Details of a Researcher - Kikuchi, Haruhisa

Papers - Kikuchi, Haruhisa

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Kinetics of the Inhibition of CYP3A4 and CYP2C19 Activity by Jabara Juice and Identification of the Responsible Inhibitory Components

Kana Koinuma, Kenji Noto, Tokio Morita, Yoshinori Uekusa, Haruhisa Kikuchi, Miyuki Shimoji, Hiroyuki Seki, Hiroshi Yamazaki, F. Peter Guengerich, Katsunori Nakamura, Koujirou Yamamoto, Ayuko Imaoka, Takeshi Akiyoshi, Hisakazu Ohtani

Journal of Pharmaceutical Sciences (Elsevier BV) 114 ( 2 ) 849 - 856 2024.10

Research paper (scientific journal), Accepted, ISSN 0022-3549

Some citrus fruits are known to cause clinically significant drug interactions by inhibiting intestinal cytochrome P450 (CYP) enzymes. This in vitro study aimed to investigate the kinetics of the inhibition of CYP3A4 and CYP2C19 by the juice of jabara, a Japanese citrus fruit that does not contain furanocoumarins such as 6′,7′-dihydroxybergamottin, and to identify the inhibitory compound(s). CYP3A4 and CYP2C19 activity levels were determined in vitro using recombinant CYP preparations and their respective substrates. The ethyl acetate extract (EAE) of jabara juice was separated to isolate and identify the compound(s) that inhibited CYP3A4. Then, the time-dependent kinetics of the inhibition of CYP3A4 and CYP2C19 by the EAE and its inhibitory compound(s) were analyzed. The EAE of jabara juice was found to inhibit CYP3A4 in a time-dependent manner. Two flavonoids, 3,3′,4′,5,6,7,8-heptamethoxyflavone (HpMF) and 3,3′,4′,5,6,7-hexamethoxyflavone (HxMF), were identified as the responsible compounds. HpMF and HxMF inhibited CYP3A4 activity in a concentration- and time-dependent manner, with inhibition constants (KI) of 10.0 and 7.90 µM and maximal inactivation rate constants (kinact,max) of 0.00856 and 0.0134 min−1, respectively. The EAE did not inhibit CYP2C19, even when preincubation was employed. These findings imply that jabara juice may cause food-drug interactions via time-dependent inhibition of intestinal CYP3A4.

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A longer-chain acylated derivative of Dictyostelium differentiation-inducing factor-1 enhances the antimalarial activity against Plasmodium parasites

Naoko Yoshida, Haruhisa Kikuchi, Makoto Hirai, Betty Balikagala, Denis A. Anywar, Hikari Taka, Naoko Kaga, Yoshiki Miura, Naoyuki Fukuda, Emmanuel I. Odongo-Aginya, Yuzuru Kubohara, Toshihiro Mita

Biochemical Pharmacology (Elsevier BV) 225 116243 - 116243 2024.07

Research paper (scientific journal), Accepted, ISSN 0006-2952

The spread of malarial parasites resistant to first-line treatments such as artemisinin combination therapies is a global health concern. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one) originally found in the cellular slime mould Dictyostelium discoideum. We previously showed that some derivatives of DIF-1, particularly DIF-1(+2) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) octan-1-one), exert potent antimalarial activities. In this study, we synthesised DIF-1(+3) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) nonan-1-one). We then evaluated the effects of DIF-1(+3) in vitro on Plasmodium falciparum and in vivo over 7 days (50–100 mg/kg/day) in a mouse model of Plasmodium berghei. DIF-1(+3) exhibited a half-maximal inhibitory concentration of approximately 20–30 % of DIF-1(+2) in three laboratory strains with a selectivity index > 263, including in strains resistant to chloroquine and artemisinin. Parasite growth and multiplication were almost completely suppressed by treatment with 100 mg/kg DIF-1(+3). The survival time of infected mice was significantly increased (P = 0.006) with no apparent adverse effects. In summary, addition of an acyl group to DIF-1(+2) to prepare DIF-1(+3) substantially enhanced antimalarial activity, even in drug-resistant malaria, indicating the potential of applying DIF-1(+3) for malaria treatment.

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Synthesis of a Library of Terpenoid Alkaloid-like Compounds Containing Medium-sized Rings via Reconstruction of the Humulene Skeleton.

Takehiro Nishimura, Kosuke Shiga, Mizuki Sekiya, Akihiro Sugawara, Takayuki Yonezawa, Haruhisa Kikuchi

Chemistry (Weinheim an der Bergstrasse, Germany) 30 ( 48 ) e202402082 2024.06

Research paper (scientific journal), Corresponding author, Accepted, ISSN 09476539

The construction of a chemical library based on natural products is a promising method for the synthesis of natural product-like compounds. In this study, we synthesized a terpenoid alkaloid-like compound library based on the humulene skeleton. Our strategy, which enables access to diverse ring systems such as 11-membered monocyclic, oxabicyclic, and medium-sized aza ring-containing scaffolds, involves the introduction of a nitrogen atom, an intermolecular C-O bond formation via Lewis acid-mediated epoxide-opening transannulation, and a ring-reconstruction strategy based on olefin metathesis. A cheminformatics analysis based on their structural and physicochemical properties revealed that the synthesized compounds have high three-dimensionality and high natural product likeness scores but with structural novelty. The usefulness of the terpenoid alkaloid-like compound library for drug discovery and the accessibility to structure-activity relationship studies were validated by performing an assay for osteoclast-specific tartrate-resistant acid phosphatase activity, resulting in the identification of a lead compound for bone-resorptive diseases such as osteoporosis.

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Isolation and Structure Determination of New Pyrones from Dictyostelium spp. Cellular Slime Molds Coincubated with Pseudomonas spp.

Takehiro Nishimura, Takuya Murotani, Hitomi Sasaki, Yoshinori Uekusa, Hiromi Eguchi, Hirotaka Ishigaki, Katsunori Takahashi, Yuzuru Kubohara, Haruhisa Kikuchi

Molecules (MDPI AG) 29 ( 9 ) 2143 - 2143 2024.05

Research paper (scientific journal), Accepted

Cellular slime molds are excellent model organisms in the field of cell and developmental biology because of their simple developmental patterns. During our studies on the identification of bioactive molecules from secondary metabolites of cellular slime molds toward the development of novel pharmaceuticals, we revealed the structural diversity of secondary metabolites. Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without using medium components. Although changing the feeding bacteria is expected to affect dramatically the secondary metabolite production, the effect of the feeding bacteria on the production of secondary metabolites is not known. Herein, we report the isolation and structure elucidation of clavapyrone (1) from Dictyostelium clavatum, intermedipyrone (2) from D. magnum, and magnumiol (3) from D. intermedium. These compounds are not obtained from usual cultural conditions with Klebsiella aerogenes but obtained from coincubated conditions with Pseudomonas spp. The results demonstrate the diversity of the secondary metabolites of cellular slime molds and suggest that widening the range of feeding bacteria for cellular slime molds would increase their application potential in drug discovery.

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Anti-trypanosomal Lignans Isolated from Salvadoran <i>Peperomia pseudopereskiifolia</i>

Ulises G. Castillo, Yoshinori Uekusa, Takehiro Nishimura, Fumiyuki Kiuchi, Morena L. Martínez, Jenny Menjívar, Junko Nakajima-Shimada, Marvin J. Núñez, Haruhisa Kikuchi

Journal of Natural Products (American Chemical Society (ACS)) 87 ( 4 ) 1067 - 1074 2024.04

Research paper (scientific journal), Corresponding author, Accepted, ISSN 0163-3864

A search for anti-trypanosomal natural compounds from plants collected in El Salvador, a country particularly endemic for Chagas disease, resulted in the isolation of five lignan-type compounds (1-5) from Peperomia pseudopereskiifolia. The lignan derivatives 1, 2, and 4 are new. Their absolute configuration was determined by chemical derivatization. Compounds 1, 5, 6, and 8 exhibited anti-trypanosomal activity against the amastigote form of T. cruzi comparable to that of the existing drug benznidazole.

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Compound–compound interaction analysis of baicalin and berberine derivatives in aqueous solution

Yoshinori Uekusa, Chiharu Tanioka, Kenjiro Nakamoto, Riina Tsutsumi, Chihiro Iida, Naoto Enshu, Takehiro Nishimura, Fumiyuki Kiuchi, Haruhisa Kikuchi

Journal of Natural Medicines (Springer Science and Business Media LLC) 78 ( 3 ) 590 - 598 2024.04

Research paper (scientific journal), Accepted, ISSN 1340-3443

Baicalin and berberine are biologically active constituents of the crude drugs Scutellaria root and Coptis rhizome/Phellodendron bark, respectively. Baicalin and berberine are reported to combine together as a 1:1 complex that forms yellow precipitates by electrostatic interaction in decoctions of Kampo formulae containing these crude drugs. However, the structural basis and mechanism for the precipitate formation of this compound–compound interaction in aqueous solution remains unclarified. Herein, we searched for berberine derivatives in the Coptis rhizome that interact with baicalin and identified the chemical structures involved in the precipitation formation. Precipitation assays showed that baicalin formed precipitates with berberine and coptisine but not with palmatine and epiberberine. Thus, the 2,3-methylenedioxy structure may be crucial to the formation of the precipitates, and electrostatic interaction is necessary but is not sufficient. In this multicomponent system experiment, palmatine formed a dissociable complex with baicalin and may competitively inhibit the formation of berberine and coptisine precipitation with baicalin. Therefore, the precipitation formed by berberine and baicalin was considered to be caused by the aggregation of the berberine–baicalin complex, and the 2,3-methylenedioxy structure is likely crucial to the aggregation of the complex. Graphic abstract: (Figure presented.)

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The inhibitory effect of DIF-3 on polyinosinic-polycytidylic acid-induced innate immunity activation in human cerebral microvascular endothelial cells.

