Novel Metabolites from the Marine-Derived Fungus Peniophora sp. SCSIO41203 Show Promising In Vitro Antitumor Activity as Methuosis Inducers in PC-3 Cells.

Two new cytochalasin derivatives, peniotrinins A (1) and B (2), three new citrinin derivatives, peniotrinins C-E (4, 5, 7), and one new tetramic acid derivative, peniotrinin F (12), along with nine structurally related known compounds, were isolated from the solid culture of Peniophora sp. SCSIO41203. Their structures, including the absolute configurations of their stereogenic carbons, were fully elucidated based on spectroscopic analysis, quantum chemical calculations, and the calculated ECD. Interestingly, 1 is the first example of a rare 6/5/5/5/6/13 hexacyclic cytochalasin. We screened the above compounds for their anti-prostate cancer activity and found that compound 3 had a significant anti-prostate cancer cell proliferation effect, while compounds 1 and 2 showed weak activity at 10 µM. We then confirmed that compound 3 exerts its anti-prostate cancer effect by inducing methuosis through transmission electron microscopy and cellular immunostaining, which suggested that compound 3 might be first reported as a potential anti-prostate methuosis inducer.

Immunosuppressive Cytochalasins from the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48.

Three new cytochalasins, phomoparagins A-C (1-3), along with five known analogs (4-8), were isolated from Phomopsis asparagi DHS-48, a mangrove-derived endophytic fungus. Their structures, including their absolute configurations, were elucidated using a combination of detailed HRESIMS, NMR, and ECD techniques. Notably, 1 possessed an unprecedented 5/6/5/8/5-fused pentacyclic skeleton. These compounds were tested for their inhibitory activity against concanavalin A (ConA)/lipopolysaccharide (LPS)-induced spleen lymphocyte proliferation and calcineurin (CN) enzyme. Several metabolites (2 and 4-6) exhibited fascinating inhibitory activities with a relatively low toxicity. Furthermore, 2 was demonstrated to inhibit ConA-stimulated activation of NFAT1 dephosphorylation and block NFAT1 translocation in vitro, subsequently inhibiting the transcription of interleukin-2 (IL-2). Our results provide evidence that 2 may, at least partially, suppress the activation of spleen lymphocytes via the CN/NFAT signaling pathway, highlighting that it could serve as an effective immunosuppressant that is noncytotoxic and natural.

Epigenetic Manipulation Induced Production of Immunosuppressive Chromones and Cytochalasins from the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48.

A mangrove endophytic fungus Phomopsis asparagi DHS-48 was found to be particularly productive with regard to the accumulation of substantial new compounds in our previous study. In order to explore its potential to produce more unobserved secondary metabolites, epigenetic manipulation was used on this fungus to activate cryptic or silent genes by using the histone deacetylase (HDAC) inhibitor sodium butyrate and the DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-Aza). Based on colony growth, dry biomass, HPLC, and 1H NMR analyses, the fungal chemical diversity profile was significantly changed compared with the control. Two new compounds, named phaseolorin J (1) and phomoparagin D (5), along with three known chromones (2-4) and six known cytochalasins (6-11), were isolated from the culture treated with sodium butyrate. Their structures, including their absolute configurations, were elucidated using a combination of detailed HRESIMS, NMR, and ECD and 13C NMR calculations. The immunosuppressive and cytotoxic activities of all isolated compounds were evaluated. Compounds 1 and 8 moderately inhibited the proliferation of ConA (concanavalin A)-induced T and LPS (lipopolysaccharide)-induced B murine spleen lymphocytes. Compound 5 exhibited significant in vitro cytotoxicity against the tested human cancer cell lines Hela and HepG2, which was comparative to the positive control adriamycin and fluorouracil. Our finding demonstrated that epigenetic manipulation should be an efficient strategy for the induction of new metabolites from mangrove endophytic fungi.

Pentacyclic Cytochalasins and Their Derivatives from the Endophytic Fungus Phomopsis sp. xz-18.

Eight new cytochalasins 1-8 and ten known analogs 9-18 were isolated from the endophytic fungus Phomopsis sp. xz-18. The planar structures of the cytochalasins were determined by HR-ESI-MS and NMR analysis. Compounds 1, 2, 9 and 10 were 5/6/6/7/5-fused pentacyclic cytochalasins; compounds 3 and 4 had conjugated diene structures in the macrocycle; and compound 6 had a ß,γ-unsaturated ketone. The absolute configuration of 6 was confirmed for the first time by the octant rule. The acid-free purification process proved that the pentacyclic system was a natural biosynthetic product and not an acid-mediated intramolecular cyclized artifact. The new compounds did not exhibit activities against human cancer cell lines in cytotoxicity bioassays or antipathogenic fungal activity, but compounds 1, 3 and 4 showed moderate antibacterial activity in disk diffusion assays.

