Influence of the fungal NF-kappaB inhibitor panepoxydone on inflammatory gene expression in MonoMac6 cells.

The fungal secondary metabolite panepoxydone has been recently described as an inhibitor of NF-kappaB activation which is a pivotal regulator of the inflammatory and immune response. These findings have led to propose that panepoxydone may be useful as anti-inflammatory agent. In this study we investigated for the first time the effects of panepoxydone on inflammatory gene expression in the monocytic cell line MonoMac6, stimulated with lipopolysaccharide (LPS) and the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA). DNA microarray analysis of 110 human genes known to be strongly regulated during inflammation, combined with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) revealed that low micromolar concentrations (12-24 microM) of panepoxydone strongly inhibited the expression of thirty-three NF-kappaB dependent pro-inflammatory genes such as the chemokines CCL3, CCL4, CCL8; CXCL8, CXCL10, CXCL20, the cytokines IL-1, IL-6, TNF-alpha, pro-inflammatory enzymes like COX-2, and components of the REL/NF-kappaB/IkappaB family without significant effects on the expression of house-keeping genes. Panepoxydone strongly inhibited hTNF-alpha, IL-8 and NF-kappaB promoter activity in LPS/TPA stimulated MonoMac6 cells with IC(50) values of 0.5-1 microg/ml by blocking the phosphorylation of IkappaB and subsequent binding of the activated NF-kappaB transcription factor to the DNA. From our data, panepoxydone may serve as lead structure for the development of transcription-based inhibitors of pro-inflammatory gene expression.

Inhibition of interleukin-6 signaling by galiellalactone.

A search for inhibitors of the IL-6-mediated signal transduction in HepG2 cells using secreted alkaline phosphatase (SEAP) as reporter gene resulted in the isolation of galiellalactone (1) from fermentations of the ascomycete strain A111-95. Galiellalactone inhibits the IL-6-induced SEAP expression with IC(50) values of 250-500 nM by blocking the binding of the activated Stat3 dimers to their DNA binding sites without inhibiting the tyrosine and serine phosphorylation of the Stat3 transcription factor. Due to its selective activity, galiellalactone may serve as a lead compound for the development of new therapeutic agents for diseases originating from the inappropriate expression of IL-6 and as molecular tool to dissect the JAK/STAT pathways.

Inhibition of interleukin-6 signaling and Stat3 activation by a new class of bioactive cyclopentenone derivatives.

The IL-6-dependent activation of the JAK/STAT pathway plays a central role in the induction of the acute phase response in the liver. In a search for new inhibitors of the IL-6-mediated signal transduction in HepG2 cells using secreted alkaline phosphatase (SEAP) as reporter gene, four novel cyclopentenones, 2-(1-chloropropenyl)-4,5-dihydroxycyclopent-2-enone (CPDHC, 1), 4, 5-dihydroxy-2-propenylcyclopent-2-enone (DHPC, 2), 5-hydroxy-2, 3-dimethylcyclopent-2-enone (HDC, 3), and 4-methyl-5-methylenecyclopent-3-en-1,2-diol (MMCD, 4) were isolated from fermentations of the ascomycete strain A23-98. CPDHC (1) inhibits the IL-6-induced SEAP expression with IC(50) values of 4. 0-5.3 microM (0.75-1 microg/ml). The compounds DHPC (2), HDC (3), and MMCD (4) which are structurally closely related to CPDHC (1) showed no inhibitory effects on the IL-6-induced SEAP expression in HepG2 cells. Studies on the mode of action revealed that CPDHC (1) affects the IL-6-dependent pathway by inhibiting the tyrosine phosphorylation of the STAT3 and STAT1 as well as the serine phosphorylation of the Stat3 transcription factor. In addition, CPDHC (1) and DHPC (2) inhibit the AP-1 and NF-kappaB mediated SEAP expression in transiently transfected HeLa S3 cells with IC(50) values of 10-15 microM (2-3 microg/ml) and 50-100 microM (8-16 microg/ml) respectively. Our results indicate that CPDHC inhibits the NF-kappaB pathway by preventing the phosphorylation and proteolytic degradation of the IkappaBalpha protein. The novel cyclopentenones may represent lead compounds for the development of new anti-inflammatory drugs.

Trichodion, a new inhibitor of inflammatory signal transduction pathways from a Trichosporiella species.

