Cyclopiazonic Acid and Okaramine Analogues, Including Chlorinated Compounds, from Chrysosporium undulatum YT-1.

Eight new cyclopiazonic acid (1-8) and five new okaramine (9-13) alkaloids together with 13 known compounds were isolated from the fungus Chrysosporium undulatum YT-1. Compounds 2, 4, 5, 7, 10, 11, and 13 were chlorinated indole alkaloids. The structures of compounds 1-13 were elucidated by HRESIMS and NMR spectroscopic data. Their relative and absolute configurations were established by J-based configuration analysis, NOESY, NOEDIFF experiments, ECD spectroscopic data, and biogenetic considerations. Compound 4 inhibited the growth of Bacillus subtilis with an MIC value of 6.3 µg/mL. Compounds 9-11 exhibited strong insecticidal capacity against the third instar larvae of silkworm and cotton bollworm (LD50: ≤7.56 µg/g). At 40 µM, compound 1 showed obvious neuroprotection to the PC12 cells with 6-OHDA treatment.

Neochrysosporazines: Precursor-Directed Biosynthesis Defines a Marine-Derived Fungal Natural Product P-Glycoprotein Inhibitory Pharmacophore.

Upregulation of ATP binding cassette (ABC) transporter efflux pumps (i.e. P-glycoprotein, P-gp) can impart multidrug resistance, rendering many chemotherapeutics ineffective and seriously limiting treatment regimes. While ABC transporters remain an attractive target for therapeutic intervention, the development of clinically useful small-molecule inhibitors has proved challenging. In this report, we describe the structure-activity relationship (SAR) analysis of a newly discovered P-gp inhibitory pharmacophore, phenylpropanoid piperazine chrysosporazines, produced by co-isolated marine-derived fungi. In the absence of any total syntheses, we apply an innovative precursor-directed biosynthesis strategy that successfully repurposed fungal biosynthetic output, allowing us to isolate, characterize, and identify the structurally diverse neochrysosporazines A-Q. SAR analysis utilizing all known (and new) neochrysosporazines, chrysosporazines, and azachrysosporazines, plus semi-synthetic analogues, established the key structure requirements for the P-gp inhibitory pharmacophore, and, in addition, identified non-essential sites that allow for the design of affinity and other conjugated probes.

Chrysosporazines F-M: P-Glycoprotein Inhibitory Phenylpropanoid Piperazines from an Australian Marine Fish Derived Fungus, Chrysosporium sp. CMB-F294.

Chemical analysis of the fungus Chrysosporium sp. CMB-F294 isolated from the gastrointestinal tract of a market-purchased specimen of Mugil mullet yielded eight new alkaloids, belonging to a rare class of phenylpropanoid piperazines. Chrysosporazines F-M (1-8) occur as an equilibrium mixture of acetamide rotamers and feature unprecedented carbocyclic and heterocyclic scaffolds. Structures inclusive of absolute configuration were assigned by detailed spectroscopic analysis, supported by biosynthetic considerations. Structure-activity relationship studies determined that selected chrysosporazines were promising noncytotoxic inhibitors of the multidrug resistance efflux pump P-glycoprotein (P-gp), capable of reversing doxorubicin resistance in P-gp-overexpressing human colon carcinoma cells (SW620 Ad300). Chrysosporazine F (1) was particularly noteworthy, with a 2.5 µM cotreatment inducing a doxorubicin gain in sensitivity (GS 14) > 2-fold that of the positive control verapamil (GS 6.1).

Identification and characterization of compounds from Chrysosporium multifidum, a fungus with moderate antimicrobial activity isolated from Hermetia illucens gut microbiota.

The gut microbiota of insects is composed of a wide range of microorganisms which produce bioactive compounds that protect their host from pathogenic attack. In the present study, we isolate and identify the fungus Chrysosporium multifidum from the gut of Hermetia illucens larvae. Extract from C. multifidum culture broth supernatant showed moderate activity against a strain of methicillin-resistant Staphylococcus aureus (MRSA). Bioguided isolation of the extract resulted in the characterization of six α-pyrone derivatives (1-6) and one diketopiperazine (7). Of these compounds, 5,6-dihydro-4-methoxy-6-(1-oxopentyl)-2H-pyran-2-one (4) showed the greatest activity (IC50 = 11.4 ± 0.7 µg/mL and MIC = 62.5 µg/mL) against MRSA.

Chrysosporazines A-E: P-Glycoprotein Inhibitory Piperazines from an Australian Marine Fish Gastrointestinal Tract-Derived Fungus, Chrysosporium sp. CMB-F214.

Chemical analysis of Chrysosporium sp. CMB-F214, yielded five new piperazines, chrysosporazines A-E (1-5), with structures assigned by spectroscopic and X-ray analyses and biosynthetic considerations. The chrysosporazines 2-5 exist as an equilibrium of major and minor N-acyl rotamers, while 1-3 incorporate an unprecedented hexahydro-6H-pyrazino[1,2-b]isoquinolin-6-one scaffold. The noncytotoxic chrysosporazines reverse doxorubicin drug resistance in P-glycoprotein overexpressing colon carcinoma cells (SW620 Ad300), with 2 delivering a comparable gain in sensitivity to the positive control, verapamil.