Zopfiellasins A-D, Two Pairs of Epimeric Cytochalasins from Kiwi-Associated Fungus Zopfiella sp. and Their Antibacterial Assessment.

In our continuous search for antibacterial agents against Pseudomonas syringae pv. actinidiae (Psa) from kiwi-associated fungi, two pairs of epimeric cytochalasins, zopfiellasins A-D (1-4), were characterized from the fungus Zopfiella sp. The structures were established on the basis of spectroscopic data analysis, while the absolute configurations were determined by single-crystal X-ray diffraction. Compounds 1 and 3 exhibited antibacterial activity against Psa with MIC values of 25 and 50 µg/mL, respectively. This is the first report of anti-Psa activity of cytochalasin derivatives.

Diversely Functionalised Cytochalasins through Mutasynthesis and Semi-Synthesis.

Mutasynthesis of pyrichalasin H from Magnaporthe grisea NI980 yielded a series of unprecedented 4'-substituted cytochalasin analogues in titres as high as the wild-type system (≈60 mg L-1 ). Halogenated, O-alkyl, O-allyl and O-propargyl examples were formed, as well as a 4'-azido analogue. 4'-O-Propargyl and 4'-azido analogues reacted smoothly in Huisgen cycloaddition reactions, whereas p-Br and p-I compounds reacted in Pd-catalysed cross-coupling reactions. A series of examples of biotin-linked, dye-linked and dimeric cytochalasins was rapidly created. In vitro and in vivo bioassays of these compounds showed that the 4'-halogenated and azido derivatives retained their cytotoxicity and antifungal activities; but a unique 4'-amino analogue was inactive. Attachment of larger substituents attenuated the bioactivities. In vivo actin-binding studies with adherent mammalian cells showed that actin remains the likely intracellular target. Dye-linked compounds revealed visualisation of intracellular actin structures even in the absence of phalloidin, thus constituting a potential new class of actin-visualisation tools with filament-barbed end-binding specificity.

Cytochalasins from an endophytic fungus Phoma multirostrata XJ-2-1 with cell cycle arrest and TRAIL-resistance-overcoming activities.

Nine new (1-9) and four known (10-13) [13]cytochalasins, along with three known 24-oxa[14]cytochalasins (14-16), were isolated from the culture of Phoma multirostrata XJ-2-1, an endophytic fungus obtained from the fibrous root of Parasenecio albus. Their structures were elucidated by interpretation of the nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS). The absolute configurations were assigned by single-crystal X-ray crystallography, modified Mosher's method, and by analysis of their experimental electronic circular dichroism (ECD) spectra. Compound 6 could induce cell cycle arrest at G2-phase in CT26 and A549 cells, and displayed moderate cytotoxicity against CT26 and A549 cell lines with IC50 values of 6.03 and 5.04 µM, respectively. Co-treatment of 7-9, 13 and 16 with tumor necrosis factor related apoptosis inducing ligand (TRAIL) could significantly decrease the cell viability of A549, which revealed that cytochalasins could possibly be a new group of TRAIL sensitizers in lung cancer therapy.

Antiplasmodial and Cytotoxic Cytochalasins from an Endophytic Fungus, Nemania sp. UM10M, Isolated from a Diseased Torreya taxifolia Leaf.

Bioassay-guided fractionation of an EtOAc extract of the broth of the endophytic fungus Nemania sp. UM10M (Xylariaceae) isolated from a diseased Torreya taxifolia leaf afforded three known cytochalasins, 19,20-epoxycytochalasins C (1) and D (2), and 18-deoxy-19,20-epoxy-cytochalasin C (3). All three compounds showed potent in vitro antiplasmodial activity and phytotoxicity with no cytotoxicity to Vero cells. These compounds exhibited moderate to weak cytotoxicity to some of the cell lines of a panel of solid tumor (SK-MEL, KB, BT-549, and SK-OV-3) and kidney epithelial cells (LLC-PK11). Evaluation of in vivo antimalarial activity of 19,20-epoxycytochalasin C (1) in a mouse model at 100 mg/kg dose showed that this compound had weak suppressive antiplasmodial activity and was toxic to animals.

