, and Biological Evaluation of Fluorinated Analogues of the Spirobisnaphthalene, Diepoxin-η.

Diepoxin-η (1) is a cytotoxic fungal metabolite belonging to the spirobisnaphthalene structural class. In this study, four mono fluorinated analogues (2-5) of diepoxin-η (1) were semisynthesized in a single-step by selectively fluorinating the naphthalene moiety with Selectfluor. The structures of 2-5 were elucidated using a set of spectroscopic and spectrometric techniques and were further confirmed by means of TDDFT-ECD and isotropic shielding tensors calculations. Compounds 2-5 showed equipotent cytotoxic activity to 1 when tested against OVCAR3 (ovarian) and MDA-MB-435 (melanoma) cancer cell lines with IC50 values that range from 5.7-8.2 µM.

Exploration of Verticillins in High-Grade Serous Ovarian Cancer and Evaluation of Multiple Formulations in Preclinical In Vitro and In Vivo Models.

Verticillins are epipolythiodioxopiperazine alkaloids isolated from a fungus with nanomolar anti-tumor activity in high-grade serous ovarian cancer (HGSOC). HGSOC is the fifth leading cause of death in women, and natural products continue to be an inspiration for new drug entities to help tackle chemoresistance. Verticillin D was recently found in a new fungal strain and compared to verticillin A. Both compounds exhibited nanomolar cytotoxic activity against OVCAR4 and OVCAR8 HGSOC cell lines, significantly reduced 2D foci and 3D spheroids, and induced apoptosis. In addition, verticillin A and verticillin D reduced tumor burden in vivo using OVCAR8 xenografts in the peritoneal space as a model. Unfortunately, mice treated with verticillin D displayed signs of liver toxicity. Tolerability studies to optimize verticillin A formulation for in vivo delivery were performed and compared to a semi-synthetic succinate version of verticillin A to monitor bioavailability in athymic nude females. Formulation of verticillins achieved tolerable drug delivery. Thus, formulation studies are effective at improving tolerability and demonstrating efficacy for verticillins.

Embellicines C-E: Macrocyclic Alkaloids with a Cyclopenta[b]fluorene Ring System from the Fungus Sarocladium sp.

Macrocyclic alkaloids with a cyclopenta[b]fluorene ring system are a relatively young structural class of fungal metabolites, with the first members reported in 2013. Bioassay-guided fractionation of a Sarocladium sp. (fungal strain MSX6737) led to a series of both known and new members of this structural class (1-5), including the known embellicine A (1), three new embellicine analogues (2, 4, and 5), and a semisynthetic acetylated analogue (3). The structures were identified by examining both high-resolution electrospray ionization mass spectrometry data and one-dimensional and two-dimensional NMR spectra. The relative configurations of these molecules were established via 1H-1H coupling constants and nuclear Overhauser effect spectroscopy, while comparisons of the experimental electronic circular dichroism (ECD) spectra with the time-dependent density functional theory ECD calculations were utilized to assign their absolute configurations, which were in good agreement with the literature. These alkaloids (1-5) showed cytotoxic activity against a human breast cancer cell line (MDA-MB-231) that ranged from 0.4 to 4.8 µM. Compounds 1 and 5 were also cytotoxic against human ovarian (OVCAR3) and melanoma (MDA-MB-435) cancer cell lines.

Probing the Cytotoxic Signaling Induced by Eupenifeldin in Ovarian Cancer Models.