Ryusei Araya, Shihu Men, Yoshinori Uekusa, Zaiqiang Yu, Haruhisa Kikuchi, Kazuyuki Daitoku, Masahito Minakawa, Shogo Kawaguchi, Ken-Ichi Furukawa, Yoshiteru Oshima, Tadaatsu Imaizumi, Kazuhiko Seya

Journal of pharmacological sciences 154 ( 3 ) 157 - 165 2024.03

Research paper (scientific journal), Accepted, ISSN 13478613

For the treatment and prevention of autoinflammatory diseases, it is essential to develop the drug, regulating the innate immune system. Although differentiation-inducing factor (DIF) derivatives, extracted from the cellular slime mold, Dictyostelium discoideum, exhibit immunomodulatory effects, their effects on the regulation of innate immunity in brain are unknown. In this study, we used the human cerebral microvascular endothelial cell line, hCMEC/D3, to investigate the effects of DIF derivatives on the generation of C-X-C motif chemokine (CXCL) 10 and interferon (IFN)-β induced by polyinosinic-polycytidylic acid (poly IC). DIF-3 (1-10 μM), but not DIF-1 and DIF-2, dose-dependently inhibited the biosynthesis of not only CXCL10 but also CXCL16 and C-C motif chemokine 2 induced by poly IC. DIF-3 also strongly decreased IFN-β mRNA expression and protein release from the cells induced by poly IC through the prohibition of p65, a subtype of NF-ĸB, not interferon regulatory transcription factor 3 phosphorylation. In the docking simulation study, we confirmed that DIF-3 had a high affinity to p65. These results suggest that DIF-3 regulates the innate immune system by inhibiting TLR3/IFN-β signaling axis through the NF-ĸB phosphorylation inhibition.

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Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake via Direct Inhibition of Mitochondrial Malate Dehydrogenase in Mouse 3T3-L1 Cells

Yuzuru Kubohara, Yuko Fukunaga, Ayako Shigenaga, Haruhisa Kikuchi

International Journal of Molecular Sciences (MDPI AG) 25 ( 3 ) 1889 - 1889 2024.02

Research paper (scientific journal), Accepted, ISSN 16616596

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography–tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10–40 μM dose-dependently suppressed growth, whereas LW6 at 20 μM, but not at 2–10 μM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10–40 μM significantly promoted glucose uptake, with the strongest effect at 20 μM DIF-1, whereas LW6 at 2–20 μM significantly promoted glucose uptake, with the strongest effect at 10 μM LW6. Western blot analyses showed that LW6 (10 μM) and DIF-1 (20 μM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.

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Pharmacological Evidence That Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake Partly via an Increase in Intracellular cAMP Content in Mouse 3T3-L1 Cells

Yuzuru Kubohara, Yuko Fukunaga, Haruhisa Kikuchi, Hidekazu Kuwayama

Molecules (MDPI AG) 28 ( 23 ) 7926 - 7926 2023.12

Research paper (scientific journal), Accepted

Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 μM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10–200 μM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40–400 μM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200–1000 μM significantly promoted glucose uptake (by 20–25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3–100 μM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1–10 μM promoted glucose uptake by 20–30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.

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Derivatives of Differentiation-Inducing Factor 1 Differentially Control Chemotaxis and Stalk Cell Differentiation in Dictyostelium discoideum

Hidekazu Kuwayama, Haruhisa Kikuchi, Yuzuru Kubohara

Biology (MDPI AG) 12 ( 6 ) 873 - 873 2023.06

Research paper (scientific journal), Accepted

Differentiation-inducing factors 1 and 2 (DIF-1 and DIF-2) are small lipophilic signal molecules that induce stalk cell differentiation but differentially modulate chemotaxis toward cAMP in the cellular slime mold Dictyostelium discoideum; DIF-1 suppresses chemotactic cell movement in shallow cAMP gradients, whereas DIF-2 promotes it. The receptor(s) for DIF-1 and DIF-2 have not yet been identified. We examined the effects of nine derivatives of DIF-1 on chemotactic cell movement toward cAMP and compared their chemotaxis-modulating activity and stalk cell differentiation–inducing activity in wild-type and mutant strains. The DIF derivatives differentially affected chemotaxis and stalk cell differentiation; for example, TM-DIF-1 suppressed chemotaxis and showed poor stalk-inducing activity, DIF-1(3M) suppressed chemotaxis and showed strong stalk-inducing activity, and TH-DIF-1 promoted chemotaxis. These results suggest that DIF-1 and DIF-2 have at least three receptors: one for stalk cell induction and two for chemotaxis modulation. In addition, our results show that the DIF derivatives can be used to analyze the DIF-signaling pathways in D. discoideum.

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<i>Dictyostelium</i> Differentiation-inducing Factor Derivatives Reduce the Glycosylation of PD-L1 in MDA-MB-231 Human Breast Cancer Cells

AIRI HIRAYAMA, HIROTAKA ISHIGAKI, KATSUNORI TAKAHASHI, YUSUKE MIURA, HARUHISA KIKUCHI, YUZURU KUBOHARA

Juntendo Medical Journal (The Jutendo Medical Journal) 2023

Research paper (scientific journal), Accepted, ISSN 2187-9737

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Elucidation of the inhibitory effect of (+)-hopeaphenol on polyinosinic-polycytidylic acid-induced innate immunity activation in human cerebral microvascular endothelial cells.

Liu Xu, Zaiqiang Yu, Yoshinori Uekusa, Shogo Kawaguchi, Haruhisa Kikuchi, Kazuyuki Daitoku, Masahito Minakawa, Shigeru Motomura, Ken-Ichi Furukawa, Yoshiteru Oshima, Kazuhiko Seya, Tadaatsu Imaizumi

Journal of pharmacological sciences (Journal of Pharmacological Sciences) 149 ( 3 ) 147 - 157 2022.07

Research paper (scientific journal), Accepted, ISSN 13478613

Drug development for regulating the innate immune system is important for the prevention and treatment of autoinflammatory and autoimmune diseases. In this context, we investigated the effect of resveratrol derivatives on the inflammatory reactions in the brain. Resveratrol, which can be found in Vitis plants in the form of oligomers, exhibits neuroprotective effects; however, its regulatory effects on innate immunity are still unclear. We examined the effects of (+)-hopeaphenol, a resveratrol tetramer, and its derivatives on the polyinosinic-polycytidylic acid (poly IC)-induced production of interferon (IFN)-β and C-X-C motif chemokine 10 (CXCL10) in the cultured human cerebral microvascular endothelial cell line hCMEC/D3. (+)-Hopeaphenol (1-10 μM) inhibited the poly IC-induced production of not only CXCL10 but also retinoic acid-inducible gene-I in a dose-dependent manner and significantly reduced the poly IC-induced IFN-β gene expression and protein release from hCMEC/D3 cells by inhibiting the phosphorylation of p65 but not that of the interferon regulatory transcription factor IRF3. A docking study indicated a high affinity of (+)-hopeaphenol for p65. These results suggest that (+)-hopeaphenol can regulate the innate immune system by inhibiting the poly IC/IFN-β/CXCL10 signaling axis via suppression of the phosphorylation of the transcription factor NF-ĸB.

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Glycidyl Silanes Enable Regioselective Hydrosilylation of In-ternal Propargyl Alcohols and Direct Transformation into Activated Silanes for Further Chemical Transformation

Akihiro Sugawara, Soya Koremura, Yusuke Sasano, Haruhisa Kikuchi

ChemRxiv (American Chemical Society (ACS)) 2022.04

Last author

Herein, we develop glycidyl silanes to facilitate highly regioselective hydrosilylation of internal propargyl alcohols, the products of which can in turn be converted to synthetically useful fluorosilanes for further chemical transfor-mations under mild conditions. Structure–selectivity studies and density functional theory calculations are consistent with high regioselectivity arising from a critical intermolecular hydrogen bond between the glycidyl and propargylic hydroxy groups. A broad substrate scope illustrates the generality of this reaction to form beta-E silylalkenes. Treatment of the beta-E silylalkenes with a fluoride salt induces simultaneous removal of the glycidyl group and activation of the silane under mild conditions. The fluorosilane products can be converted into vinyl arene and ketone derivatives via Hiyama coupling and Tamao–Fleming oxidation reactions, respectively. The discovery that glycidyl silane improves hydrosilylation regioselectivity and is compatible with expedient silylalkene derivatization may prove applicable to a variety of similar alkyne hydrofunctionalization reactions.

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Mg, K-containing microparticle: A possible active principle of a culture extract produced by a microbial consortium

Toru Higashinakagawa, Haruhisa Kikuchi, Hidekazu Kuwayama

PLOS ONE (Public Library of Science (PLoS)) 16 ( 11 ) e0259297 - e0259297 2021.11

Research paper (scientific journal), Joint Work, Accepted

A synthetic microbial consortium called Effective Microorganisms (EM) consists mainly of photosynthetic bacteria, lactic acid bacteria and yeast. Various effects of EM∙XGOLD, a health drink produced by EM, on life cycle of <italic>Dictyostelium discoideum</italic> were described previously. Here, we report our attempt to identify the active principle, termed EMF, that brought about the observed effects. Throughout the purification processes, the presence of the active principle was monitored by promoted fruiting body formation. By liquid-liquid separation the activity was recovered in aqueous phase, which, after concentration, was further subjected to reverse-phase column chromatography. No activity was detected in any eluant, while almost all the activity was recovered in residual insoluble material. The application of conventional organic chemistry procedures to the residual fraction did not lead to any informative results. Acid treatment of the insoluble material produced air bubbles, suggesting it to be composed of some inorganic carbonate. Viewed under scanning electronmicroscope, the residue revealed spherical particles of <italic>μ</italic>m size range. Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence, on the surface of the particles, of magnesium and, to a certain extent, of potassium. In separate experiments, acid treatment and alkali neutralization of EM∙XGOLD completely wiped out the stimulatory activity of fruiting body formation. These lines of evidence indicate these Mg, K-containing microparticles to be an active principle of EM culture extract. How these particles exert their effect is currently under intensive investigation.

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A Phenylfurocoumarin Derivative Reverses ABCG2-Mediated Multidrug Resistance In Vitro and In Vivo.

