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During our ongoing research on fungal strains from unexplored sources, the reinvestigation of the CHCl(3)−MeOH extract of the marine-facultative Aspergillus sp. MEXU 27854 yielded a new N-methyl cyclic pentapeptide (1) along with known butyrolactone II and PF1233 A. In addition, from the marine-facultative Gymnoascus hyalinosporus MEXU 29901, a new alternariol glucoside, 10-O-[β-D-(4-methoxyl-glucopyranosyl)]-4-O-methylalternariol (2) and known alternariol 4-O-methyl ether, alternariol and beauvericin, were isolated. The structures of 1 and 2 were established by detailed spectroscopic data, and their absolute configuration was ascertained by Marfey’s analysis and HRESIMS-MS/MS data for 1, and by chemical degradation and optical rotation analysis for 2.

A new 8,8ˈ-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-4ˈ was assigned through a modified Mosher’s method, while the absolute configuration at C-3/C-3ˈ was deduced based on analysis of the 3JH-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.

Studies on an organic extract of a marine fungus, Periconia sp. (strain G1144), led to the isolation of three halogenated cyclopentenes along with the known and recently reported rhytidhyester D; a series of spectrometric and spectroscopic techniques were used to elucidate these structures. Interestingly, two of these compounds represent tri-halogenated cyclopentene derivatives, which have been observed only rarely from Nature. The relative and absolute configurations of the compounds were established via mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, Mosher’s esters method, optical rotation and GIAO NMR calculations, including correlation coefficient calculations and the use of both DP4+ and dJ DP4 analyses. Several of the isolated compounds were tested for activity in anti-parasitic, antimicrobial, quorum sensing inhibition, and cytotoxicity assays and were shown to be inactive.

Three previously undescribed diketomorpholine natural products, along with the known phenalenones, herqueinone and norherqueinone, were isolated from the mycoparasitic fungal strain G1071, which was identified as a Penicillium sp. in the section Sclerotiora. The structures were established by analyzing NMR data and mass spectrometry fragmentation patterns. The absolute configurations of deacetyl-javanicunine A, javanicunine C, and javanicunine D, were assigned by examining ECD spectra and Marfey's analysis. The structural diversity generated by this fungal strain was interesting, as only a few diketomorpholines (~17) have been reported from nature.

[2017-2018 UNCG University Libraries Open Access Publishing Fund Grant Winner.] Griseofulvin is a fungal metabolite and antifungal drug used for the treatment of dermatophytosis in both humans and animals. Recently, griseofulvin and its analogues have attracted renewed attention due to reports of their potential anticancer effects. In this study griseofulvin (1) and related analogues (2–6, with 4 being new to literature) were isolated from Xylaria cubensis. Six fluorinated analogues (7–12) were synthesized, each in a single step using the isolated natural products and Selectflour, so as to examine the effects of fluorine incorporation on the bioactivities of this structural class. The isolated and synthesized compounds were screened for activity against a panel of cancer cell lines (MDA-MB-435, MDA-MB-231, OVCAR3, and Huh7.5.1) and for antifungal activity against Microsporum gypseum. A comparison of the chemical space occupied by the natural and fluorinated analogues was carried out by using principal component analysis, documenting that the isolated and fluorinated analogues occupy complementary regions of chemical space. However, the most active compounds, including two fluorinated derivatives, were centered around the chemical space that was occupied by the parent compound, griseofulvin, suggesting that modifications must preserve certain attributes of griseofulvin to conserve its activity.

Chemical investigation of an organic extract of a fungus isolated from submerged wood collected from fresh water (strain G173), identified as a Talaromyces amestolkiae (Eurotiales; Trichocomaceae), led to the isolation of three coumarins, three dihydroisocoumarins, a dibenzo-α-pyrone, a meroterpenoid, and a merodrimane. Three of the isolated compounds, namely 7-chloropestalasin A (3), 4-hydroxyaspergillumarin (6), and ent-thailandolide B (9) were new. The structures were elucidated using a combination of spectroscopic and spectrometric techniques. The absolute configurations of 2, 3, 5, and 6 were established via a modified Mosher’s ester method, whereas for 9 a combination of TDDFT ECD and ORD calculations were employed. Compounds 1–9 were evaluated for antimicrobial activity against a group of bacteria and fungi.