High-grade serous ovarian cancer (HGSOC) is the most common and lethal ovarian cancer histotype. Lack of early detection methods, limited therapeutic agents, and low 5-year survival rate reflect the urgent need to develop new therapies. Eupenifeldin, a bistropolone, originally isolated from Eupenicillium brefeldianum, is a cytotoxic fungal metabolite. In three HSGOC cell lines (OVCAR3, OVCAR5, OVCAR8), eupenifeldin was found to have an IC50 value less than 10 nM, while 10 times higher concentrations were required for cytotoxicity in nontumorigenic fallopian tube secretory epithelial cell lines (FTSEC). An in vivo hollow fiber assay showed significant cytotoxicity in OVCAR3. Eupenifeldin significantly increased Annexin V staining in OVCAR3 and -8, but not OVCAR5. Eupenifeldin activated caspases 3/7 in OVCAR3, OVCAR5, and OVCAR8; however, cleaved PARP was only detected in OVCAR3. Quantitative proteomics performed on OVCAR3 implicated ferroptosis as the most enriched cell death pathway. However, validation experiments did not support ferroptosis as part of the cytotoxic mechanism of eupenifeldin. Autophagic flux and LC3B puncta assays found that eupenifeldin displayed weak autophagic induction in OVCAR3. Inhibition of autophagy by cotreatment with bafilomycin reduced the toxicity of eupenifeldin, supporting the idea that induction of autophagy contributes to the cytotoxic mechanism of eupenifeldin.

Anticancer activity of Neosetophomone B by targeting AKT/SKP2/MTH1 axis in leukemic cells.

Neosetophomone B (NSP-B), a meroterpenoid fungal secondary metabolite, was investigated for its anticancer potential in leukemic cell lines (K562 and U937). NSP-B treatment of leukemic cells suppressed cell viability by triggering apoptotic cell death. Apoptosis induced by NSP-B is triggered by mitochondrial signaling and caspase activation. Additionally, NSP-B treatment of leukemic cells causes AKT's inactivation accompanied by downregulation of SKP2 oncogene and MTH1 with a concomitant increase of p21Cip1and p27Kip1. Furthermore, NSP-B causes suppression of antiapoptotic proteins, including cIAP1, cIAP2, XIAP, survivin and BCl-XL. Overall, NSP-B reduces cell viability by mitochondrial and caspase-dependent apoptosis. The inhibition of AKT and SKP2 axis could be a promising therapeutic target for leukemia treatment.

Structural Diversity of Perylenequinones Is Driven by Their Redox Behavior.

Hypocrellins and hypomycins are two subclasses of fungal perylenequinones with unique structural, biological, and photochemical properties. With the growing interest in these naturally occurring photosensitizers, more studies were warranted to better understand the structural relationships between these two subclasses of perylenequinones. In this study, the long-postulated biosynthetic precursor (7) of class B fungal perylenequinones was isolated and characterized from a Shiraia-like sp. (strain MSX60519). Furthermore, the electrochemical and chemical redox behaviors of hypocrellins and hypomycins were investigated under aerobic and anaerobic conditions. These studies served to define the structural relationship within hypocrellins (1-3), which was further supported by X-ray crystallography, and between hypocrellins and hypomycins (4-6). Chemical reductions of hypocrellins under anaerobic conditions identified the origin of hypomycin A (4), hypomycin C (5), and hypomycin E (6), which in turn served to confirm 4 and revise the absolute configurations of 5 and 6. Hypocrellins were shown to undergo reversible reduction and reoxidation under aerobic conditions, while in an anaerobic environment and longer time scale, the fully reduced form can, to some extent, undergo an intramolecular ring closing metathesis. This may impart a means of reductive pathway for self-protection against these phototoxins and explain the chemical diversity observed in the fungal metabolites.

Dereplication of Fungal Metabolites by NMR-Based Compound Networking Using MADByTE.

Strategies for natural product dereplication are continually evolving, essentially in lock step with advances in MS and NMR techniques. MADByTE is a new platform designed to identify common structural features between samples in complex extract libraries using two-dimensional NMR spectra. This study evaluated the performance of MADByTE for compound dereplication by examining two classes of fungal metabolites, the resorcylic acid lactones (RALs) and spirobisnaphthalenes. First, a pure compound database was created using the HSQC and TOCSY data from 19 RALs and 10 spirobisnaphthalenes. Second, this database was used to assess the accuracy of compound class clustering through the generation of a spin system feature network. Seven fungal extracts were dereplicated using this approach, leading to the correct prediction of members of both families from the extract set. Finally, NMR-guided isolation led to the discovery of three new palmarumycins (20-22). Together these results demonstrate that MADByTE is effective for the detection of specific compound classes in complex mixtures and that this detection is possible for both known and new natural products.

Semisynthesis of Hypothemycin Analogues Targeting the C8-C9 Diol.

