Yumiko Kubota

Decalpenic acid, a novel small molecule from Penicillium verruculosum CR37010, induces early osteoblastic markers in pluripotent mesenchymal cells

Osteoblasts are the cells responsible for bone formation during embryonic development and adult life. Small compounds that could induce osteoblast differentiation might be promising sources of therapies for bone diseases such as osteoporosis. During screening for inducers of osteoblast differentiation of mouse pluripotent mesenchymal C3H10T1/2 cells, we isolated a small compound from the fermentation broth of Penicillium verruculosum CR37010. This compound, named decalpenic acid, bears a decalin moiety with a tetraenoic acid side chain. Treatment of C3H10T1/2 cells with decalpenic acid alone induced the expression of early osteoblast markers, such as alkaline phosphatase activity and osteopontin mRNA, but did not induce the late osteoblast marker osteocalcin mRNA or adipocyte markers under our experimental conditions.

Asteltoxins from the Entomopathogenic Fungus Pochonia bulbillosa 8-H-28

New asteltoxins C (3) and D (4) were found in the extract of the entomopathogenic fungus Pochonia bulbillosa 8-H-28. Compound 2, which was spectroscopically identical with the known asteltoxin B, was isolated, and structural analysis led to a revision of the structure of asteltoxin B. Compounds 2 and 4 have a novel tricyclic ring system connected to a dienyl α-pyrone structure. Compound 3 has a 2,8-dioxabicyclo[3.3.0]octane ring similar to that of asteltoxin (1). Compound 3 showed potent antiproliferative activity against NIAS-SL64 cells derived from the fat body of Spodoptera litura larvae, while 2 and 4 were inactive.

Discovery and characterization of NK13650s, naturally occurring p300-selective histone acetyltransferase inhibitors.

The histone acetyltransferase (HAT) activity of p300 is essential for androgen receptor (AR) function. Androgen-independent prostate cancer cells require AR-mediated transcriptional activation for their growth. These observations indicate that p300 HAT is a promising target to overcome such hormone-resistant cancer cells. We sought p300 HAT inhibitors among microbial metabolites. By culturing a production strain belonging to Penicillium, we identified two new compounds, NK13650A and NK13650B, which were obtained as specific p300 HAT inhibitors. Structural analyses of these compounds elucidated that NK13650s have novel chemical structures comprising several amino acids and citrate. We applied a newly developed biosynthesis-based method to reveal the absolute configuration at the citrate quaternary carbon. This was accomplished by feeding a (13)C-labeled biosynthetic precursor of citrate. NK13650s selectively inhibited the activity of p300 HAT but not that of Tip60 HAT. NK13650s showed inhibitory activity against agonist-induced AR transcriptional activation, and NK13650A treatment inhibited hormone-dependent and -independent growth of prostate cancer cells.

A New Teleocidin Analog from Streptomyces sp. MM216-87F4 Induces Substance P Release from Rat Dorsal Root Ganglion Neurons

A new teleocidin analog was isolated from the fermentation medium of Streptomyces sp. MM216-87F4 and its structure was elucidated as 14-O-(N-acetylglucosaminyl) teleocidin A (GlcNAc-TA). GlcNAc-TA induces the translocation of protein kinases Cα and θ fused with enhanced green fluorescent protein (PKCα-EGFP and PKCθ-EGFP) to the plasma membrane in stable transfectants, and reduces intracellular calcium mobilization induced by agonists of G-protein coupled receptors in various cell lines without causing irritation of the mouse ear. Further, GlcNAc-TA sensitizes the release of excitatory neuropeptides substance P induced by capsaicin from primary-cultured dorsal root ganglion (DRG) neurons of the rat and GlcNAc-TA alone also triggers substance P release in a dose-dependent manner. This study provides the first observation that a teleocidin analog without a free hydroxyl group at C-14 acts as a PKC activator and directly induces the release of excitatory neuropeptide.