Kokubo, S, Ohnuma, S, Murakami, M, Kikuchi, H, Funayama, S, Suzuki, H, Kajiwara, T, Yamamura, A, Karasawa, H, Sugisawa, N, Ohsawa, K, Kano, K, Aoki, J, Doi, T, Naitoh, T, Ambudkar, S. V, Unno, M

Int. J. Mol. Sci. (MDPI AG) 22 ( 22 ) 12502 - 12502 2021.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 16616596

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.

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Derivatives of Dictyostelium differentiation-inducing factors suppress the growth of Plasmodium parasites in vitro and in vivo

Toshihiro Mita, Makoto Hirai, Yoshiko Maki, Saifun Nahar, Naoko Yoshida, Yoshiteru Oshima, Haruhisa Kikuchi, Yuzuru Kubohara

Biochemical Pharmacology (Elsevier BV) 194 114834 - 114834 2021.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-2952

Malaria, which is caused by protozoa of the genus Plasmodium, remains a major endemic public health problem worldwide. Since artemisinin combination therapies are used as a first-line treatment in all endemic regions, the emergence of parasites resistant to these regimens has become a serious problem. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone originally found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivatives exhibit a range of biological activities. In the present study, we investigated the effects of 41 DIF derivatives on the growth of Plasmodium falciparum in vitro using four laboratory strains and 12 field isolates. Micromolar concentrations of several DIF derivatives strongly suppressed the growth of the four laboratory strains, including strains that exhibited resistance to chloroquine and artemisinin, as well as strains that were susceptible to these drugs. In addition, DIF-1(+2), the most potent derivative, strongly suppressed the growth of 12 field isolates. We also examined the effects of DIF-1(+2) on the activity of the rodent malarial parasite Plasmodium berghei in mice. Intraperitoneal administration of DIF-1(+2) over 4 days (50 or 70 mg/kg/day) significantly suppressed the growth of the parasite in the blood with no apparent adverse effects, and a dose of 70 mg/kg/day significantly prolonged animal survival. These results suggest that DIF derivatives, such as DIF-1(+2), could serve as new lead compounds for the development of antimalarial agents.

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Development of Indole Alkaloid-Type Dual Immune Checkpoint Inhibitors against CTLA-4 and PD-L1 Based on Diversity-Enhanced Extracts

Suzuki, Y, Ichinohe, K, Sugawara, A, Kida, S, Murase, S, Zhang, J, Yamada, O, Hattori, T, Oshima, Y, Kikuchi, H

Frontiers in Chemistry (Frontiers Media SA) 9 2021.10

Research paper (scientific journal), Joint Work, Accepted

Cancer immunotherapy involves the use of the immune system for cancer treatment. Recently, immune checkpoint-blocking antibodies have become integral for the treatment of some cancers. However, small molecules exhibit advantages over monoclonal antibody drugs, such as cell penetration, long half-life, and low manufacturing costs, and the possibility of oral administration. Thus, it is imperative to develop small-molecule immune checkpoint inhibitors. Previously, we have screened a library of synthetic indole-alkaloid-type compounds, which are produced by diversity-enhanced extracts of Japanese cornelian cherry, and reported that an unnatural pentacyclic compound inhibits CTLA-4 gene expression. In this study, immune checkpoint inhibitors with increased potency were developed by introducing substituents and conversion of functional groups based on the unnatural pentacyclic compound. The developed compounds suppressed not only CTLA-4 and PD-L1 gene expression but also protein expression on the cell surface. Their efficacy was not as potent as that of the existing small-molecule immune checkpoint inhibitors, but, to the best of our knowledge, the developed compounds are the first reported dual small-molecule inhibitors of CTLA-4 and PD-L1.

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Inhibition of Plasmodium falciparum Lysyl‐tRNA synthetase via a piperidine‐ring scaffold inspired Cladosporin analogues

Palak Babbar, Mizuki Sato, Yogavel Manickam, Siddhartha Mishra, Karl Harlos, Swati Gupta, uhel Parvez, Haruhisa Kikuchi, Amit Sharma

ChemBioChem (Wiley) 22 ( 14 ) 2468 - 2477 2021.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 14394227

Plasmodium falciparum lysyl-tRNA synthetase (PfKRS) represents a promising therapeutic anti-malarial target. Cladosporin was identified as a selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report chemical synthesis, biological evaluation and structural characterization of analogues where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine ring bearing functional group variations. Thermal binding, enzymatic, kinetic and parasitic assays complemented with X-ray crystallography reveal compounds that are moderate in potency. Co-crystals of Cla−B and Cla−C with PfKRS reveal key atomic configurations that allow drug binding to and inhibition of the enzyme. Collectively these piperidine ring scaffold inhibitors lay a framework for further structural editing and functional modifications of the cladosporin scaffold to obtain a potent lead.

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Dictyostelium Differentiation-Inducing Factor-1 Promotes Glucose Uptake, at Least in Part, via an AMPK-Dependent Pathway in Mouse 3T3-L1 Cells

Yuzuru Kubohara, Yoshimi Homma, Hiroshi Shibata, Yoshiteru Oshima, Haruhisa Kikuchi

International Journal of Molecular Sciences (MDPI AG) 22 ( 5 ) 1 - 13 2021.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 16616596

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (a polyketide) found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivative, DIF-1(3M) promote glucose consumption in vitro in mammalian cells and in vivo in diabetic rats; they are expected to be the leading antiobesity and antidiabetes compounds. In this study, we investigated the mechanisms underlying the actions of DIF-1 and DIF-1(3M). In isolated mouse liver mitochondria, these compounds at 2–20 μM promoted oxygen consumption in a dose-dependent manner, suggesting that they act as mitochondrial uncouplers, whereas CP-DIF-1 (another derivative of DIF-1) at 10–20 μM had no effect. In confluent mouse 3T3-L1 fibroblasts, DIF-1 and DIF-1(3M) but not CP-DIF-1 induced phosphorylation (and therefore activation) of AMP kinase (AMPK) and promoted glucose consumption and metabolism. The DIF-induced glucose consumption was reduced by compound C (an AMPK inhibitor) or AMPK knock down. These data suggest that DIF-1 and DIF-1(3M) promote glucose uptake, at least in part, via an AMPK-dependent pathway in 3T3-L1 cells, whereas cellular metabolome analysis revealed that DIF-1 and DIF-1(3M) may act differently at least in part.

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Effect of mushroom polysaccharides from Pleurotus eryngii on obesity and gut microbiota in mice fed a high-fat diet.

Nakahara, D, Nan, C, Mori, K, Hanayama, M, Kikuchi, H, Hirai, S, Egashira, Y

European Journal of Nutrition (European Journal of Nutrition) 59 ( 7 ) 3231 - 3244 2020.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 14366207

PURPOSE: Mushrooms are reported to have a variety of health-promoting activities. However, little information is available on the effects of intake of polysaccharides from Pleurotus eryngii on obesity. In this study, we investigated the effects of P. eryngii polysaccharides on obesity and gut microbiota in mice fed a high-fat diet. METHODS: Soluble polysaccharides were extracted from P. eryngii using hot water. C57BL/6J mice were fed a standard diet (ST), a high-fat diet (HF), or HF with 1% or 5% P. eryngii polysaccharide fraction (LP or HP) for 16 weeks. Adipose tissues were weighed and blood parameters were measured. Expression of genes involved in fatty acid and cholesterol metabolism was assessed by real-time quantitative PCR. The gut microbiota composition was analysed by 16S rRNA gene sequencing. RESULTS: Body weight gain and mesenteric fat tissue were lower in the HP group than in the HF group. In the HP group, serum total cholesterol and LDL cholesterol levels decreased, and lipid and total bile acids in faeces increased. Mice in the HP group showed increased expression of the LDLR gene in the liver and GPR43 in fat. The relative abundance of Firmicutes was significantly higher in the HF and HP groups than in the ST group. The abundance of some short-chain fatty acid-producing gut bacteria was altered by P. eryngii polysaccharides. CONCLUSIONS: These results provide the first evidence that P. eryngii polysaccharides have anti-obesity and LDL cholesterol-lowering effects in obese mice through increased excretion of bile acids and lipids and altered microbiota.

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Two New Terpenes Isolated from Dictyostelium Cellular Slime Molds

Hitomi Sasaki, Yuzuru Kubohara, Hirotaka Ishigaki, Katsunori Takahashi, Hiromi Eguchi, Akihiro Sugawara, Yoshiteru Oshima, Haruhisa Kikuchi

Molecules (MDPI AG) 25 ( 12 ) 2895 - 2895 2020.06

Research paper (scientific journal), Joint Work, Accepted

We report a protoilludane-type sesquiterpene, mucoroidiol, and a geranylated bicyclogermacranol, firmibasiol, isolated from Dictyostelium cellular slime molds. The methanol extracts of the fruiting bodies of cellular slime molds were separated by chromatographic methods to give these compounds. Their structures have been established by several spectral means. Mucoroidiol and firmibasiol are the first examples of more modified and oxidized terpenoids isolated from cellular slime molds. Mucoroidiol showed moderate osteoclast-differentiation inhibitory activity despite demonstrating very weak cell-proliferation inhibitory activity. Therefore, cellular slime molds produce considerably diverse secondary metabolites, and they are promising sources of new natural product chemistry.

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Downregulation of PD‑L1 via amide analogues of brefelamide: Alternatives to antibody‑based cancer immunotherapy

Jing Zhang, Osamu Yamada, Shinya Kida, Shinya Murase, Toshio Hattori, Yoshiteru Oshima, Haruhisa Kikuchi

Experimental and Therapeutic Medicine (Spandidos Publications) 2020.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 1792-0981

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An automated microliter-scale high-throughput screening system (MSHTS) for real-time monitoring of protein aggregation using quantum-dot nanoprobes.

