Cryo-EM structure of a fungal mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU orthologue from the fungus Neosartorya fischeri (NfMCU) determined to 3.8 Å resolution by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and separated by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.

Soil-Borne Neosartorya spp.: A Heat-Resistant Fungal Threat to Horticulture and Food Production-An Important Component of the Root-Associated Microbial Community.

Soil-borne Neosartorya spp. are the highly resilient sexual reproductive stage (teleomorph) of Aspergillus spp. Fungi of this genus are relevant components of root-associated microbial community, but they can also excrete mycotoxins and exhibit great resistance to high temperatures. Their ascospores easily transfer between soil and crops; thus, Neosartorya poses a danger to horticulture and food production, especially to the postharvest quality of fruits and vegetables. The spores are known to cause spoilage, mainly in raw fruit produce, juices, and pulps, despite undergoing pasteurization. However, these fungi can also participate in carbon transformation and sequestration, as well as plant protection in drought conditions. Many species have been identified and included in the genus, and yet some of them create taxonomical controversy due to their high similarity. This also contributes to Neosartorya spp. being easily mistaken for its anamorph, resulting in uncertain data within many studies. The review discusses also the factors shaping Neosartorya spp.'s resistance to temperature, preservatives, chemicals, and natural plant extracts, as well as presenting novel solutions to problems created by its resilient nature.

Bioactive Compounds from Terrestrial and Marine-Derived Fungi of the Genus Neosartorya †.

Fungi comprise the second most species-rich organism group after that of insects. Recent estimates hypothesized that the currently reported fungal species range from 3.5 to 5.1 million types worldwide. Fungi can grow in a wide range of habitats, from the desert to the depths of the sea. Most develop in terrestrial environments, but several species live only in aquatic habitats, and some live in symbiotic relationships with plants, animals, or other fungi. Fungi have been proved to be a rich source of biologically active natural products, some of which are clinically important drugs such as the ß-lactam antibiotics, penicillin and cephalosporin, the immunosuppressant, cyclosporine, and the cholesterol-lowering drugs, compactin and lovastatin. Given the estimates of fungal biodiversity, it is easy to perceive that only a small fraction of fungi worldwide have ever been investigated regarding the production of biologically valuable compounds. Traditionally, fungi are classified primarily based on the structures associated with sexual reproduction. Thus, the genus Neosartorya (Family Trichocomaceae) is the telemorphic (sexual state) of the Aspergillus section known as Fumigati, which produces both a sexual state with ascospores and an asexual state with conidiospores, while the Aspergillus species produces only conidiospores. However, according to the Melbourne Code of nomenclature, only the genus name Aspergillus is to be used for both sexual and asexual states. Consequently, the genus name Neosartorya was no longer to be used after 1 January 2013. Nevertheless, the genus name Neosartorya is still used for the fungi that had already been taxonomically classified before the new rule was in force. Another aspect is that despite the small number of species (23 species) in the genus Neosartorya, and although less than half of them have been investigated chemically, the chemical diversity of this genus is impressive. Many chemical classes of compounds, some of which have unique scaffolds, such as indole alkaloids, peptides, meroterpenes, and polyketides, have been reported from its terrestrial, marine-derived, and endophytic species. Though the biological and pharmacological activities of a small fraction of the isolated metabolites have been investigated due to the available assay systems, they exhibited relevant biological and pharmacological activities, such as anticancer, antibacterial, antiplasmodial, lipid-lowering, and enzyme-inhibitory activities.

Metabolites from Marine-Derived Fungi as Potential Antimicrobial Adjuvants.

Marine-derived fungi constitute an interesting source of bioactive compounds, several of which exhibit antibacterial activity. These acquire special importance, considering that antimicrobial resistance is becoming more widespread. The overexpression of efflux pumps, capable of expelling antimicrobials out of bacterial cells, is one of the most worrisome mechanisms. There has been an ongoing effort to find not only new antimicrobials, but also compounds that can block resistance mechanisms which can be used in combination with approved antimicrobial drugs. In this work, a library of nineteen marine natural products, isolated from marine-derived fungi of the genera Neosartorya and Aspergillus, was evaluated for their potential as bacterial efflux pump inhibitors as well as the antimicrobial-related mechanisms, such as inhibition of biofilm formation and quorum-sensing. Docking studies were performed to predict their efflux pump action. These compounds were also tested for their cytotoxicity in mouse fibroblast cell line NIH/3T3. The results obtained suggest that the marine-derived fungal metabolites are a promising source of compounds with potential to revert antimicrobial resistance and serve as an inspiration for the synthesis of new antimicrobial drugs.