Hypothemycin, an epoxide derivative of (5Z)-7-oxozeaenol, was used in the semisynthesis of a series of C8-C9 diol derivatives, with many inhibiting TAK1 at submicromolar concentrations. A step-economical approach was chosen, whereby nonselective reactions functionalized the diol to generate multiple analogues in a single reaction. Using this approach, 35 analogues were synthesized using 12 reactions, providing a wealth of information about the role that the C8-C9 diol plays in TAK1 inhibition and cytotoxicity in ovarian and breast cancer cell lines. Monofunctionalized analogues exhibited strong inhibition of TAK1, showing potential for modification of this section of the molecule to assist with solubility, formulation, and other desirable properties. Most analogues were cytotoxic, and three compounds had similar or slightly increased potency with >100-fold improvement in solubility profiles.

Discovery of Anticancer Agents of Diverse Natural Origin.

Research progress from mainly over the last five years is described for a multidisciplinary collaborative program project directed toward the discovery of potential anticancer agents from a broad range of taxonomically defined organisms. Selected lead compounds with potential as new antitumor agents that are representative of considerable structural diversity have continued to be obtained from each of tropical plants, terrestrial and aquatic cyanobacteria, and filamentous fungi. Recently, a new focus has been on the investigation of the constituents of U.S. lichens and their fungal mycobionts. A medicinal chemistry and pharmacokinetics component of the project has optimized structurally selected lead natural products, leading to enhanced cytotoxic potencies against selected cancer cell lines. Biological testing has shown several compounds to have in vivo activity, and relevant preliminary structure-activity relationship and mechanism of action studies have been performed. Several promising lead compounds worthy of further investigation have been identified from the most recent collaborative work performed.

Cytotoxic Naphthoquinone Analogues, Including Heterodimers, and Their Structure Elucidation Using LR-HSQMBC NMR Experiments.

Approximately 1700 naphthoquinones have been reported from a range of natural product source materials, but only 283 have been isolated from fungi, fewer than 75 of those were dimers, and only 2 were heterodimers with a head-to-tail linkage. During a search for anticancer leads from fungi, a series of new naphthoquinones (1-4), including two heterodimers (3 and 4), were isolated from Pyrenochaetopsis sp. (strain MSX63693). In addition, the previously reported 5-hydroxy-6-(1-hydroxyethyl)-2,7-dimethoxy-1,4-naphthalenedione (5), misakimycin (6), 5-hydroxy-6-[1-(acetyloxy)ethyl]-2,7-dimethoxy-1,4-naphthalenedione (7), 6-ethyl-2,7-dimethoxyjuglone (8), and kirschsteinin (9) were isolated. While the structure elucidation of 1-9 was achieved using procedures common for natural products chemistry studies (high-resolution electrospray ionization mass spectrometry (HRESIMS), 1D and 2D NMR), the elucidation of the heterodimers was facilitated substantially by data from the long-range heteronuclear single quantum multiple bond correlation (LR-HSQMBC) experiment. The absolute configuration of 1 was established by analysis of the measured vs calculated ECD data. The racemic mixture of 4 was established via X-ray crystallography of an analogue that incorporated a heavy atom. All compounds were evaluated for cytotoxicity against the human cancer cells lines MDA-MB-435 (melanoma), MDA-MB-231 (breast), and OVCAR3 (ovarian), where the IC50 values ranged between 1 and 20 µM.

Opportunities and Limitations for Assigning Relative Configurations of Antibacterial Bislactones using GIAO NMR Shift Calculations.

Four new bislactones, dihydroacremonol (1), clonostachyone (2), acremodiol B (3), and acremodiol C (4), along with one known compound, hymeglusin (5), were isolated from cultures of two fungal strains (MSX59876 and MSX59260). Both strains were identified based on phylogenetic analysis of molecular data as Clonostachys spp.; yet, they biosynthesized a suite of related, but different, secondary metabolites. Given the challenges associated with elucidating the structures and configurations of bislactones, GIAO NMR calculations were tested as a complement to traditional NMR and HRESIMS experiments. Fortuitously, the enantiomer of the new natural product (4) was known as a synthetic compound, and the predicted configuration from GIAO NMR calculations (i.e., for the relative configuration) and optical rotation calculations (i.e., for the absolute configuration) matched those of the synthesis product. These results engendered confidence in using similar procedures, particularly the mixture of GIAO NMR shift calculations coupled with an orthogonal technique, to predict the configuration of 1-3; however, there were important limitations, which are discussed for each of these. The metabolites displayed antimicrobial activities, with compounds 1 and 4 being the most potent against Staphylococcus aureus with MICs of 1 and 4 µg/mL, respectively.