In a search for new inhibitors of the IFN-gamma mediated signal transduction in HeLa S3 cells using secreted alkaline phosphatase (SEAP) as reporter gene, the novel pyran-dione trichodion was isolated from fermentations of the imperfect fungus Trichosporiella sp. 20-95. The compound inhibits the IFN-gamma mediated expression of the reporter gene with IC(50) values of 21-42 microM (5-10 microgram/ml). The NF-kappaB and AP-1 mediated expression of the reporter gene are inhibited with IC(50) values of 42-84 microM (10-20 microgram/ml) and 21 microM (5 microgram/ml) respectively. Western blotting with COX-2 and NOS II antibodies showed that the expression of both proinflammatory enzymes is almost completely inhibited at 21-42 microM (5-10 microgram/ml) in LPS/IFN-gamma stimulated J774 mouse macrophages. Studies on the mode of action of the compound revealed that the inhibition of the NF-kappaB dependent pathway is due to the stabilization of the IkappaB protein and the inhibition of the IFN-gamma dependent signaling is caused by an inhibition of the phosphorylation of the STAT1alpha transcription factor.

The irpexans, a new group of biologically active metabolites produced by the basidiomycete Irpex sp. 93028.

Five novel antibiotics described as irpexans (1, 2, 3a, 3b, 4) were isolated from fermentations of an Irpex species in the course of a screening for new inhibitors of AP-1 and NF-kappaB mediated signal transduction pathways in COS-7 cells using secreted alkaline phosphatase (SEAP) as a reporter gene. The expression of an AP-1 and NF-kappaB driven SEAP reporter gene was inhibited in a dose dependent manner with 14-acetoxy-15-hydroxyirpexan (3b) being the most potent compound, followed by 14,15-irpexanoxide (2), 14,15-dihydroxyirpexan (3a) and 14-acetoxy-22,23-dihydro-15,23-dihydroxyirpexan (4). Irpexan (1) exhibited no activity. The irpexans (1, 2, 3a, 3b, 4) are characterized by weak cytotoxic but neither antibacterial nor antifungal activities. All five compounds are terpenoids with a mannose moiety. The structures were elucidated by spectroscopic methods.

Cycloepoxydon, 1-hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene and 1-hydroxy-2-hydroxymethyl-3-pent-1,3-dienylbenzene, new inhibitors of eukaryotic signal transduction.

In a screening for new inhibitors of NF-KB and AP-1 mediated signal transduction pathways in COS-7 cells using secreted alkaline phosphatase (SEAP) as a reporter gene three novel compounds, cycloepoxydon (1), 1-hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene (2) and 1-hydroxymethyl-3-pent-1,3-dienylbenzene (3) were isolated from fermentations of the deuteromycete strain 45-93. Cycloepoxydon inhibits the TPA-induced NF-KB and AP-1 mediated SEAP expression with an IC50 of 1-2 micrograms/ml (4.2-8.4 microns) and 3-5 micrograms/ml (12.6-21 microns) respectively. 1-Hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene (2) inhibits the TPA-induced NF-KB and AP-1 mediated SEAP expression with an IC50 of 7 micrograms/ml (36.4 microns) and 5 micrograms/ml (26 microns). 3 showed only a weak inhibition of the AP-1 and no influence on NF-KB dependent reporter gene expression. In COS-7 and HeLa S3 cells electrophoretic mobility shift assays showed that cycloepoxydon strongly reduced the TPA and TNF- alpha mediated binding of NF-KB to a high affinity consensus sequence which was due to the inhibition of phosphorylation of the protein IKB.

Nitidon, a new bioactive metabolite from the basidiomycete Junghuhnia nitida (Pers.: Fr.) Ryv.

Nitidon, a highly oxidised pyranone derivative produced by the basidiomycete Junghuhnia nitida, has been isolated and its biological activities evaluated. The structure was determined by spectroscopic methods. Nitidon exhibits antibiotic and cytotoxic activities and induces morphological and physiological differentiation of tumor cells at nanomolar concentrations.

Pulvinatal, a novel bioactive metabolite from the basidiomycete Nidularia pulvinata (Schw.) Fr.

Pulvinatal (1) was isolated from fermentations of Nidularia pulvinata as an inducer of differentiation of HL-60 promyelocytic leukemia cells. Its structure was elucidated by spectroscopic methods. In addition, N. pulvinata was found to produce 2,4,5-trihydroxy-6-methyl-benzenecarbaldehyde (2) and orsellinic acid (3). Pulvinatal exhibits weak antifungal and only marginal cytotoxic activities.

Nidulal, a novel inducer of differentiation of human promyelocytic leukemia cells from Nidula candida.