Rina Sasaki, Reina Tainaka, Yuichi Ando, Yurika Hashi, Hadya V Deepak, Yoshiko Suga, Yuta Murai, Masaki Anetai, Kenji Monde, Kiminori Ohta, Ikuko Ito, Haruhisa Kikuchi, Yoshiteru Oshima, Yasuyuki Endo, Hitomi Nakao, Masafumi Sakono, Koji Uwai, Kiyotaka Tokuraku

Scientific reports (Scientific Reports) 9 ( 1 ) 2587 - 2587 2019.12

Research paper (scientific journal), Joint Work, Accepted

Protein aggregation is the principal component of numerous protein misfolding pathologies termed proteinopathies, such as Alzheimer's disease, Parkinson's disease, prion disease, and AA amyloidosis with unmet treatment needs. Protein aggregation inhibitors have great potential for the prevention and treatment of proteinopathies. Here we report the development of an automated real-time microliter-scale high throughput screening (MSHTS) system for amyloid aggregation inhibitors using quantum-dot nanoprobes. Screening 504 crude extracts and 134 low molecular weight aromatic compounds revealed the relationship of amyloid-β (Aβ) aggregation inhibitory activities of plant extracts using a plant-based classification. Within the eudicots, rosids, Geraniales and Myrtales showed higher activity. Screening low molecular weight aromatic compounds demonstrated that the structure of tropolone endows it with potential Aβ aggregation inhibitory activity. The activity of the most active tropolone derivative was higher than that of rosmarinic acid. MSHTS also identified three chaperone molecules as tau aggregation inhibitors. These results demonstrate that our automated MSHTS system is a novel and robust tool that can be adapted to a wide range of compounds and aggregation-prone polypeptides.

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Structure and biological activity of metarhizin C, a stereoisomer of BR-050 from Tolypocladium album RK17-F0007.

Nogawa, T, Kawatani, M, Okano, A, Futamura, Y, Aono, H, Shimizu, T, Kato, N, Kikuchi, H, Osada, H

The Journal of Antibiotics (Journal of Antibiotics) 72 ( 12 ) 996 - 1000 2019.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 00218820

Metarhizin C, a stereoisomer of BR-050 was isolated from a fungus Tolypocladium album RK17-F0007 through a screening program to search for new antitumor compounds. A structure of the isomer was determined by spectroscopic methods including detailed analysis of NOESY correlation and mass spectrometry, and found to be identical to that of 3-desacylmetarhizin A with the absolute structure. Previously, it had been isolated by Kikuchi et al and proposed as BR-050 including the stereo-structure. However, detailed analysis for the newly isolated isomer confirmed that 3-desacylmetarhizin A was not identical to BR-050. Therefore, we assigned it metarhizin C as a new BR-050 isomer. Metarhizin C showed selective cytotoxicity against osteosarcoma MG-63 cells in a glucose independent condition with IC50 value of 0.79 µg/ml, while > 30 µg/ml of IC50 value in a normal condition, and inhibited a mitochondrial respiration.

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Differentiation-inducing factor-1 prevents hepatic stellate cell activation through inhibiting GSK3β inactivation.

Furukawa, S, Yamaguchi, M, Ooka, A, Kikuchi, H, Ishikawa, T, Saito, S

Biochemical and Biophysical Research Communications (Elsevier BV) 520 ( 1 ) 140 - 144 2019.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

Differentiation-inducing factor-1 (DIF-1), a morphogen produced by the cellular slime mold Dictyostelium discoideum, is a natural product that has attracted considerable attention for its antitumor properties. Here, we report a novel inhibitory effect of DIF-1 on the activation of hepatic stellate cells (HSCs) responsible for liver fibrosis. DIF-1 drastically inhibited transdifferentiation of quiescent HSCs into myofibroblastic activated HSCs in a concentration-dependent manner, thus conferring an antifibrotic effect against in the liver. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, showed any effect on the inhibition of HSC activation by DIF-1. In contrast, TWS119, a glycogen synthase kinase 3β (GSK3β) inhibitor, attenuated the inhibitory effect of DIF-1. Moreover, the level of inactive GSK3β (phosphorylated at Ser9) was significantly reduced by DIF-1. DIF-1 also inhibited nuclear translocation of β-catenin and reduced the level of non-phospho (active) β-catenin. These results suggest that DIF-1 inhibits HSC activation by disrupting the Wnt/β-catenin signaling pathway through dephosphorylation of GSK3β. We propose that DIF-1 is a possible candidate as a therapeutic agent for preventing liver fibrosis.

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Conformational heterogeneity in apo and drug-bound structures of Toxoplasma gondii proryl tRNA synthase.

Mishra S, Malhotra, N, Kumari, S, Sato M, Kikuchi, H, Yogavel M, Sharma A

Acta Crystallographica Section F (Acta Crystallographica Section F: Structural Biology Communications) 75 714 - 724 2019.11

Research paper (scientific journal), Joint Work, Accepted

Prolyl-tRNA synthetase (PRS) is a member of the aminoacyl-tRNA synthetase family that drives protein translation in cells. The apicomplexan PRSs are validated targets of febrifugine (FF) and its halogenated derivative halofuginone (HF). PRSs are of great interest for drug development against Plasmodium falciparum and Toxoplasma gondii. In this study, structures of apo and FF-bound T. gondii (TgPRS) are revealed and the dynamic nature of the conformational changes that occur upon FF binding is unraveled. In addition, this study highlights significant conformational plasticity within two different crystal structures of apo PRSs but not within drug-bound PRSs. The apo PRSs exist in multi-conformational states and manifest pseudo-dimeric structures. In contrast, when FF is bound the PRS dimer adopts a highly symmetrical architecture. It is shown that TgPRS does not display extant fold switching, in contrast to P. falciparum PRS, despite having over 65% sequence identity. Finally, structure-comparison analyses suggest the utility of r.m.s.d. per residue (r.m.s.d./res) as a robust tool to detect structural alterations even when the r.m.s.d. is low. Apo TgPRS reveals FF/HF-induced rigidity and this work has implications for drug-design studies that rely on the apo structures of target proteins.

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Inhibition of inflammatory-molecule synthesis in THP-1 cells stimulated with phorbol 12-myristate 13-acetate by brefelamide derivatives

Gaowa Bai, Takashi Matsuba, Haruhisa Kikuchi, Haorile Chagan-Yasutan, Hirotoshi Motoda, Ryo Ozuru, Osamu Yamada, Yoshiteru Oshima, Toshio Hattori

International Immunopharmacology (Elsevier BV) 75 105831 - 105831 2019.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 15675769

Plasma osteopontin (OPN) levels are elevated in tuberculosis patients and may involve granuloma formation. New inhibitors using brefelamide, an aromatic amide isolated from Dictyostelium cellular slime molds that may inhibit OPN transcription in A549 cells at 1 μM concentration, were synthesized as compounds C, D, and E. Their inhibitory activity against OPN synthesis in phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 cells was confirmed using enzyme-linked immunosorbent assay (ELISA), a multicolor immune-fluorescent microscope, and western blot. In the ELISA performed using full-length OPN, each compound showed significant inhibition in culture supernatants with half maximal inhibitory concentration (IC50) values of 1.6, 1.8, and 2.2 μM for C, D, and E, respectively. In another ELISA to detect the immune-related form of OPN, IC50 values were 0.6, 1.2, and 2.5 μM for compounds C, D, and E, respectively. The decreases in OPN expression and synthesis were confirmed using immunofluorescence and western blot studies using compound-treated cells or cell lysates. Luminex assay of the supernatants of PMA-treated THP-1 cells showed significant reduction in the synthesis of interleukin (IL)-1β, galectin-9, and tumor necrosis factor (TNF)-α. Elucidation of the detailed mechanisms of the biological activities of these compounds would be necessary; however, they may be used in clinical trials for infectious diseases, inflammatory disorders, and cancer.

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Identification of Celastramycin as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension.

Ryo Kurosawa, Kimio Satoh, Nobuhiro Kikuchi, Haruhisa Kikuchi, Daisuke Saigusa, Md Elias Al-Mamun, Mohammad A H Siddique, Junichi Omura, Taijyu Satoh, Shinichiro Sunamura, Masamichi Nogi, Kazuhiko Numano, Satoshi Miyata, Akira Uruno, Kuniyuki Kano, Yotaro Matsumoto, Takayuki Doi, Junken Aoki, Yoshiteru Oshima, Masayuki Yamamoto, Hiroaki Shimokawa

Circulation research (Circulation Research) 125 ( 3 ) 309 - 327 2019.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 00097330

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) accompanying increased production of inflammatory factors and adaptation of the mitochondrial metabolism to a hyperproliferative state. However, all the drugs in clinical use target pulmonary vascular dilatation, which may not be effective for patients with advanced PAH. OBJECTIVE: We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. METHODS AND RESULTS: We screened 5562 compounds from original library using high-throughput screening system to discover compounds which inhibit proliferation of PASMCs from patients with PAH (PAH-PASMCs). We found that celastramycin, a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PAH-PASMCs in a dose-dependent manner with relatively small effects on PASMCs from healthy donors. Then, we made 25 analogs of celastramycin and selected the lead compound, which significantly inhibited cell proliferation of PAH-PASMCs and reduced cytosolic reactive oxygen species levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of HIF-1α (hypoxia-inducible factor 1α), which impairs aerobic metabolism, and κB (nuclear factor-κB), which induces proinflammatory signals, in PAH-PASMCs, leading to reduced secretion of inflammatory cytokine. Importantly, celastramycin treatment reduced reactive oxygen species levels in PAH-PASMCs with increased protein levels of Nrf2 (nuclear factor erythroid 2-related factor 2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. Moreover, these celastramycin-mediated effects were regulated by ZFC3H1 (zinc finger C3H1 domain-containing protein), a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in 3 experimental animal models, accompanied by reduced inflammatory changes in the lungs. CONCLUSIONS: These results indicate that celastramycin ameliorates pulmonary hypertension, reducing excessive proliferation of PAH-PASMCs with less inflammation and reactive oxygen species levels, and recovered mitochondrial energy metabolism. Thus, celastramycin is a novel drug for PAH that targets antiproliferative effects on PAH-PASMCs.