From solid rice-based cultures of Malbranchea albolutea, three undescribed ardeemins and sartoryglabrins analogs were discovered and named alboluteins A-C. 1H-Indole-3-carbaldehyde, and anthranilic acid were also isolated. 1D and 2D-NMR techniques, as well as DFT-calculated chemical shifts, allowed characterizing alboluteins A-C. Testing these compounds against PTP1B indicated their inhibitory activity with IC50's ranging from 19 to 129 μM (ursolic acid IC50 = 29.8 μM, positive control). Kinetic analysis revealed that albolutein C behaved as a non-competitive inhibitor. Docking studies of alboluteins A-C into the crystal structure of PTP1B (PDB ID: 1T49 ) predicted that all compounds prefer to bind at the allosteric site of the enzyme, with Ki values of 2.02 × 10−4, 1.31 × 10−4, and 2.67 × 10−4 mM, respectively. Molecular dynamic studies indicated that the active compounds remained tied to the enzyme with good binding energy.

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), along with 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 previously reported 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.

One challenge in the dietary supplement industry is confirmation of species identity for processed raw materials, i.e. those modified by milling, drying, or extraction, which move through a multilevel supply chain before reaching the finished product. This is particularly difficult for samples containing fungal mycelia, where processing removes morphological characteristics, such that they do not present sufficient variation to differentiate species by traditional techniques. To address this issue, we have demonstrated the utility of DNA barcoding to verify the taxonomic identity of fungi found commonly in the food and dietary supplement industry; such data are critical for protecting consumer health, by assuring both safety and quality. By using DNA barcoding of nuclear ribosomal internal transcribed spacer (ITS) of the rRNA gene with fungal specific ITS primers, ITS barcodes were generated for 33 representative fungal samples, all of which could be used by consumers for food and/or dietary supplement purposes. In the majority of cases, we were able to sequence the ITS region from powdered mycelium samples, grocery store mushrooms, and capsules from commercial dietary supplements. After generating ITS barcodes utilizing standard procedures accepted by the Consortium for the Barcode of Life, we tested their utility by performing a BLAST search against authenticate published ITS sequences in GenBank. In some cases, we also downloaded published, homologous sequences of the ITS region of fungi inspected in this study and examined the phylogenetic relationships of barcoded fungal species in light of modern taxonomic and phylogenetic studies. We anticipate that these data will motivate discussions on DNA barcoding based species identification as applied to the verification/certification of mushroom-containing dietary supplements.

The potential of fungal endophytes to alter or contribute to plant chemistry and biology has been the topic of a great deal of recent interest. For plants that are used medicinally, it has been proposed that endophytes might play an important role in biological activity. With this study, we sought to identify antimicrobial fungal endophytes from the medicinal plant goldenseal (Hydrastis canadensis L., Ranunculaceae), a plant used in traditional medicine to treat infection. A total of 23 fungal cultures were obtained from surface-sterilized samples of H. canadensis roots, leaves and seeds. Eleven secondary metabolites were isolated from these fungal endophytes, five of which had reported antimicrobial activity. Hydrastis canadensis plant material was then analyzed for the presence of fungal metabolites using liquid chromatography coupled to high resolving power mass spectrometry. The antimicrobial compound alternariol monomethyl ether was detected both as a metabolite of the fungal endophyte Alternaria spp. isolated from H. canadensis seeds, and as a component of an extract from the H. canadensis seed material. Notably, fungi of the Alternaria genus were isolated from three separate accessions of H. canadensis plant material collected in a time period spanning 5 years. The concentration of alternariol monomethyl ether (991 mg/kg in dry seed material) was in a similar range to that previously reported for metabolites of ecologically important fungal endophytes. The seed extracts themselves, however, did not possess antimicrobial activity.

Summary Interspecific hybridization substantially alters genotypes and phenotypes and can give rise to new lineages. Hybrid isolates that differ from their parental species in infection-relevant traits have been observed in several human-pathogenic yeasts and plant-pathogenic filamentous fungi but have yet to be found in human-pathogenic filamentous fungi. We discovered 6 clinical isolates from patients with aspergillosis originally identified as Aspergillus nidulans (section Nidulantes) that are actually allodiploid hybrids formed by the fusion of Aspergillus spinulosporus with an unknown close relative of Aspergillus quadrilineatus, both in section Nidulantes. Evolutionary genomic analyses revealed that these isolates belong to Aspergillus latus, an allodiploid hybrid species. Characterization of diverse infection-relevant traits further showed that A. latus hybrid isolates are genomically and phenotypically heterogeneous but also differ from A. nidulans, A. spinulosporus, and A. quadrilineatus. These results suggest that allodiploid hybridization contributes to the genomic and phenotypic diversity of filamentous fungal pathogens of humans.