The Neosartorya fischeri Antifungal Protein 2 (NFAP2): A New Potential Weapon against Multidrug-Resistant Candida auris Biofilms.

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312-0.5, 0.155-0.5, 0.037-0.375, 0.064-0.375, and 0.064-0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 µM2%, 2.16 µM2%, 33.31 µM2%, 10.72 µM2%, and 111.19 µM2% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.

Effect of storage temperature, water activity, oxygen headspace concentration and pasteurization intensity on the time to growth of Aspergillus fischerianus (teleomorph Neosartorya fischeri).

This study aims to assess, by means of a full factorial design, the effect of storage temperature (10-30 °C), water activity (aw, 0.87-0.89), headspace oxygen (O2) level (0.15-0.80%) and pasteurization intensity (95 °C-105 °C/15sec) on the time to visible growth (tv, days) of Aspergillus fischerianus on acidified Potato Dextrose Agar (aPDA, pH 3.6) for up to 90 days. Moreover, in order to validate the results obtained on aPDA, 12 conditions were selected and assessed in concentrate strawberry-puree based medium. Overall, storage temperature had the greatest effect on the tv of A. fischerianus on the evaluated conditions. At 10 °C, no visible growth was observed over the 90 day incubation period, whilst visible mycelia (diameter ≥ 2 mm) were present in 37% and 89% of the conditions at 22 °C and 30 °C, respectively. Pasteurization intensity had only a minor effect on the outgrowth of A. fischerianus. Growth inhibition was observed when aw was reduced to 0.870 ± 0.005 in combination with very low headspace O2 levels (0.15% ± 0.10) in both, aPDA and concentrate strawberry-based media, regardless of the incubation temperature and heat pasteurization intensity. Overall, longer tv's were required when incubation was done at 22 °C compared to 30 °C. Ultimately, the effect of O2 (0.05 and 1%) and pasteurization intensity (95 °C and 105 °C/15sec) were evaluated on totally 22 fruit purees (un-concentrates and concentrates) over a 60 day storage period. None of the concentrates purees (aw ≤0.860) evaluated in this study supported the growth of A. fischerianus. On the other hand, A. fischerianus growth inhibition was only observed when the O2 levels were ≤0.05% on un-concentrates fruit purees (aw ≥ 0.980) stored at ambient temperature (22 °C). Combination of multiple stress factors effectively inhibited growth of A. fischerianus. In general, storage of fruit purees at low temperatures (<10 °C) or distribution in the form of concentrates can be considered as important strategies to prevent the growth of spoilage associated heat-resistant moulds.

Can marine-derived fungus Neosartorya siamensis KUFA 0017 extract and its secondary metabolites enhance antitumor activity of doxorubicin? An in vitro survey unveils interactions against lung cancer cells.

Doxorubicin (Dox) is one of the most successful anticancer drugs in use. However, chemoresistance is one of the main limitations that patients face. Therefore, development of new strategies to improve the efficacy of Dox is needed. Marine-derived fungi are especially promising sources of new anticancer compounds. In this work, antitumor activity of crude ethyl extract of the cultures of the marine-derived fungus Neosartorya siamensis KUFA 0017 (NS), combined with Dox, was evaluated in six cancer cell lines. To evaluate possible mechanisms involved in the eventual improvement of Dox's cytotoxicity by NS extract, effects on DNA damage, cell death, ultrastructural modifications, and intracellular accumulation of Dox were assessed. The NS extract demonstrated a significant enhancement of Dox's cytotoxic activity in A549 cells, inducing DNA damage, cell death, and intracellular accumulation of Dox. Additionally, the cytotoxic effect of eight compounds, isolated from this extract, that is, 2,4-dihydroxy-3-methylacetophenone-(C1), nortryptoquivaline-(C2), chevalone C-(C3), tryptoquivaline H-(C4), fiscalin A-(C5), epi-fiscalin-C (C6), epi-neofiscalin A-(C7), and epi-fiscalin A-(C8), alone and combined with Dox was also evaluated in lung cancer cells. The cytotoxic effect of Dox was potentiated by all the isolated compounds (except C1) in A549 cells. Therefore, we concluded that NS extract potentiated cytotoxicity by inhibiting cell proliferation, increasing intracellular accumulation of Dox, and inducing cell death (possibly by an autophagic process). The isolated compounds also enhanced the activity of Dox, supporting the potential of this sort of combination. These data call for further studies to characterize drug interactions and underlying mechanisms.