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Halogen-substituted derivatives of Dictyostelium differentiation-inducing factor-1 suppress serum-induced cell migration of human breast cancer MDA-MB-231 cells in vitro.

Totsuka, K, Makioka, Y, Iizumi, K, Takahashi, K, Oshima, Y, Kikuchi, H, Kubohara, Y

Biomolecules (Biomolecules) 9 ( 7 ) 256 2019.07

Research paper (scientific journal), Joint Work, Accepted

Triple-negative breast cancer (TNBC) is highly proliferative and metastatic, and because it lacks three major molecular targets for chemotherapy (estrogen receptor, progesterone receptor, and human epidermal receptor 2), it is extremely refractory. Differentiation-inducing factor 1 (DIF-1) and DIF-3, which are chlorinated alkylphenones, are lead anticancer compounds found in the cellular slime mold Dictyostelium discoideum. Here, we examined the in vitro effects of DIF-1, DIF-3, and 25 DIF derivatives on cell proliferation and serum-induced cell migration in human MDA-MB-231 cells, a model TNBC cell line. We found that Br-DIF-1, a chlorine-to-bromine-substituted derivative of DIF-1, strongly suppressed cell migration (IC50, 3.8 µM) with negligible effects on cell proliferation (IC50, >20 µM). We then synthesized 18 derivatives of Br-DIF-1 and examined the in vitro effects of these derivatives on cell proliferation and serum-induced cell migration in MDA-MB-231 cells. Among the derivatives, Br-DIF-1(+1), Br-DIF-1(+2), and Br-DIF-3(+2) exhibited strong anti-cell migration activities with IC50 values of 1.5, 1.0, and 3.1 µM, respectively, without affecting cell proliferation (IC50, >20 µM). These results suggest that these Br-DIF derivatives are good lead compounds for the development of anti-metastatic drugs against TNBC.

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Antimicrobial Activities of Dictyostelium Differentiation-Inducing Factors and Their Derivatives.

Kubohara, Y, Shiratsuchi Y, Ishigaki, H, Takahashi, K, Oshima, Y, Kikuchi, H

Biomolecules (Biomolecules) 9 ( 5 ) 163 2019.05

Research paper (scientific journal), Joint Work, Accepted

At the end of its life cycle, the cellular slime mold Dictyostelium discoideum forms a fruiting body consisting of spores and a multicellular stalk. Originally, the chlorinated alkylphenone differentiation-inducing factors (DIFs) -1 and -3 were isolated as stalk cell inducers in D. discoideum. Later, DIFs and their derivatives were shown to possess several biologic activities including antitumor and anti-Trypanosoma properties. In this study, we examined the antibacterial activities of approximately 30 DIF derivatives by using several bacterial species. Several of the DIF derivatives strongly suppressed the growth of the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis and Enterococcus faecium, at minimum inhibitory concentrations (MICs) in the sub-micromolar to low-micromolar range. In contrast, none of the DIF derivatives evaluated had any noteworthy effect on the growth of the Gram-negative bacterium Escherichia coli (MIC, >100 µM). Most importantly, several of the DIF derivatives strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant E. faecalis and E. faecium. Transmission electron microscopy revealed that treatment with DIF derivatives led to the formation of distinct multilayered structures consisting of cell wall or plasma membrane in S. aureus. The present results suggest that DIF derivatives are good lead compounds for developing novel antimicrobials.

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Prenylated quinolinecarboxylic acid derivative prevents neuronal cell death through inhibition of MKK4.

Ogura, M, Kikuchi, H, Shakespear N. B, Suzuki, T, Yamaki, J, Homma, M. K, Oshima, Y, Homma, Y

Biochemical Pharmacology (Biochemical Pharmacology) 162 109 - 122 2019.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 00062952

The development of neuroprotective agents is necessary for the treatment of neurodegenerative diseases. Here, we report PQA-11, a prenylated quinolinecarboxylic acid (PQA) derivative, as a potent neuroprotectant. PQA-11 inhibits glutamate-induced cell death and caspase-3 activation in hippocampal cultures, as well as inhibits N-Methyl-4-phenylpyridinium iodide- and amyloid β 1-42 -induced cell death in SH-SY5Y cells. PQA-11 also suppresses mitogen-activated protein kinase kinase 4 (MKK4) and c-jun N-terminal kinase (JNK) signaling activated by these neurotoxins. Quartz crystal microbalance analysis and in vitro kinase assay reveal that PQA-11 interacts with MKK4, and inhibits its sphingosine-induced activation. The administration of PQA-11 by intraperitoneal injection alleviates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of nigrostriatal dopaminergic neurons in mice. These results suggest that PQA-11 is a unique MKK4 inhibitor with potent neuroprotective effects in vitro and in vivo. PQA-11 may be a valuable lead for the development of novel neuroprotectants.

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Construction of a Meroterpenoid-like Compounds Library Based on Diversity-Enhanced Extracts.

Kikuchi, H, Kawai, K, Nakashiro, Y, Yonezawa, T, Kawaji, K, Kodama, E. N, Oshima, Y

Chemistry - A European Journal (Chemistry - A European Journal) 25 ( 4 ) 1106 - 1112 2019.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 09476539

The structural diversity of natural products and their derivatives have long contributed to the development of new drugs. However, the difficulty in obtaining compounds bearing skeletally novel structures has recently led to a decline of pharmaceutical research into natural products. This paper reports the construction of a meroterpenoid-like library containing 25 compounds with diverse molecular scaffolds obtained from diversity-enhanced extracts. This method constitutes an approach for increasing the chemical diversity of natural-product-like compounds by combining natural product chemistry and diversity-oriented synthesis. Extensive pharmacological screening of the library revealed promising compounds for anti-osteoporotic and anti-lymphoma/leukemia drugs. This result indicates that the use of diversity-enhanced extracts is an effective methodology for producing chemical libraries for the purpose of drug discovery.

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H-1. Mg, K-containing microparticle: A possible active principle of EM fermentation product

Higashinakagawa T., Kiku-Chi H., Kuwayama H.

Biopolymers and Cell (Biopolymers and Cell) 35 ( 3 ) 2019

ISSN 02337657

EM represents Effective Microorganisms, a microbial consortium consisting mainly of photosynthetic bacteria, lactic acid bacteria and yeast. Various effects on cells of EM fermentation product were reported in the previous WBW25 Meeting in 2017. Here, we report our attempt to identify its active principle and the ongoing preliminary results. The activity was assayed by promoted formation of fruiting body of Dictyostelium discoideum. The EM fermentation product was first subjected to liquid-liquid separation, with the activity being recovered in aqueous phase. Concentrated aqueous fraction was further subjected to column chromatography. No activity was detected in any eluant, while almost all activity was recovered in residual insoluble material. The application of conventional organic chemistry procedures did not lead to any informative results. Acid treatment of the insoluble material produced air bubbles, suggesting it to be composed of some inorganic carbonate. Viewed under scanning electronmicroscope, these residues revealed spherical particles of μm range. Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence of magnesium and, to a certain extent, potassium. In a separate experiment, acid treatment and alkali neutralization of EM fermentation product completely wiped out the stimulatory activity of fruiting body formation. These lines of evidence indicate these Mg, K-containing microparticle to be active principle of EM fermentation product, at least, in regard to fruiting body formation in Dictyostelium discoideum. How these particles exert their effect is currently under extensive investigation.

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Dictyostelium: An Important Source of Structural and Functional Diversity in Drug Discovery (Review)

Kubohara, Y, Kikuchi, H

Cells 8 ( 1 ) 6 2019

Research paper (scientific journal), Joint Work, Accepted

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Removal of the E-olefin Barrier of Humulene Leading to Unnatural Terpenoid-like Skeletons.

Nishimura, T, Kawai, J, Oshima, Y, Kikuchi, H

Organic Letters (AMER CHEMICAL SOC) 20 ( 22 ) 7317 - 7320 2018.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 15237060

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合成化合物を対象とした新規免疫制御剤の探索

石垣宏尚, 高橋克典, 久保原禅, 菊地晴久, 藤田清貴

臨床化学 ((一社)日本臨床化学会) 47 ( Suppl.1 ) 312 - 312 2018.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0370-5633

Developments toward the Production of Diverse Natural-Product-Like Compounds: Diversity-Oriented Synthesis and Diversity-Enhanced Extracts (Review)

Kikuchi H, Oshima Y

Heterocycles (Heterocycles) 96 ( 9 ) 1509 - 1527 2018

Research paper (scientific journal), Joint Work, Accepted, ISSN 03855414

Natural products and their derivatives have proven very useful in the search for biologically active compounds and in the development of new drugs because of their structural diversity. However, new approaches that may increase the chemical diversity of such natural products must be developed in order to retain the future usefulness of these compounds. Diversity-oriented synthesis (DOS) has recently emerged as an efficient methodology for constructing complex and diverse compounds from simple and similar precursors. Through the combination of natural product chemistry and DOS, we therefore propose a new approach, diversity-enhanced extracts, for increasing the diversity of natural-product-like compounds. This review describes recent developments toward the production of diverse natural-product-like compounds by DOS based on natural products and direct chemical derivatization of natural extracts, including diversity-enhanced extracts.