Structural analyses of mannose pentasaccharide of high mannose type oligosaccharides by 1D and 2D NMR spectroscopy

NMR spectroscopy is a very important and useful method for the structural analysis of oligosaccharides, despite its low sensitivity. We first applied conventional measuring methods, 2D DQF COSY, 1H–13C HSQC, and 1H–13C HMBC, and also the Double Pulsed Field Gradient Spin Echo (DPFGSE)‐TOCSY and DPFGSE‐NOESY/ROESY techniques to analyze a branched mannose pentasaccharide as a model of high mannose type N‐glycans in natural abundance. The NMR spectra of the model compound are very complex and difficult to analyze owing to overlapping signals. The superior selective irradiation capability of the DPFGSE technique is useful for fine structural and conformational analyses of such complex oligosaccharides.

Sacchathridine A, a prostaglandin release inhibitor from Saccharothrix sp.

Sacchathridine A (1) was isolated from the fermentation broth of strain Saccharothrix sp. MI559-46F5. The structure was determined as a new naphthoquinone derivative with an acetylhydrazino moiety by a combination of NMR, MS spectral analyses, and chemical degradation. Compound 1 showed inhibitory activity of prostaglandin E2 release in a concentration-dependent manner from human synovial sarcoma cells, SW982, with an IC50 value of 1.0 μM, but had no effect on cell growth up to 30 μM.

Novel autophagy inducers lentztrehaloses A, B and C

Trehalose has widespread use as a sweetener, humectant and stabilizer, and is now attracting attention as a promising candidate for the treatment of neurodegenerative diseases as it is an autophagy inducer and chemical chaperone. However, the bioavailability of trehalose is low because it is digested by the hydrolyzing enzyme trehalase, expressed in the intestine and kidney. Enzyme-stable analogs of trehalose would potentially solve this problem. We have previously reported an enzyme-stable analog of trehalose, lentztrehalose, and herein report two new analogs. The original lentztrehalose has been renamed lentztrehalose A and the analogs named lentztrehaloses B and C. Lentztrehalose B is a di-dehydroxylated analog and lentztrehalose C is a cyclized analog of lentztrehalose A. All the lentztrehaloses are only minimally hydrolyzed by mammalian trehalase. The production of the lentztrehaloses is high in rather dry conditions and low in wet conditions. Lentztrehalose B shows a moderate antioxidative activity. These facts suggest that the lentztrehaloses are produced as humectants or protectants for the producer microorganism under severe environmental conditions. All the lentztrehaloses induce autophagy in human cancer cells at a comparable level to trehalose. Considering the enzyme-stability, these lentztrehaloses can be regarded as promising new drug candidates for the treatment of neurodegenerative diseases and other autophagy-related diseases, such as diabetes, arteriosclerosis, cancer and heart disease.