In Vivo Applicability of Neosartorya fischeri Antifungal Protein 2 (NFAP2) in Treatment of Vulvovaginal Candidiasis.

As a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-forming Candida species, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective antifungal therapeutic strategies. The small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of several Candida spp. in vitro; however, no information is available about their in vivo antifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis, Neosartorya fischeri antifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistant Candida albicans strain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilm in vitro We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MIC in vitro. In vivo murine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistant C. albicans cells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.

Methylsulfonylated Polyketides Produced by Neosartorya udagawae HDN13-313 via Exogenous Addition of Small Molecules.

Two new polyketides modified with a rare methylsulfonyl group, 3-methoxy-6-methyl-5-(methylsulfonyl)benzene-1,2,4-triol (1) and neosartoryone A (2), along with a biogenetically related compound (3), were isolated from Neosartorya udagawae HDN13-313 cultivated with the DNA methyltransferase inhibitor 5-azacytidine. The methylsulfonyl group of 1 and 2 was proven to be derived from DMSO, which was used as the solvent to dissolve 5-azacytidine. This is the first report of a fungus that can achieve a sulfonylation-like modification of natural products utilizing DMSO as a sulfur source. Compound 2 showed lipid-lowering activity in vitro comparable to simvastatin.

Studies on the Chemical Diversities of Secondary Metabolites Produced by Neosartorya fischeri via the OSMAC Method.

The One Strain Many Compounds (OSMAC) method was applied to explore the chemical diversities of secondary metabolites produced by Neosartorya fischeri NRRL 181. Four pyripyropenes 1⁻4, eight steroids 5⁻11, and four prenylated indole alkaloids 12⁻15, were obtained from the fungus cultured in petri dishes containing potato dextrose agar (PDA). 1,7,11-trideacetylpyripyropene A (1) and 1,11-dideacetyl pyripyropene A (2) were obtained and spectroscopically characterized (1D, 2D NMR, and HR-ESI-MS) from a natural source for the first time. It offered a sustainable source of these two compounds, which were usually used as starting materials in preparing pyripyropene derivatives. In addition, as compared with all the other naturally occurring pyripyropenes, 1 and 2 possessed unique acetylation patterns that did not follow the established late-step biosynthetic rules of pyripyropenes. The natural occurrence of 1 and 2 in the fungus implied that the timing and order of hydroxylation and acetylation in the late-step biosynthetic pathway of pyripyropenes remained to be revealed. The isolation and identification of 1⁻15 indicated that the OSMAC method could remarkably alter the metabolic profile and enrich the chemical diversities of fungal metabolites. Compounds 1⁻4 exhibited no obvious cytotoxicity against the triple-negative breast cancer cell line MDA-MB-231 as compared with taxol.

The Fungal Metabolite Eurochevalierine, a Sequiterpene Alkaloid, Displays Anti-Cancer Properties through Selective Sirtuin 1/2 Inhibition.