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Effect of brefelamide on proliferation of 1321N1 human astrocytoma cells induced by glial cell line-derived neurotrophic factor

S. Honma, K. Kouno, S. Takasaka, S. Mitazaki, S. Abe, H. Kikuchi, Y. Oshima, M. Yoshida

Pharmazie (Govi-Verlag Pharmazeutischer Verlag GmbH) 73 ( 1 ) 22 - 28 2018

Research paper (scientific journal), Joint Work, Accepted, ISSN 00317144

Malignant gliomas are highly resistant to chemotherapy and radiation and more effective options for treatment are urgently needed. We reported previously that the aromatic amide brefelamide, which is isolated from methanolic extracts of the cellular slime molds Dictyostelium giganteum and D. brefeldianum, hinders cellular proliferation in a glioma model utilizing 1321N1 human astrocytoma cells. Herein, we examined the mechanisms underlying the inhibition of 1321N1 cell proliferation by brefelamide. Glial cell line-derived neurotrophic factor (GDNF) was found to enhance the rate of proliferation of serum-free cultured 1321N1 cells, but did not affect proliferation in PC12 cells. Brefelamide pretreatment inhibited GDNF-induced cell proliferation and expression of rearranged during transfection (RET). GDNF enhanced the phosphorylation of extracellular signal-regulated kinase (ERK), AKT, and c-jun-N-terminal kinase (JNK)
however, brefelamide pretreatment inhibited these effects. Brefelamide also reduced the expression of GDNF mRNA and GDNF secretion. Together, the findings from this study indicate that brefelamide inhibits the proliferation of 1321N1 cell via several mechanisms including reduced GDNF receptor expression and GDNF secretion, and reduced phosphorylation of ERK, AKT, and JNK.

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Biological Activities of Novel Derivatives of Differentiation-Inducing Factor 3 from Dictyostelium discoideum

Katsunori Takahashi, Haruhisa Kikuchi, Van Hai Nguyen, Yoshiteru Oshima, Hirotaka Ishigaki, Junko Nakajima-Shimada, Yuzuru Kubohara

BIOLOGICAL & PHARMACEUTICAL BULLETIN (PHARMACEUTICAL SOC JAPAN) 40 ( 11 ) 1941 - 1947 2017.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0918-6158

Differentiation-inducing factor-3 (DIF-3; 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), which is found in the cellular slime mold Dictyostelium discoideum, is a potential candidate compound for the development of new medicines; DIF-3 and its derivatives possess several beneficial biological activities, including anti-tumor, anti-Trypanosoma cruzi, and immunoregulatory effects. To assess the relationship between the biological activities of DIF-3 and its chemical structure, particularly in regard to its alkoxy group and the length of the alkyl chains at the acyl group, we synthesized two derivatives of DIF-3, 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)octan-1-one (DIF-3(+3)) and 1-(3-chloro-2,6-dihydroxy-4-butoxyphenyl)-hexan1-one (Hex-DIF-3), and investigated their biological activities in vitro. At micro-molar levels, DIF-3(+3) and Hex-DIF-3 exhibited strong anti-proliferative effects in tumor cell cultures, but their anti-T. cruzi activities at 1 mu m in vitro were not as strong as those of other known DIF derivatives. In addition, Hex-DIF-3 at 5 mu m significantly suppressed mitogen-induced interleukin-2 production in vitro in Jurkat T cells. These results suggest that DIF-3(+3) and Hex-DIF-3 are promising leads for the development of anti-cancer and immunosuppressive agents.

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Targeting Prolyl-tRNA Synthetase to Accelerate Drug Discovery against Malaria, Leishmaniasis, Toxoplasmosis, Cryptosporidiosis, and Coccidiosis

Vitul Jain, Manickam Yogavel, Haruhisa Kikuchi, Yoshiteru Oshima, Norimitsu Hariguchi, Makoto Matsumoto, Preeti Goel, Bastien Touquet, Rajiv S. Jumani, Fabienne Tacchini-Cottier, Karl Harlos, Christopher D. Huston, Mohamed-Ali Hakimi, Amit Sharma

STRUCTURE (CELL PRESS) 25 ( 10 ) 1495 - + 2017.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0969-2126

Developing anti-parasitic lead compounds that act on key vulnerabilities are necessary for new anti-infectives. Malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis together kill > 500,000 humans annually. Their causative parasites Plasmodium, Leishmania, Toxoplasma, Cryptosporidium and Eimeria display high conservation in many housekeeping genes, suggesting that these parasites can be attacked by targeting invariant essential proteins. Here, we describe selective and potent inhibition of prolyl-tRNA synthetases (PRSs) from the above parasites using a series of quinazolinone-scaffold compounds. Our PRS-drug co-crystal structures reveal remarkable active site plasticity that accommodates diversely substituted compounds, an enzymatic feature that can be leveraged for refining drug-like properties of quinazolinones on a per parasite basis. A compound we termed In-5 exhibited a unique double conformation, enhanced drug-like properties, and cleared malaria in mice. It thus represents a new lead for optimization. Collectively, our data offer insights into the structure-guided optimization of quinazolinone-based compounds for drug development against multiple human eukaryotic pathogens.

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Isolation, Synthesis, and Biological Activity of Chlorinated Alkylresorcinols from Dictyostelium Cellular Slime Molds

Haruhisa Kikuchi, Ikuko Ito, Katsunori Takahashi, Hirotaka Ishigaki, Kyoichi Iizumi, Yuzuru Kubohara, Yoshiteru Oshima

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 80 ( 10 ) 2716 - 2722 2017.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0163-3864

Eight chlorinated alkylresorcinols, monochasiol A-H (1-8), were isolated from the fruiting bodies of Dictyostelium monochasioides. Compounds 1-8 were synthesized to confirm their structures and to obtain sufficient material for performing biological tests. Monochasiol A (1) selectively inhibited the concanavalin A-induced interleukin-2 production in Jurkat cells, a human T lymphocyte cell line. Monochasiols were biogenetically synthesized by the combination of biosynthetic enzymes relating to the principal polyketides, MPBD and DIF-1, produced by Dictyostelium discoideum.

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Evidence that differentiation-inducing factor-1 controls chemotaxis and cell differentiation, at least in part, via mitochondria in D-discoideum

Yuzuru Kubohara, Haruhisa Kikuchi, Van Hai Nguyen, Hidekazu Kuwayama, Yoshiteru Oshima

BIOLOGY OPEN (COMPANY OF BIOLOGISTS LTD) 6 ( 6 ) 741 - 751 2017.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 2046-6390

Differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one (DIF-1)] is an important regulator of cell differentiation and chemotaxis in the development of the cellular slime mold Dictyostelium discoideum. However, the entire signaling pathways downstream of DIF-1 remain to be elucidated. To characterize DIF-1 and its potential receptor(s), we synthesized two fluorescent derivatives of DIF-1, boron-dipyrromethene (BODIPY)-conjugated DIF-1 (DIF-1-BODIPY) and nitrobenzoxadiazole (NBD)conjugated DIF-1 (DIF-1-NBD), and investigated their biological activities and cellular localization. DIF-1-BODIPY (5 mu M) and DIF-1 (2 nM) induced stalk cell differentiation in the DIF-deficient strain HM44 in the presence of cyclic adenosine monosphosphate (cAMP), whereas DIF-1-NBD (5 mu M) hardly induced stalk cell differentiation under the same conditions. Microscopic analyses revealed that the biologically active derivative, DIF-1-BODIPY, was incorporated by stalk cells at late stages of differentiation and was localized to mitochondria. The mitochondrial uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), at 25-50 nM, and dinitrophenol (DNP), at 2.5-5 mu M, induced partial stalk cell differentiation in HM44 in the presence of cAMP. DIF-1-BODIPY (1-2 mu M) and DIF-1 (10 nM), as well as CCCP and DNP, suppressed chemotaxis in the wild-type strain Ax2 in shallow cAMP gradients. These results suggest that DIF-1-BODIPY and DIF-1 induce stalk cell differentiation and modulate chemotaxis, at least in part, by disturbing mitochondrial activity.

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Development of Natural Product-Like Compound Library for Drug Discovery Based on Diversity-Enhanced Extracts

Haruhisa Kikuchi, Yoshiteru Oshima

JOURNAL OF SYNTHETIC ORGANIC CHEMISTRY JAPAN (SOC SYNTHETIC ORGANIC CHEM JPN) 75 ( 4 ) 349 - 359 2017.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0037-9980

Natural products and their derivatives have been very useful in the search for biologically active compounds and for the development of new drugs because of their structural diversity However, to retain their usefulness in the future, new approaches to increase the chemical diversity of such natural products must be developed. Diversity-oriented synthesis has recently emerged as an efficient methodology to construct complex and diverse compounds from simple and similar precursors. Thus, through the combination of natural products chemistry and diversity-oriented synthesis, we propose a new approach, diversity-enhanced extracts, for increasing the diversity of natural product-like compounds. Diversity-enhanced extracts are prepared from chemical reactions that remodel molecular scaffolds directly on extracts of natural resources. The subsequent isolation of each compound produced from such reactions affords a diverse natural product like library. We applied this method of diversification on several medicinal plants. From diversity enhanced extracts of the medicinal plants, we obtained new and diverse sesquiterpenoids, indole alkaloids, meroterpenoids, and biaryls with some containing new molecular skeletons.

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Induction of Osteopontin by Dengue Virus-3 Infection in THP-1 Cells: Inhibition of the Synthesis by Brefelamide and Its Derivative

Dyshelly N. Pascapurnama, Hannah K. M. Labayo, Isolde Dapat, Divya D. Nagarajegowda, Jingge Zhao, Jing Zhang, Osamu Yamada, Haruhisa Kikuchi, Shinichi Egawa, Yoshiteru Oshima, Haorile Chagan-Yasutan, Toshio Hattori

FRONTIERS IN MICROBIOLOGY (FRONTIERS MEDIA SA) 8 ( MAR ) 2017.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 1664-302X

Osteopontin (OPN) is a multifunctional matricellular protein produced by a broad range of cells including osteoclasts, macrophages, T cells, endothelial cells, and vascular smooth muscle cells. OPN modulates various physiological and pathological events such as inflammation, wound healing, and bone formation and remodeling. Dengue virus (DENV) infection causes an increase in plasma OPN levels, which is correlated with the severity of symptoms and coagulation abnormalities. DENV infection also induces OPN gene expression in human macrophages. This study investigated the inhibitory effects of brefelamide and its methyl ether derivative on DENV-3 by measuring changes in OPN levels in human THP-1 and 293T cell lines infected at different multiplicities of infection and post-infection time points. OPN mRNA expression and viral RNA were detected by reverse transcriptase quantitative real-time PCR, whereas protein level was determined by enzyme-linked immunosorbent assay. We found that viral copy number was higher in 293T than in THP-1 cells. However, THP-1 constitutively expressed higher levels of OPN mRNA and protein, which were enhanced by DENV-3 infection. Brefelamide and its derivative suppressed OPN production in DENV-3 infected THP-1 cells; the effective doses of these compounds had no effect on uninfected cells, indicating low cytotoxicity. These results suggest that brefelamide and its methyl ether derivative have therapeutic effects in preventing inflammation, coagulopathy, and fibrinolysis caused by OPN upregulation induced by DENV-3 infection.