Cytotoxic and antimicrobial drimane meroterpenoids from a fungus of the Stictidaceae (Ostropales, Ascomycota).

As part of our ongoing research on bioactive fungal metabolites, two new metabolites were isolated from a fungus of the Stictidaceae (strain MSX62440), dasyscyphins F and G (1 and 3), and the known dasyscyphin C (2). Compound 1 was characterized by HRMS and 1D and 2D NMR data, and its absolute configuration established by ECD spectroscopy. A structural revision of dasyscyphin C (2) was based on NMR data and verified by ECD calculations. Compound 3 was reported previously as a synthetic product, and its identity confirmed by comparison with NMR data in the literature, and its absolute configuration was established by ECD spectroscopy. Compounds 1 and 2 showed moderate cytotoxicity and antimicrobial activity.

Enhanced Production and Anticancer Properties of Photoactivated Perylenequinones.

Hypocrellins and hypomycins are naturally occurring fungal perylenequinones with potential photodynamic activity against cancer and microbial diseases. This project pursued three lines of research. First, the production of perylenequinones was enhanced by investigating the effect of culture medium and light exposure on their biosynthesis. Solid-fermentation cultures on rice medium allowed for enhanced production of hypocrellins as compared to Cheerios or oatmeal medium. Alternatively, increased production of hypomycins, which are structurally related to the hypocrellins, was observed on oatmeal medium. In both cases, light exposure was an essential factor for the enhanced biosynthesis. In addition, this led to the discovery of two new perylenequinones, ent-shiraiachrome A (5) and hypomycin E (8), which were elucidated based on spectroscopic data. Finally, the photocytotoxic effects of both classes of compounds were evaluated against human skin melanoma, with EC50 values at nanomolar levels for hypocrellins and micromolar levels for hypomycins. In contrast, both classes of compounds showed reduced dark toxicity (EC50 values >100 µM), demonstrating promising phototherapeutic indices.

Engineering Fluorine into Verticillins (Epipolythiodioxopiperazine Alkaloids) via Precursor-Directed Biosynthesis.

Precursor-directed biosynthesis was used to generate a series of fluorinated verticillins. The biosynthesis of these epipolythiodioxopiperazine alkaloids was monitored in situ via the droplet liquid microjunction surface sampling probe (droplet probe), and a suite of NMR and mass spectrometry data were used for their characterization. All analogues demonstrated nanomolar IC50 values vs a panel of cancer cell lines. This approach yielded new compounds that would be difficult to generate via synthesis.

Mycopyranone: a 8,8'-binaphthopyranone with potent anti-MRSA activity from the fungus Phialemoniopsis sp.

A new 8,8'-binaphthopyranone (mycopyranone, 1) was isolated from a solid fermentation of Phialemoniopsis sp. (fungal strain MSX61662), and the structure was elucidated via analysis of the NMR and HRESIMS data. The axial chirality of 1 was determined to be M by ECD. The central chirality at C-4/C-4' was assigned through a modified Mosher's method, while the absolute configuration at C-3/C-3' was deduced based on analysis of the 3 J H-3-H-4 values and NOESY correlations. Compound 1 was evaluated for its antimicrobial properties against Staphylococcus aureus SA1199 and a clinically relevant methicillin-resistant S. aureus strain (MRSA USA300 LAC strain AH1263). Compound 1 inhibited the growth of both strains in a concentration dependent manner with IC50 values in the low µM range. Molecular docking indicated that compound 1 binds to the FtsZ (tubulin-like) protein in the same pocket as viriditoxin (2), suggesting that 1 targets bacterial cell division.