NAD⁺-dependent histone deacetylases (sirtuins) are implicated in cellular processes such as proliferation, DNA repair, and apoptosis by regulating gene expression and the functions of numerous proteins. Due to their key role in cells, the discovery of small molecule sirtuin modulators has been of significant interest for diverse therapeutic applications. In particular, it has been shown that inhibition of sirtuin 1 and 2 activities is beneficial for cancer treatment. Here, we demonstrate that the fungal metabolite eurochevalierine from the fungus Neosartorya pseudofischeri inhibits sirtuin 1 and 2 activities (IC50 about 10 µM) without affecting sirtuin 3 activity. The binding modes of the eurochevalierine for sirtuin 1 and 2 have been identified through computational docking analyses. Accordingly, this sequiterpene alkaloid induces histone H4 and α-tubulin acetylation in various cancer cell models in which it induces strong cytostatic effects without affecting significantly the viability of healthy PBMCs. Importantly, eurochevalierine targets preferentially cancer cell proliferation (selectivity factor ≫ 7), as normal human primary CD34⁺ stem/progenitor cells were less affected by the treatment. Finally, eurochevalierine displays suitable drug-likeness parameters and therefore represent a promising scaffold for lead molecule optimization to study the mechanism and biological roles of sirtuins and potentially a basis for development into therapeutics.

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency.

Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4) from Aspergillus niger Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (kcat/Km , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (kcat/Km ) of these enzymes (in descending order, NfEG12A [100%], NfEG12A-FN [140%], and NfEG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (kcat). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the -1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (kcat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.

A New Dihydrochromone Dimer and Other Secondary Metabolites from Cultures of the Marine Sponge-Associated Fungi Neosartorya fennelliae KUFA 0811 and Neosartorya tsunodae KUFC 9213.

A previously unreported dihydrochromone dimer, paecilin E (1), was isolated, together with eleven known compounds: ß-sitostenone, ergosta-4,6,8 (14), 22-tetraen-3-one, cyathisterone, byssochlamic acid, dehydromevalonic acid lactone, chevalone B, aszonalenin, dankasterone A (2), helvolic acid, secalonic acid A and fellutanine A, from the culture filtrate extract of the marine sponge-associated fungus Neosartorya fennelliae KUFA 0811. Nine previously reported metabolites, including a chromanol derivative (3), (3ß, 5α, 22E), 3,5-dihydroxyergosta-7,22-dien-6-one (4), byssochlamic acid, hopan-3ß,22-diol, chevalone C, sartorypyrone B, helvolic acid, lumichrome and the alkaloid harmane were isolated from the culture of the marine-sponge associated fungus Neosartorya tsunodae KUFC 9213. Paecilin E (1), dankasterone A (2), a chromanol derivative (3), (3ß, 5α, 22E)-3,5-dihydroxyergosta-7,22-dien-6-one (4), hopan-3ß,22-diol (5), lumichrome (6), and harmane (7) were tested for their antibacterial activity against Gram-positive and Gram-negative reference and multidrug-resistant strains isolated from the environment. While paecilin E (1) was active against S. aureus ATCC 29213 and E. faecalis ATCC 29212, dankastetrone A (2) was only effective against E. faecalis ATCC 29212 and the multidrug-resistant VRE E. faecalis A5/102. Both compounds neither inhibit biofilm mass production in any of the strains at the concentrations tested nor exhibit synergistic association with antibiotics.

Functional analysis of the gene controlling hydroxylation of festuclavine in the ergot alkaloid pathway of Neosartorya fumigata.

Bioactive ergot alkaloids produced by several species of fungi are important molecules in agriculture and medicine. Much of the ergot alkaloid pathway has been elucidated, but a few steps, including the gene controlling hydroxylation of festuclavine to fumigaclavine B, remain unsolved. Festuclavine is a key intermediate in the fumigaclavine branch of the ergot alkaloid pathway of the opportunistic pathogen Neosartorya fumigata and also in the dihydrolysergic acid-based ergot alkaloid pathway of certain Claviceps species. Based on several lines of evidence, the N. fumigata gene easM is a logical candidate to encode the festuclavine-hydroxylating enzyme. To test this hypothesis we disrupted easM function by replacing part of its coding sequences with a hygromycin resistance gene and transforming N. fumigata with this construct. High-pressure liquid chromatography analysis demonstrated that easM deletion mutants were blocked in the ergot alkaloid pathway at festuclavine, and downstream products were eliminated. An additional alkaloid, proposed to be a prenylated form of festuclavine on the basis of mass spectral data, also accumulated to higher concentrations in the easM knockout. Complementation with the wild-type allele of easM gene restored the ability of the fungus to produce downstream compounds. These results indicate that easM encodes an enzyme required for fumigaclavine B synthesis likely by hydroxylating festuclavine. The festuclavine-accumulating strain of N. fumigata may facilitate future investigations of the biosynthesis of dihydrolysergic acid derivatives, which are derived from festuclavine and are the basis for several important drugs.