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Production of novel bispyrone metabolites in the cellular slime mold Dictyostelium giganteum induced by zinc(II) ion

Van Hai Nguyen, Haruhisa Kikuchi, Hiraku Sasaki, Kyoichi Iizumi, Yuzuru Kubohara, Yoshiteru Oshima

TETRAHEDRON (PERGAMON-ELSEVIER SCIENCE LTD) 73 ( 5 ) 583 - 588 2017.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4020

In this study, Zn2+ induced the production of two new bispyrone-type metabolites, dictyobispyrone B and E, in the cellular slime mold Dictyostelium giganteum. Their structures were proposed on the basis of spectroscopic analysis and confirmed by chemical synthesis. They possess a novel alpha,alpha-bispyrone skeleton modified with long carbon chains. Both could be formed from two different polyketide chains through a novel biosynthetic pathway. Our results indicate that cultivation of cellular slime molds in the presence of Zn2+ is a useful technique for discovering other structurally unique compounds. (C) 2016 Elsevier Ltd. All rights reserved.

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Halofuginone enhances the chemo-sensitivity of cancer cells by suppressing NRF2 accumulation

Kouhei Tsuchida, Tadayuki Tsujita, Makiko Hayashi, Asaka Ojima, Nadine Keleku-Lukwete, Fumiki Katsuoka, Akihito Otsuki, Haruhisa Kikuchi, Yoshiteru Oshima, Mikiko Suzuki, Masayuki Yamamoto

FREE RADICAL BIOLOGY AND MEDICINE (ELSEVIER SCIENCE INC) 103 236 - 247 2017.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0891-5849

The KEAP1-NRF2 system regulates the cellular defence against oxidative and xenobiotic stresses. NRF2 is a transcription factor that activates the expression of cytoprotective genes encoding antioxidative, detoxifying and metabolic enzymes as well as transporters. Under normal conditions, KEAP1 represses NRF2 activity by degrading the NRF2 protein. When cells are exposed to stresses, KEAP1 stops promoting NRF2 degradation, and NRF2 rapidly accumulates and activates the transcription of target genes. Constitutive accumulation of NRF2 via a variety of mechanisms that disrupt KEAP1-mediated NRF2 degradation has been observed in various cancer types. Constitutive NRF2 accumulation confers cancer cells with a proliferative advantage as well as resistance to anti-cancer drugs and radiotherapies. To suppress the chemo-and radio-resistance of cancer cells caused by NRF2 accumulation, we conducted high-throughput chemical library screening for NRF2 inhibitors and identified febrifugine derivatives. We found that application of the less-toxic derivative halofuginone in a low dose range rapidly reduced NRF2 protein levels. Halofuginone induced a cellular amino acid starvation response that repressed global protein synthesis and rapidly depleted NRF2. Halofuginone treatment ameliorated the resistance of NRF2-addicted cancer cells to anti-cancer drugs both in vitro and in vivo. These results provide preclinical proof-of-concept evidence for halofuginone as an NRF2 inhibitor applicable to treatment of chemoand radio-resistant forms of cancer.

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Alkaloids from aerial parts of Annona senegalensis against Streptococcus mutans

N. Lall, N. Kishore, D. Bodiba, G. More, E. Tshikalange, H. Kikuchi, Y. Oshima

NATURAL PRODUCT RESEARCH (TAYLOR & FRANCIS LTD) 31 ( 16 ) 1944 - 1947 2017

Research paper (scientific journal), Joint Work, Accepted, ISSN 1478-6419

Antimicrobial potential of medicinal plants have been explored extensively these days. This study was carried out to evaluate the antibacterial potential from aerial parts of plant, called Annona senegalensis' and its constituents. Bioassay guided fractionation led to the isolation of four metabolites, (+)-catechin (1), (-)-anonaine (2), (-)-asimilobine (3) and (+)-nornantenine (4). This is the first report on the isolation of compounds 1, 3 and 4 from this plant. Compounds 2 and 4 showed good activity, whereas 1 and 3 displayed weak inhibition against Streptococcus mutans (ATCC 25175). The results showed that compound 2 and 3 showed significant activity with a minimum inhibition concentration (MIC) of 0.12 and 0.25 mg/mL, respectively. The present study reports for the first time the antibacterial activity of the extract of A. senegalensis and its constituents. As S. mutans is a rather resistant bacteria, the MIC obtained during the present study is significant.
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Glutathione S-transferase 4 is a putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium discoideum

Hidekazu Kuwayama, Haruhisa Kikuchi, Yoshiteru Oshima, Yuzuru Kubohara

Biochemistry and Biophysics Reports (Elsevier B.V.) 8 219 - 226 2016.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 2405-5808

In the development of the cellular slime mold Dictyostelium discoideum, two chlorinated compounds, the differentiation-inducing factors DIF-1 and DIF-2, play important roles in the regulation of both cell differentiation and chemotactic cell movement. However, the receptors of DIFs and the components of DIF signaling systems have not previously been elucidated. To identify the receptors for DIF-1 and DIF-2, we here performed DIF-conjugated affinity gel chromatography and liquid chromatography–tandem mass spectrometry and identified the glutathione S-transferase GST4 as a major DIF-binding protein. Knockout and overexpression mutants of gst4 (gst4– and gst4OE, respectively) formed fruiting bodies, but the fruiting bodies of gst4– cells were smaller than those of wild-type Ax2 cells, and those of gst4OE cells were larger than those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell formation by DIFs in the gst4 mutants were similar to those of Ax2 cells. These results suggest that GST4 is a DIF-binding protein that regulates the sizes of cell aggregates and fruiting bodies in D. discoideum.

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Monoterpene Indole Alkaloid-Like Compounds Based on Diversity-Enhanced Extracts of Iridoid-Containing Plants and Their Immune Checkpoint Inhibitory Activity

Haruhisa Kikuchi, Keisuke Ichinohe, Shinya Kida, Shinya Murase, Osamu Yamada, Yoshiteru Oshima

ORGANIC LETTERS (AMER CHEMICAL SOC) 18 ( 22 ) 5948 - 5951 2016.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 1523-7060

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Development of a Terpenoid Alkaloid-like Compound Library Based on the Humulene Skeleton

Haruhisa Kikuchi, Takehiro Nishimura, Eunsang Kwon, Junya Kawai, Yoshiteru Oshima

CHEMISTRY-A EUROPEAN JOURNAL (WILEY-V C H VERLAG GMBH) 22 ( 44 ) 15819 - 15825 2016.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0947-6539

Many natural terpenoid alkaloid conjugates show biological activity because their structures contain both sp(3)-rich terpenoid scaffolds and nitrogen-containing alkaloid scaffolds. However, their biosynthesis utilizes a limited set of compounds as sources of the terpenoid moiety. The production of terpenoid alkaloids containing various types of terpenoid moiety may provide useful, chemically diverse compound libraries for drug discovery. Herein, we report the construction of a library of terpenoid alkaloid-like compounds based on Lewis-acid-catalyzed transannulation of humulene diepoxide and subsequent sequential olefin metathesis. Cheminformatic analysis quantitatively showed that the synthesized terpenoid alkaloid-like compound library has a high level of three-dimensional-shape diversity. Extensive pharmacological screening of the library has led to the identification of promising compounds for the development of antihypolipidemic drugs. Therefore, the synthesis of terpenoid alkaloid-like compound libraries based on humulene is well suited to drug discovery. Synthesis of terpenoid alkaloid-like compounds based on several natural terpenoids is an effective strategy for producing chemically diverse libraries.

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Identification of brefelamide as a novel inhibitor of osteopontin that suppresses invasion of A549 lung cancer cells

Jing Zhang, Osamu Yamada, Shinya Kida, Yoshihisa Matsushita, Shinya Murase, Toshio Hattori, Yuzuru Kubohara, Haruhisa Kikuchi, Yoshiteru Oshima

ONCOLOGY REPORTS (SPANDIDOS PUBL LTD) 36 ( 4 ) 2357 - 2364 2016.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 1021-335X

The contribution of aberrant osteopontin (OPN) expression to tumor progression and metastasis has been documented in a wide spectrum of malignancies, and targeted inhibition of OPN has therefore emerged as an attractive strategy for cancer therapy. Transcription of OPN is regulated by various transcription factors, and our recently published study demonstrated that downregulation of OPN is an important event in the TGF-beta cytostatic program. We report here that brefelamide exerts an inhibitory effect on OPN expression and function in A549 human lung carcinoma cells. The promoter, RNA, and protein levels of OPN were decreased in brefelamide-treated A549 cells, which was accompanied by reduced invasive ability in vitro. OPN inhibition by brefelamide was largely abrogated by disruption of a putative TGF-beta inhibitory element in the OPN promoter. Treatment with brefelamide induced Smad4 expression, and knockdown of Smad4 by RNA interference partially diminished the inhibitory effect of brefelamide on OPN. These results indicate that brefelamide inhibited OPN-mediated cell invasion through restoration of the OPN repression by TGF-beta/Smad signaling. Together with the reported antiproliferative property, our findings suggest that brefelamide might serve as a potential candidate for the development of a new antitumor and antimetastatic agent.