Antifungal activities of cytochalasins produced by Diaporthe miriciae, an endophytic fungus associated with tropical medicinal plants.

In the present study, we evaluated the antifungal potential of cytochalasins produced by Diaporthe taxa against phytopathogenic fungi. Using molecular methods, seven endophytic fungal strains from the medicinal plants Copaifera pubiflora and Melocactus ernestii were identified as Diaporthe miriciae, while two isolates were identified to the genus level (Diaporthe sp.). All crude extracts of Diaporthe species produced via solid-state fermentation were evaluated by 1H NMR analyses. Crude extracts of the isolates D. miriciae UFMGCB 6350, 7719, 7646, 7653, 7701, 7772, and 7770 and Diaporthe sp. UFMGCB 7696 and 7720 were demonstrated to produce highly functionalized compounds. The extracts of D. miriciae UFMGCB 7719 and 6350 were selected as representative Diaporthe samples and subjected to bioassay-directed fractionation to isolate cytochalasins H and J. Cytochalasins H and J were evaluated for activities against the fungal plant pathogens Colletotrichum fragariae, Colletotrichum gloeosporioides, Colletotrichum acutatum, Botrytis cinerea, Fusarium oxysporum, Phomopsis obscurans, and Phomopsis viticola using microdilution broth assays. Cytochalasins H and J exhibited the most potent activities against the Phomopsis species tested. Our results showed that Diaporthe species were potential producers of different cytochalasins, which exhibit potential for controlling fungal diseases in planta and (or) maintaining antagonism.

Antibacterial and cytotoxic cytochalasins from the endophytic fungus Phomopsis sp. harbored in Garcinia kola (Heckel) nut.

BACKGROUND: The continuous emergence of multidrug-resistant (MDR) bacteria drastically reduced the efficacy of our antibiotic armory and consequently, increased the frequency of therapeutic failure. The search for bioactive constituents from endophytic fungi against MDR bacteria became a necessity for alternative and promising strategies, and for the development of novel therapeutic solutions. We report here the isolation and structure elucidation of antibacterial and cytotoxic compounds from Phomopsis sp., an endophytic fungus associated with Garcinia kola nuts. METHODS: The fungus Phomopsis sp. was isolated from the nut of Garcinia kola. The crude extract was prepared from mycelium of Phomopsis sp. by maceration in ethyl acetate and sequentially fractionated by column chromatography. The structures of isolated compounds were elucidated on the basis of spectral studies and comparison with published data. The isolated compounds were evaluated for their antibacterial and anticancer properties by broth microdilution and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide methods respectively. The samples were also tested spectrophotometrically for their hemolytic properties against human red blood cells. RESULTS: The fractionation of the crude extract afforded three known cytochalasins including 18-metoxycytochalasin J (1), cytochalasins H (2) and J (3) together with alternariol (4). The cytochalasin compounds showed different degrees of antibacterial activities against the tested bacterial pathogens. Shigella flexneri was the most sensitive microorganism while Vibrio cholerae SG24 and Vibrio cholerae PC2 were the most resistant. Ampicillin did not show any antibacterial activity against Vibrio cholerae NB2, Vibrio cholerae PC2 and Shigella flexneri at concentrations up to 512 µg/mL, but interestingly, these multi-drug resistant bacterial strains were sensitive to the cytochalasin metabolites. These compounds also showed significant cytotoxic properties against human cancer cells (LC50 = 3.66-35.69 µg/mL) with low toxicity to normal non-cancer cells. CONCLUSION: The three cytochalasin compounds isolated from the Phomopsis sp. crude extract could be a clinically useful alternative for the treatment of cervical cancer and severe infections caused by MDR Shigella and Vibrio cholerae.

Bioactive tyrosine-derived cytochalasins from fungus Eutypella sp. D-1.

Three new cytochalasins Z24 , Z25 , Z26 (1-3, resp.) and one known compound, scoparasin B (4), were isolated from the fungus Eutypella sp. D-1 isolated from the soil of high latitude of the Arctic. The structures of 1-3 were elucidated from spectroscopic data (NMR, MS). These compounds were evaluated for cytotoxic activities against several human tumor cell lines. Among them, compound 1 exhibited moderate cytotoxicity toward human breast cancer MCF-7 cell line with IC50 of 9.33 µM.

Rosellichalasins A-H, cytotoxic cytochalasans from the endophytic fungus Rosellinia sp. Glinf021.