Revisiting overexpression of a heterologous ß-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.

BACKGROUND: The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in ß-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous ß-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. RESULTS: The thermophilic ß-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed ß-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different ß-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a ß-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. CONCLUSIONS: We report herein the successful overexpression of a thermophilic N. fischeri ß-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved ß-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.

Five New Cytotoxic Metabolites from the Marine Fungus Neosartorya pseudofischeri.

The marine fungus Neosartorya pseudofischeri was isolated from Acanthaster planci from the South China Sea. In a preliminary bioactivity screening, the crude methanol extract of the fungal mycelia showed significant inhibitory activity against the Sf9 cell line from the fall armyworm Spodoptera frugiperda. Five novel compounds, including 5-olefin phenylpyropene A (1), 13-dehydroxylpyripyropene A (4), deacetylsesquiterpene (7), 5-formyl-6-hydroxy-8-isopropyl-2- naphthoic acid (9) and 6,8-dihydroxy-3-((1E,3E)-penta-1,3-dien-1-yl)isochroman-1-one (10), together with eleven known compounds, phenylpyropene A (2) and C (3), pyripyropene A (5), 7-deacetylpyripyropene A (6), (1S,2R,4aR,5R,8R,8aR)-1,8a-dihydroxy-2-acetoxy-3,8-dimethyl-5- (prop-1-en-2-yl)-1,2,4a, 5,6,7,8,8a-octahydronaphthalene (8), isochaetominine C (11), trichodermamide A (12), indolyl-3-acetic acid methyl ester (13), 1-acetyl-ß-carboline (14), 1,2,3,4-tetrahydro-6-hydroxyl-2-methyl-l,3,4-trioxopyrazino[l,2-a]-indole (15) and fumiquinazoline F (16), were obtained. The structures of these compounds were determined mainly by MS and NMR data. The absolute configuration of 9 was assigned by the single-crystal X-ray diffraction studies. Compounds 1-11 and 15 showed significant cytotoxicity against the Sf9 cells from S. frugiperda.

New Cyclotetrapeptides and a New Diketopiperzine Derivative from the Marine Sponge-Associated Fungus Neosartorya glabra KUFA 0702.

Two new cyclotetrapeptides, sartoryglabramides A (5) and B (6), and a new analog of fellutanine A (8) were isolated, together with six known compounds including ergosta-4, 6, 8 (14), 22-tetraen-3-one, ergosterol 5, 8-endoperoxide, helvolic acid, aszonalenin (1), (3R)-3-(1H-indol-3-ylmethyl)-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (2), takakiamide (3), (11aR)-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (4), and fellutanine A (7), from the ethyl acetate extract of the culture of the marine sponge-associated fungus Neosartorya glabra KUFA 0702. The structures of the new compounds were established based on extensive 1D and 2D spectral analysis. X-ray analysis was also used to confirm the relative configuration of the amino acid constituents of sartoryglabramide A (5), and the absolute stereochemistry of the amino acid constituents of sartoryglabramide A (5) and sartoryglabramides B (6) was determined by chiral HPLC analysis of their hydrolysates by co-injection with the d- and l- amino acids standards. Compounds 1-8 were tested for their antibacterial activity against Gram-positive (Escherichia coli ATCC 25922) and Gram-negative (Staphyllococus aureus ATCC 25923) bacteria, as well as for their antifungal activity against filamentous (Aspergillus fumigatus ATCC 46645), dermatophyte (Trichophyton rubrum ATCC FF5) and yeast (Candida albicans ATCC 10231). None of the tested compounds exhibited either antibacterial (MIC > 256 µg/mL) or antifungal activities (MIC > 512 µg/mL).

New Polyketides and New Benzoic Acid Derivatives from the Marine Sponge-Associated Fungus Neosartorya quadricincta KUFA 0081.