Acremopeptin, a new peptaibol from Acremonium sp. PF1450

The inhibitor of apoptosis protein (IAP) plays important roles in cancer cells, apoptosis control, tumorgenesis and chemotherapy resistance.1 The expression and function of IAP are deregulated in many human cancers because of genetic aberrations, an increase in their protein expression or the loss of endogenous inhibitors such as second mitochondria-derived activator of caspase (SMAC).2 In cell cytoplasm, survivin, a member of IAP family, plays a role as a suppressor of apoptosis, together with X-chromosome-linked IAP (XIAP). Furthermore, survivin controls cell division by its association with Aurora B and inner centromere protein (INCENP). Because apoptosis induced by suppression of IAP can lead to antitumor effects, an IAP such as survivin is considered as a promising molecular target for cancer chemotherapy. YM155, a survivin suppressant, suppresses survivin expression through direct binding to its promoter,3 and induces regression of established human hormone-refractory prostate tumor xenografts.4 An SMAC-mimicking IAP antagonist, AT-406 binds to XIAP, c-IAP1 and c-IAP2, and prevents the association of these proteins with caspases and SMAC.5 An XIAP antisense oligonucleotide, AEG35156, efficiently decreases XIAP mRNA and XIAP protein amounts.6 In addition, their combination with other cytotoxic agents, small molecule signal transduction inhibitors, proteasome inhibitors and death receptor ligands potentiated their effects.2 We screened for the microbial products to identify new candidate compounds that enhance doxorubicin-induced apoptosis in murine monocyte/macrophage RAW264.7 cells. Furthermore, we selected a culture broth that could suppress protein levels of survivin and/or XIAP. As a result of the purification of active compounds, we discovered the new peptide acremopeptin (1), which suppressed survivin and XIAP, and inhibited the growth of hormone-refractory prostate cancer PC-3 cells and colorectal adenocarcinoma HT-29 cells in vitro. Here we describe the isolation, structural elucidation and biological properties of 1 (Figure 1a). Acremonium sp. PF1450 was isolated from a soil sample collected from Ishigaki Island in Okinawa prefecture, Japan. The strain was inoculated to 500-ml Erlenmeyer flasks each containing 100 ml of a medium comprising of 2% soluble starch, 1% glucose, 0.5% polypeptone (Nihon Seiyaku), 0.6% wheat germ, 0.3% yeast extract (Nihon Seiyaku), 0.2% soybean meal (Ajinomoto), 0.2% CaCO3, with a pH of 7.0 before sterilization for seed culture. A loop of the slant culture of Acremonium sp. PF1450 was inoculated into 20 ml of the seed medium and was cultured on a rotary shaker at 25 °C for 3 days. Two ml of the seed cultured broth was transferred to 100 ml of the same medium and was cultured on a rotary shaker at 25 °C for 4 days. The culture filtrate (1 l) obtained from the fermented broth (1.2 l) was adjusted to pH 8.0 with 4% NaHCO3, and was applied to a Diaion HP-20 column (200 ml). After washing with 80% aqueous MeOH, the fraction containing the active compounds was eluted with methanol. The active fractions were concentrated in vacuo, and further purification was carried out by HPLC (Shiseido, Tokyo, Japan, CapcellPak C18 UG120, φ 20× 250 mm, flow rate: 5 ml min− 1) using a solvent system of 40% aqueous CH3CN containing 0.1% acetic acid. The new compound, acremopeptin (1, 16 mg), was eluted at 24– 25 min and a known compound, adenopeptin7 (2, 80 mg), was eluted at 22–23 min (Figures 1a and b). Acremopeptin (1) was obtained as white powder. The specific rotation of 1 was [α]D–3.0° (c 0.10, MeOH). The melting point exhibited 127 °C–132 °C. The molecular formula of 1 was determined to be C70H121N16O14 by high-resolution ESI mass spectrometer; (found, m/z 1409.9242 [M]+, calcd for C70H121N16O14, 1409.9256). The UV spectrum of 1 exhibited an absorption maximum at 203.5 nm (ε 28 900) in MeOH. The IR spectrum of 1 showed strong absorptions at 3330, 1664 and 1536 cm− 1, suggesting the existence of an amide group. The analysis of the partial structures via 1Hand 13C-NMR spectroscopy suggested that 1 belongs to the peptaibol family.8 The fragment ion pattern of 1 by LC/MS/MS experiments was similar to that of 2. By the LC/MS/MS experiment, the fragment ions of b-series (m/z 140, 239, 338, 409, 466, 551, 636 and 735) from 1 were in accordance with those of 2. Similarly, the fragment ions of y-series (m/z 224 and 323) from 1 were also in accordance with those of 2 (Figures 1c and d and Supplementary Figure S1). However, the fragment ion (m/z 420) of 1 was not observed in 2. Furthermore, the

Vegfrecine, an inhibitor of VEGF receptor tyrosine kinases isolated from the culture broth of Streptomyces sp.

A new inhibitor of VEGF receptor tyrosine kinases, vegfrecine (1), was isolated from the culture broth of Streptomyces sp. MK931-CF8. The molecular structure of 1 was determined by NMR and MS analysis combined with synthesis. Compound 1 showed potent inhibitory activity against vascular endothelial growth factor receptor (VEGFR) tyrosine kinases in in vitro enzyme assays, but platelet-derived growth factor receptors (PDGFRs), fibroblast growth factor receptor (FGFR), and epidermal growth factor receptor (EGFR) responded only weakly. Compound 1 is a promising new selective VEGFR inhibitor for investigating new treatments of cancer and inflammatory diseases.

Antipain Y, a new antipain analog that inhibits neurotransmitter release from rat dorsal root ganglion neurons

Antipain Y, a new antipain analog that inhibits neurotransmitter release from rat dorsal root ganglion neurons