Eight new cytochalasans rosellichalasins A-H (1-8), as well as two new shunt metabolites rosellinins A (9) and B (10) before intramolecular Diels-Alder cycloaddition reaction in cytochalasan biosynthesis, along with nine known cytochalsans (11-19) were isolated from the endophytic fungus Rosellinia sp. Glinf021, which was derived from the medicinal plant Glycyrrhiza inflata. Their structures were characterized by extensive analysis of 1D and 2D NMR as well as HRESIMS spectra and quantum chemical ECD calculations. The cytotoxic activities of these compounds were evaluated against four human cancer cell lines including HCT116, MDA-MB-231, BGC823, and PANC-1 with IC50 values ranging from 0.5 to 58.2 µM.

Cytochalasins as Modulators of Stem Cell Differentiation.

Regenerative medicine aims to identify new research strategies for the repair and restoration of tissues damaged by pathological or accidental events. Mesenchymal stem cells (MSCs) play a key role in regenerative medicine approaches due to their specific properties, such as the high rate of proliferation, the ability to differentiate into several cell lineages, the immunomodulatory potential, and their easy isolation with minimal ethical issues. One of the main goals of regenerative medicine is to modulate, both in vitro and in vivo, the differentiation potential of MSCs to improve their use in the repair of damaged tissues. Over the years, much evidence has been collected about the ability of cytochalasins, a large family of 60 metabolites isolated mainly from fungi, to modulate multiple properties of stem cells (SCs), such as proliferation, migration, and differentiation, by altering the organization of the cyto- and the nucleo-skeleton. In this review, we discussed the ability of two different cytochalasins, cytochalasins D and B, to influence specific SC differentiation programs modulated by several agents (chemical or physical) or intra- and extra-cellular factors, with particular attention to human MSCs (hMSCs).

Unreported cytochalasins from an acid-mediated transformation of cytochalasin J isolated from Diaporthe cf. ueckeri.

Chemical investigation of an endophytic fungus herein identified as Diaporthe cf. ueckeri yielded four known compounds, named cytochalasins H and J and dicerandrols A and B. Reports of acid sensitivity within the cytochalasan family inspired an attempt of acid-mediated conversion of cytochalasins H and J, resulting in the acquisition of five polycyclic cytochalasins featuring 5/6/5/8-fused tetracyclic and 5/6/6/7/5-fused pentacyclic skeletons. Two of the obtained polycyclic cytochalasins constituted unprecedented analogues, for which the trivial names cytochalasins J4 and J5 were proposed, whereas the others were identified as the known phomopchalasin A, phomopchalasin D and 21-acetoxycytochalasin J3. The structures of the compounds were determined by extensive spectral analysis, namely HR-ESIMS, ESIMS and 1D/2D NMR. The stereochemistry of cytochalasins J4 and J5 was proposed using their ROESY data, biosynthetic and mechanistic considerations and by comparison of their ECD spectra with those of related congeners. All compounds except for cytochalasins H and J were tested for antimicrobial and cytotoxic activity. Cytochalasins J4 and J5 showed neither antimicrobial nor cytotoxic activity in the tested concentrations, with only weak antiproliferative activity observable against KB3.1 cells. The actin disruptive properties of all cytochalasins obtained in this study and of the previously reported cytochalasins RKS-1778 and phomopchalasin N were examined, and monitored by fluorescence microscopy using human osteo-sarcoma (U2-OS) cells. Compared to their precursor molecules (cytochalasins H and J), phomopchalasins A and D, 21-acetoxycytochalasin J3, cytochalasins J4 and J5 revealed a strongly reduced activity on the F-actin network, highlighting that the macrocyclic ring is crucial for bioactivity.

Six Unprecedented Cytochalasin Derivatives from the Potato Endophytic Fungus Xylaria curta E10 and Their Cytotoxicity.

Six previously undescribed cytochalasins, Curtachalasins X1-X6 (1-6), together with six known compounds (7-12) were isolated from the endophytic fungus Xylaria curta E10 harbored in the plant Solanum tuberosum. The structures were elucidated by the interpretation of HRESIMS, UV, and NMR data. The absolute configurations of Curtachalasins X1-X6 were determined by comparison of their experimental and calculated electronic circular dichroism (ECD) spectra. In bioassays, Curtachalasin X1 (1) and X5 (5) showed cytotoxic activity against the MCF-7 cell line with IC50 values of 2.03 µM and 0.85 µM, respectively.

Two new cytochalasins from the endophytic fungus Xylaria sp. GDGJ-77B.