Two new pentaketides, including a new benzofuran-1-one derivative (1) and a new isochromen-1-one (5), and seven new benzoic acid derivatives, including two new benzopyran derivatives (2a, b), a new benzoxepine derivative (3), two new chromen-4-one derivatives (4b, 7) and two new benzofuran derivatives (6a, b), were isolated, together with the previously reported 2,3-dihydro-6-hydroxy-2,2-dimethyl-4H-1-benzopyran-4-one (4a), from the culture of the marine sponge-associated fungus Neosartorya quadricincta KUFA 0081. The structures of the new compounds were established based on 1D and 2D NMR spectral analysis, and in the case of compounds 1, 2a, 4b, 5, 6a and 7, the absolute configurations of their stereogenic carbons were determined by an X-ray crystallographic analysis. None of the isolated compounds were active in the tests for antibacterial activity against Gram-positive and Gram-negative bacteria, as well as multidrug-resistant isolates from the environment (MIC > 256 µg/mL), antifungal activity against yeast (Candida albicans ATTC 10231), filamentous fungus (Aspergillus fumigatus ATTC 46645) and dermatophyte (Trichophyton rubrum FF5) (MIC > 512 µg/mL) and in vitro growth inhibitory activity against the MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma) cell lines (GI50 > 150 µM) by the protein binding dye SRB method.

Cytotoxic activity of Secondary Metabolites from Marine-derived Fungus Neosartorya siamensis in Human Cancer Cells.

Compounds isolated from the marine sea fan-derived fungus Neosartorya siamensis (KUFA 0017), namely, 2,4-dihydroxy-3-methylacetophenon (1), chevalone C (2), nortryptoquivaline (4), tryptoquivaline H (6), tryptoquivaline F (7), fiscalin A (8), epi-fiscalin A (9), epi-neofiscalin A (11) and epi-fiscalin C (13) were tested for anti-proliferative activity by MTT assay, DNA damage induction by comet assay, and induction of cell death by nuclear condensation assay on colon HCT116, liver HepG2 and melanoma A375 cancer cell lines. Compounds 2, 4, 8, 9, 11 and 13 presented IC50 values ranging from 24 to 153 µM in the selected cell lines. Cell death was induced in HCT116 by compounds 2, 4 and 8. In HepG2, compounds 4, 8, 9 and 11 were able to induce significant cell death. This induction of cell death is possibly not related to genotoxicity because none of the compounds induced significant DNA damage. These results suggest that selected compounds present an interesting anti-proliferative activity and cell death induction, consequently showing potential (specifically epi-fiscalin C) as future leads for chemotherapeutic agents. Further studies on mechanisms of action should ensue. Copyright © 2016 John Wiley & Sons, Ltd.

Avenaciolides: potential MurA-targeted inhibitors against peptidoglycan biosynthesis in methicillin-resistant Staphylococcus aureus (MRSA).

Discovery of new antibiotics for combating methicillin-resistant Staphylococcus aureus (MRSA) is of vital importance in the post-antibiotic era. Here, we report four avenaciolide derivatives (1-4) isolated from Neosartorya fischeri, three of which had significant antimicrobial activity against MRSA. The morphology of avenaciolide-treated cells was protoplast-like, which indicated that cell wall biosynthesis was interrupted. Comparing the structures and minimum inhibitory concentrations of 1-4, the α,ß-unsaturated carbonyl group seems to be an indispensable moiety for antimicrobial activity. Based on a structural similarity survey of other inhibitors with the same moiety, we revealed that MurA was the drug target. This conclusion was validated by (31)P NMR spectroscopy and MS/MS analysis. Although fosfomycin, which is the only clinically used MurA-targeted antibiotic, is ineffective for treating bacteria harboring the catalytically important Cys-to-Asp mutation, avenaciolides 1 and 2 inhibited not only wild-type but also fosfomycin-resistant MurA in an unprecedented way. Molecular simulation revealed that 2 competitively perturbs the formation of the tetrahedral intermediate in MurA. Our findings demonstrated that 2 is a potent inhibitor of MRSA and fosfomycin-resistant MurA, laying the foundation for the development of new scaffolds for MurA-targeted antibiotics.