Isolation, optimization, and partial purification of amylase from Chrysosporium asperatum by submerged fermentation.

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at 30 degrees C and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at 50 degrees C for 20 min, and no inhibitory effect of Ca+2 ions on amylase production was observed.

Microbial mannosidation of bioactive chlorogentisyl alcohol by the marine-derived fungus Chrysosporium synchronum.

The biological transformation of the biologically active chlorogentisyl alcohol (1), isolated from the marine-derived fungus Aspergillus sp., was studied. Preparative-scale fermentation of chlorogentisyl alcohol with marine-derived fungus Chrysosporium synchronum resulted in the isolation of a new glycosidic metabolite, 1-O-(α-D-mannopyranosyl)chlorogentisyl alcohol (2). The stereostructure of the new metabolite obtained was assigned on the basis of detailed spectroscopic data analyses, chemical reaction, and chemical synthesis. Compounds 1 and 2 exhibited significant radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) with IC(50) values of 1.0 and 4.7 µM, respectively. The compounds 1 and 2 were more active than the positive control, L-ascorbic acid (IC(50), 20.0 µM).

Efficacy of Chrysosporium tropicum metabolite against mixed population of adult mosquito (Culex quinquefasciatus, Anopheles stephensii, and Aedes aegypti) after purification with flash chromatography.

Chrysosporium tropicum is a keratinophilic fungus and an effective mosquito control agent. This fungus was grown in Richards broth at 27 +/- 2 degrees C and a relative humidity of 75% +/- 5% for 15 +/- 2 days. Filtration was done with Whatman number 1 filter paper, column chromatography, and flash chromatography. Adulticidal efficacy was performed against a mixed population of mosquitoes including Culex quinquefasciatus, Anophelese stephensii, Aedes aegypti at five different concentrations 5:5, 6:4, 7:3, 8:2, 9:1 by adding fungal filtrate of flash chromatography to methanol in different ratio (metabolite/methanol). The experiment was conducted in the cage with an area of 2 x 2 x 3 ft. The mortality in mosquito population was recorded after 8 hours of exposure, and adulticidal activity was tested by probit analysis. The LC(50) was determined to be 4.9921 ml. Results of present study confirm that metabolites of C. tropicum can be utilized as alternative biological control agents for adult mosquitoes.

Caryophyllenes from a fungal culture of Chrysosporium pilosum.

Four new caryophyllene derivatives, Sch 725432 (1), Sch 601253 (2), Sch 601254 (3), and Sch 725434 (4), were isolated from the fungal fermentation broth of Chrysosporium pilosum by reversed-phase HPLC purification. The structure elucidation of trioxygenated caryophyllenes 1-4 was accomplished on the basis of spectroscopic data interpretation. Sch 725434 (4) possesses a dihydrofuran-3-one ring, forming a tricyclic ring skeleton, which represents an unprecedented ring skeleton for the caryophyllene-type of sesquiterpenes. Compounds 1-4 were evaluated for their antifungal activity.

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) were used to identify iron-responsive proteins in the white-rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron-sufficient and iron-deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS-PAGE for P. chrysosporium and twelve for L. edodes. In 2-DE, the numbers of iron-restricted induced proteins were 12 and 9, respectively, in a resolution range of 15-60 kDa and pI 4.5-8.1. SDS-PAGE for the plasma membrane protein did not show differences, whereas the outer-membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron-complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.

[A fungus-derived novel nucleoside transport inhibitor potentiates the activity of antitumor drugs].

Antibiotic C3368-B (CB), identified as 3,9-dihydroxy-1-methoxy-7-methylanthraquinone, is produced by a fungus strain, Chrysosporium verrucosum Tubaki, isolated from a soil sample collected from Antarctica. CB was found to be a highly-active nucleoside transport inhibitor. By radiolabelled nucleoside assay, CB was shown to markedly inhibit thymidine and uridine transport in Ehrlich carcinoma cells, with IC50 values of 7.5 and 9.6 mumol.L-1 respectively. CB showed fairly low cytotoxicity to tumor cells. The IC50 values for epidermoid cancer KB cells and hepatoma BEL-7402 cells in clonogenic assay was 77 and 69 mumol.L-1. At relatively noncytotoxic concentrations, CB markedly enhanced the cytotoxicity of methotrexate, 5-fluorouracil, mitomycin C against KB cells and BEL-7402 cells. CB was also found to partly reverse the multi-drug resistance to vincristine and actinomycin D in leukemia L1210/MDR cells. The IC50 values were reduced by 4.9-fold (1.75 to 0.36 mumol.L-1) for vincristine and 3.3-fold (0.39 to 0.12 mumol.L-1) for actinomycin D. These results suggest that CB, as a newly-found nucleoside transport inhibitor, may be potentially useful in cancer chemotherapy.