Two new open-chain cytochalasins, xylarchalasins A and B (1 and 2), together with six known analogues (3-8), were isolated from the endophytic fungus Xylaria sp. GDGJ-77B from the Chinese medicinal plant Sophora tonkinensis. Their structures were elucidated on the basis of comprehensive spectroscopic analysis. Compound 2 displayed moderate antibacterial activities against Bacillus subtilis and Escherichia coli with MIC values of 25 and 12.5 µg/mL, respectively.

Influencing the Actin Dynamics in Plant Cells by Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latrunculins.

This chapter presents an overview of the most common F-actin influencing substances, used to study actin dynamics in living plant cells for studies on morphogenesis, motility, organelle movement, apoptosis, or abiotic stress. These substances can be divided into two major subclasses-F-actin-stabilizing and F-actin-polymerizing substances like jasplakinolide and chondramides and F-actin-severing compounds like cytochalasins and latrunculins. Jasplakinolide, which may have anti-cancer activities, was originally isolated from a marine sponge and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane-permeable F-actin-stabilizing and F-actin-polymerizing agent. Recently an acyclic derivate of jasplakinolide was isolated. Cytochalasins, derived from fungi, show an F-actin-severing function, and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from Red Sea sponges; however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide, isolated from red algae or the cyanobacterium Nostoc, a stable complex with actin dimers is formed, resulting in severing F-actin.For influencing F-actin dynamics in plant cells, only membrane permeable drugs are useful in a broad range. We, however, introduce also the phallotoxins and synthetic derivatives thereof, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes but has also been described in siphonal giant algae. The focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; moreover fluorescence methods for phalloidin- and antibody staining as well as techniques for immunoelectron microscopy are explained.

New curtachalasin from the Xylaria sp. DO 1801.

One new cytochalasin, named curtachalasin Q (1), together with 8 known cytochalasins were isolated from the ethyl acetate extract of the Xylaria sp. DO1801. The structure of the new compound was elucidated on the basis of IR, UV, HR-ESI-MS, NMR spectra and X-ray diffraction. Compound 1 and 2 were not cytotoxic (IC50>50uM) against four tumour cell lines (SW-1990, ASPC-1, HepG2 and Hep3B).

Identification of the Antifungal Metabolite Chaetoglobosin P From Discosia rubi Using a Cryptococcus neoformans Inhibition Assay: Insights Into Mode of Action and Biosynthesis.

Cryptococcus neoformans is an important human pathogen with limited options for treatments. We have interrogated extracts from fungal fermentations to find Cryptococcus-inhibiting natural products using assays for growth inhibition, differential thermosensitivity, and synergy with existing antifungal drugs. Extracts from fermentations of strains of Discosia rubi from eastern Texas showed anticryptococcal bioactivity with preferential activity in agar zone of inhibition assays against C. neoformans at 37°C versus 25°C. Assay-guided fractionation led to the purification and identification of chaetoglobosin P as the active component of these extracts. Genome sequencing of these strains revealed a biosynthetic gene cluster consistent with chaetoglobosin biosynthesis and ß-methylation of the tryptophan residue. Proximity of genes of the actin-binding protein twinfilin-1 to the chaetoglobosin P and K gene clusters suggested a possible self-resistance mechanism involving twinfilin-1 which is consistent with the predicted mechanism of action involving interference with the polymerization of the capping process of filamentous actin. A C. neoformans mutant lacking twinfilin-1 was hypersensitive to chaetoglobosin P. Chaetoglobosins also potentiated the effects of amphotericin B and caspofungin on C. neoformans.

Cytotoxic seco-cytochalasins from an endophytic Aspergillus sp. harbored in Pinellia ternata tubers.

Six new seco-cytochalasins A-F (1-6), two new asperlactones G-H (7-8) along with three known cytochalasins (9-11) were isolated from the solid cultures of an endophytic fungus Aspergillus sp. Their structures were elucidated by comprehensive spectral analysis, and their absolute configurations were determined through Mo2(OCOCH3)4-induced electronic circular dichroism (ECD) spectra and Rh2(OCOCF3)4-induced ECD experiment. Compounds 5 and 6 were rare seco-cytochalasins possessing an α, ß-unsaturated furanone structure in their side-chains. These isolates exhibited cytotoxicity against human lung cancer A-549 cell line with IC50 values ranging from 7.8 to 70.2 µM. At the concentration of 16 µM, compound 4 also exerted a 3-fold enhancement of doxorubicin susceptibility on doxorubicin-resistant human breast cancer (MCF-7/DOX) cell line.