Spiroscytalin (1), a new tetramic acid that possesses an uncommon spiro-ring fusion between a polyketide-derived octalin ring system and a 2,4-pyrrolidinedione, along with two known compounds, leporin B (2) and purpactin A (3), were isolated from a solid phase culture of the fungus Scytalidium cuboideum (MSX 68345). The molecular connectivity of 1–3 was determined using NMR spectroscopy and mass spectrometry. The relative configurations of 1 and 2 were determined by NOESY experiments. The absolute configuration of 1 was determined by electronic circular dichroism (ECD) via a combination of experimental measurements and computational calculations. While leporin B was known, it displayed activities that had not been reported previously, including cytotoxicity against three human tumor cell lines and antibacterial activity against Candida albicans and Staphylococcus aureus.

With this study, we explored the identity and chemistry of fungal endophytes from the roots of yerba mansa [Anemopsis californica (Nutt.) Hook. & Arn. (Saururaceae)], a botanical traditionally used to treat infection. We compared the diversity of fungal endophytes isolated from a wild-harvested A. californica population, and those from plants cultivated for one year in a greenhouse environment. The wild-harvested population yielded thirteen fungal strains (eleven unique genotypes). Of the extracts prepared from these fungi, four inhibited growth of Staphylococcus aureus by >25% at 20 µg/mL, and three inhibited growth of Pseudomonas aeruginosa by =20% at 200 µg/mL. By comparison, A. californica roots after one year of cultivation in the greenhouse produced only two unique genotypes, neither of which displayed significant antimicrobial activity. The fungus Chaetomium cupreum isolated from wild-harvested A. californica yielded a new antimicrobial spirolactone, chaetocuprum (1). An additional 14 known compounds were identified using LC–MS dereplication of the various fungal endophytes. This study provides new insights into the identity and chemistry of A. californica fungal endophytes, and demonstrates the importance of considering growing conditions when pursuing natural product drug discovery from endophytic fungi.

Natural products are a valuable source of anticancer agents, with many naturally derived compounds currently used in clinical and preclinical treatments. This study aims to investigate the antiproliferative activity and potential mechanism of action of the xanthoquinodin JBIR-99, isolated from fungi Parengyodontium album MEXU 30,054 and identified by single-crystal X-ray crystallography. Cytotoxicity of xanthoquinodin was evaluated in a panel of human cancer cells lines and CCD-112-CoN normal colon cells, using the sulforhodamine B assay. PC-3 prostate cancer cells were used in biochemical assays including cell cycle, mitochondrial transmembrane potential (MTP), reactive oxygen species (ROS) and caspase activity. Expression levels of apoptosis-pathway-related proteins were analyzed by Western blot. The in vivo toxicity of xanthoquinodin was determined using a zebrafish model. Xanthoquinodin showed cytotoxicity in all cancer cell lines but demonstrated relative selective potency against PC-3 cells with an IC50 1.7 μM. In CCD-112-CoN cells, xanthoquinodin was non-cytotoxic at 100 μM. In PC-3 cells, the compound induced loss of MTP, production of ROS, and cell cycle arrest in S phase. The expression and activity of caspase-3 was increased, which correlates with the upregulation of Cyt c, Bax, nuclear factor kappa-B (NF-κB) (p65) and IKKβ, and downregulation of poly ADP ribose polymerase (PARP-1) and Bcl-2. Lastly, xanthoquinodin did not cause any visible developmental toxicity in zebrafish at 50 μM. These results demonstrate xanthoquinodin induces apoptosis in PC-3 prostate cancer cells by activation of both intrinsic and extrinsic apoptotic pathways. In addition, the non-toxic effect in vivo indicates that xanthoquinodin could be a useful lead in the development of a novel, anti-cancer agent that is selective for prostate cancer.

An organic extract of a filamentous fungus (MSX 58801), identified as a Volutella sp. (Hypocreales, Ascomycota), displayed moderate cytotoxic activity against NCI-H460 human large cell lung carcinoma. Bioactivity-directed fractionation led to the isolation of three γ-lactones having the furo[3,4-b]pyran-5-one bicyclic ring system [waol A (1), trans-dihydrowaol A (2), and cis-dihydrowaol A (3)]. The structures were elucidated using a set of spectroscopic and spectrometric techniques; the absolute configuration of 2 was established via a modified Mosher’s ester method. Compounds 1 and 2 were evaluated for cytotoxicity against a human cancer cell panel.