Nidulal (1), a novel inducer of differentiation of human HL-60 promyelocytic leukemia cells, was isolated from fermentations of the basidiomycete Nidula candida together with low amounts of niduloic acid (2). Both compounds are bisabolane sesquiterpenes. Their structures were elucidated by spectroscopic methods. In reporter gene assays nidulal (1) preferentially activated the transcription factor complex AP-1-mediated expression of secreted alkaline phosphatase in COS-7 cells. In addition nidulal (1) and niduloic acid (2) exhibited weak cytotoxic and antibiotic activities.

Inhibition of NF-kappa B activation by panepoxydone.

In a search for new inhibitors of NF-kappa B mediated signal transduction in COS-7 cells using the Secreted Alkaline Phosphatase as reporter gene, panepoxydone has been isolated from fermentations of the basidiomycete Lentinus crinitus. Panepoxydone inhibits the NF-kappa B activated expression of the SEAP with an IC50 of 1.5-2 micrograms/ml (7.15-9.52 microM). No inhibition of AP-1 mediated expression of the reporter gene could be observed at a concentration up to 5 micrograms/ml panepoxydone. Panepoxydone strongly reduced the TPA, TNF-alpha and ocadaic acid mediated binding of NF-kappa B to the high affinity consensus sequence in COS-7 and HeLa S3 cells as confirmed by EMSA's. Panepoxydone inhibits the I kappa B phosphorylation and therefore sequesteres the NF-kappa B complex in an inactive form.

Kuehneromycins A and B, two new biological active compounds from a Tasmanian Kuehneromyces sp. (Strophariaceae, Basidiomycetes).

In a search for new inhibitors of RNA-directed DNA-polymerases kuehneromycin A (1) was isolated from fermentations of a Tasmanian Kuehneromyces species. Its structure was elucidated by spectroscopic methods. Kuehneromycin A (1) is a non-competitive inhibitor of avian myeloblastosis virus (Ki 200 microM) and moloney murine leukemia virus (Ki 40 microM) reverse transcriptases. The second compound, kuehneromycin B (2) is a strong inhibitor of platelet aggregation stimulated with different inducers. In addition, both compounds exhibit cytotoxic and antimicrobial activities.

Hyphodontal, a new antifungal inhibitor of reverse transcriptases from Hyphodontia sp. (Corticiaceae, Basidiomycetes).

In a search for inhibitors of RNA-directed DNA polymerases a new isolactarane sesquiterpenoid, hyphodontal (1), was isolated from fermentations of a Canadian Hyphodontia species. Its structure was elucidated by spectroscopic methods. Hyphodontal strongly inhibits the growth of several yeasts and is a non-competitive inhibitor of avian myeloblastosis virus (Ki 346 microM) and Moloney murine leukemia virus (Ki 112 microM) reverse transcriptases. In addition, cytotoxic and antifungal activities were observed.

Antibiotics from basidiomycetes. XLI. Clavicoronic acid, a novel inhibitor of reverse transcriptases from Clavicorona pyxidata (Pers. ex Fr.) Doty.

A novel inhibitor of RNA-directed DNA-polymerases was isolated from fermentations of Clavicorona pyxidata. Its structure was elucidated by spectroscopic methods. Clavicoronic acid (1) is a noncompetitive inhibitor of avian myeloblastosis virus (Ki 130 microM) and Moloney murine leukemia virus (Ki 68 microM) reverse transcriptases. In permeabilized cells and isolated nucleic DNA- and RNA-synthesis are not affected. Clavicoronic acid markedly inhibits the multiplication of vesicular stomatitis virus in baby hamster kidney cells by interfering with this virus's RNA-directed RNA-polymerase. 1 exhibits no cytotoxic and very weak antimicrobial activities.

Podoscyphic acid, a new inhibitor of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptase from a Podoscypha species.

A novel enzyme inhibitor of RNA-directed DNA-polymerases of avian myeloblastosis and murine leukemia virus was isolated from fermentations of an tasmanian Podoscypha species. Its structure was elucidated by spectroscopic methods and oxidative degradation as (E)-4,5-dioxo-2-hexadecenoic acid (1). The enzyme inhibitor, which was named podoscyphic acid, did not inhibit DNA and RNA synthesis in permeabilized L 1210 cells nor did it affect RNA synthesis in isolated nuclei of L 1210 cells. 1 inhibits protein synthesis in whole L 1210 cells and rabbit reticulocyte lysate and shows very weak antimicrobial and cytotoxic properties. The testing of ethyl (E)-4,5-dioxo-2-hexadecenoate (2) and (E)-4-oxo-2-tetradecenoic acid (11) revealed the importance of the free gamma-oxoacrylic acid unit for the biological activities of 1.