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Oral administration of Dictyostelium differentiation-inducing factor 1 lowers blood glucose levels in streptozotocin-induced diabetic rats

Ritsuko Kawaharada, Akio Nakamura, Katsunori Takahashi, Haruhisa Kikuchi, Yoshiteru Oshima, Yuzuru Kubohara

LIFE SCIENCES (PERGAMON-ELSEVIER SCIENCE LTD) 155 56 - 62 2016.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0024-3205

Aims: Differentiation-inducing factor 1 (DIF-1), originally discovered in the cellular slime mold Dictyostelium discoideum, and its derivatives possess pharmacological activities, such as the promotion of glucose uptake in non-transformed mammalian cells in vitro. Accordingly, DIFs are considered promising lead candidates for novel anti-diabetic drugs. The aim of this study was to assess the anti-diabetic and toxic effects of DIF-1 in mouse 3T3-L1 fibroblast cells in vitro and in diabetic rats in vivo.
Main methods
We investigated the in vitro effects of DIF-1 and DIF-1(3M), a derivative of DIF-1, on glucose metabolism in 3T3-L1 cells by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). We also examined the effects of DIF-1 on blood glucose levels in streptozotocin (STZ)-induced rats.
Key findings.
CE-TOF-MS revealed that 20 mu M DIF-1 and 20 mu M DIF-1(3M) promoted glucose uptake and metabolismin 3T3-L1 cells. Oral administration of DIF-1 (30 mg/kg) significantly lowered basal blood glucose levels in STZ-treated rats and promoted a decrease in blood glucose levels after oral glucose loading (2.5 g/kg) in the rats. In addition, daily oral administration of DIF-1 (30 mg/kg/day) for 1 wk significantly lowered the blood glucose levels in STZ-treated rats but did not affect their body weight and caused only minor alterations in the levels of other blood analytes.
Significance: These results indicate that DIF-1 may be a good lead compound for the development of anti-diabetic drugs. (C) 2016 Elsevier Inc. All rights reserved.

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1-Methyl-2-undecyl-4(1H)-quinolone, a derivative of quinolone alkaloid evocarpine, attenuates high phosphate-induced calcification of human aortic valve interstitial cells by inhibiting phosphate cotransporter PiT-1

Kazuhiko Seya, Ken-Ichi Furukawa, Mari Chiyoya, Zaiqiang Yu, Haruhisa Kikuchi, Kazuyuki Daitoku, Shigeru Motomura, Manabu Murakami, Yoshiteru Oshima, Ikuo Fukuda

JOURNAL OF PHARMACOLOGICAL SCIENCES (JAPANESE PHARMACOLOGICAL SOC) 131 ( 1 ) 51 - 57 2016.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 1347-8613

An abnormally high serum phosphate level induces calcific aortic stenosis (CAS), which is characterized by ectopic valve calcification and stenosis of the orifice area. Inhibition of ectopic calcification is a critical function of any internal medical therapy for CAS disease. The aim of the present study was to investigate the inhibitory effects of several derivatives of evocarpine, methanolic extracts from the fruits of Evodia rutaecarpa Bentham (Japanese name: Go-Shu-Yu) on the high phosphate-induced calcification of human aortic valve interstitial cells (HAVICs) obtained from patients with CAS. High phosphate (3.2 mM) concentrations significantly increased the calcification of HAVICs after 7 days of culture. This calcification was completely inhibited in the presence of sodium phosphonoformate (PFA), a selective inhibitor of the type III sodium-dependent phosphate cotransporter (PiT-1). PiT-1 contributes to phosphate uptake, resulting in calcification. 1-Methyl-2-undecyl-4(1H)-quinolone (MUQ; 30-300 nM), but not evocarpine or its derivatives dihydroevocarpine and 1-methyl-2-nonyl-4(1H)-quinolone, inhibited the high phosphate-induced HAVICs calcification in a concentration-dependent manner. Although all of the evocarpine derivatives attenuated alkaline phosphatase activity, only MUQ also decreased PiT-1 gene expression with cellular PiT-1 protein diminution. These results suggest that MUQ mitigated high phosphate-induced HAVICs calcification by inhibiting PiT-1 gene expression. (C) 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Design and Synthesis of Structure-Simplified Derivatives of Gonytolide for the Promotion of Innate Immune Responses

Haruhisa Kikuchi, Tsuyoshi Hoshikawa, Shoichiro Kurata, Yasuhiro Katou, Yoshiteru Oshima

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 79 ( 5 ) 1259 - 1266 2016.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0163-3864

Gonytolide A (1), a dimeric chromanone substituted with the gamma-lactone, shows promoting activity of innate immune responses. However, biological studies on this compound have been limited by the low amounts of 1 available from natural resources and the difficulty of its synthesis. In this study, we designed and synthesized structure-simplified gonytolide derivatives. Bischromone 10 and biflavone 13 both promoted the mammalian TNF-alpha signaling pathway and Drosophila innate immunity. They did not contain a chiral center and were easy to synthesize. Hence, they can be used as lead compounds for a new type of immunostimulating drugs and as research reagents.

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Prenylated quinolinecarboxylic acid derivative suppresses immune response through inhibition of PAK2

Masato Ogura, Haruhisa Kikuchi, Toshiyuki Suzuki, Junko Yamaki, Miwako K. Homma, Yoshiteru Oshima, Yoshimi Homma

BIOCHEMICAL PHARMACOLOGY (PERGAMON-ELSEVIER SCIENCE LTD) 105 55 - 65 2016.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-2952

Development of new immunosuppressing agents is necessary in organ transplantation or immune diseases. Because Ppc-1 exhibits a suppressing effect on interleukin-2 (IL2) production in Jurkat cells, we synthesized and screened Ppc-1 derivatives that preserve prenylated quinolinecarboxylic acid (PQA) structure, and identified compound 18 (PQA-18) as a novel molecule with immunosuppressing effect. PQA-18 suppressed not only 112 but also IL4, IL6, and tumor necrosis factor -alpha production in human peripheral lymphocytes without affecting cell viability. Two-dimensional gel electrophoresis analysis and in vitro kinase assay revealed that PQA-18 inhibits kinase activity of p21-activated kinase 2 (PAK2). Administration of PQA-18 by intraperitoneal injection suppressed the population of a subset of regulatory T cells and the immunoglobulin (Ig) production against T cell-dependent antigens in mice. Treatment with the PQA-18 ointment on Nc/Nga mice, a model of human atopic dermatitis, improved skin lesions and serum IgE levels. These results suggest that PQA-18 is a unique PAK2 inhibitor with potent immunosuppressing effects in vitro and in vivo. PQA-18 may be a valuable lead for the development of novel immunosuppressants. (C) 2016 Elsevier Inc. All rights reserved.

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Agarol, an ergosterol derivative from Agaricus blazei, induces caspase-independent apoptosis in human cancer cells

Takamitsu Shimizu, Junya Kawai, Kenji Ouchi, Haruhisa Kikuchi, Yoshiteru Osima, Rikiishi Hidemi

INTERNATIONAL JOURNAL OF ONCOLOGY (SPANDIDOS PUBL LTD) 48 ( 4 ) 1670 - 1678 2016.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 1019-6439

Agaricus blazei (A. blazei) is a mushroom with many biological effects and active ingredients. We purified a tumoricidal substance from A. blazei, an ergosterol derivative, and named it 'Agarol'. Cytotoxic effects of Agarol were determined by the MTT assay using A549, MKN45, HSC-3, and HSC-4 human carcinoma cell lines treated with Agarol. Apoptosis was detected by flow cytometry analysis. Reactive oxygen species (ROS) levels and mitochondria membrane potential (Delta Psi m) were also determined by flow cytometry. Western blot analysis was used to quantify the expression of apoptosis-related proteins. Agarol predominantly induced apoptosis in two p53-wild cell lines (A549 and MKN45) compared to the other p53-mutant cell lines (HSC-3 and HSC-4). Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of ROS, reduced Delta Psi m, release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol, upregulation of Bax, and downregulation of Bcl-2. Caspase-3 activities did not increase, and z-VAD-fmk, a caspase inhibitor, did not inhibit the Agarol-induced apoptosis. These findings indicate that Agarol induces caspase-independent apoptosis in human carcinoma cells through a mitochondrial pathway. The in vivo anticancer activity of Agarol was confirmed in a xenograft murine model. This study suggests a molecular mechanism by which Agarol induces apoptosis in human carcinoma cells and indicates the potential use of Agarol as an anticancer agent.

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Isolation of a Cyclic Depsipetide, Aspergillicin F, and Synthesis of Aspergillicins with Innate Immune-Modulating Activity

Haruhisa Kikuchi, Tsuyoshi Hoshikawa, Shimpei Fujimura, Noriaki Sakata, Shoichiro Kurata, Yasuhiro Katou, Yoshiteru Oshima

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 78 ( 8 ) 1949 - 1956 2015.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0163-3864

Innate immunity is the front line of self-defense against microbial infection. After searching for natural compounds that regulate innate immunity using an ex vivo Drosophila culture system, we identified a new cyclic depsipeptide, aspergillicin F, from the fungus Aspergillus sp., as an innate immune suppressor. The total synthesis and biological evaluation of the aspergillicin family, including aspergillicin F, were performed, revealing that slight structural differences in the side chains of amino acid residues alter innate immunity-regulating activity.

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Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acid-stimulated migration of murine osteosarcoma LM8 cells

Yuzuru Kubohara, Mayumi Komachi, Yoshimi Homma, Haruhisa Kikuchi, Yoshiteru Oshima

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (ACADEMIC PRESS INC ELSEVIER SCIENCE) 463 ( 4 ) 800 - 805 2015.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), -2, and -3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5-20 mu M) dose-dependefitly suppressed LPA-induced cell migration with associated IC50 values of 5.5, 4.6, and 4.2 mu M, respectively. On the other hand, the IC50 values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 mu M, respectively, in LM8 cells, and >20, 14.8, and 4.3 mu M, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis. (C) 2015 Elsevier Inc. All rights reserved.

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Development of novel DIF-1 derivatives that selectively suppress innate immune responses

Van Hai Nguyen, Haruhisa Kikuchi, Yuzuru Kubohara, Katsunori Takahashi, Yasuhiro Katou, Yoshiteru Oshima

BIOORGANIC & MEDICINAL CHEMISTRY (PERGAMON-ELSEVIER SCIENCE LTD) 23 ( 15 ) 4311 - 4315 2015.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0968-0896

The multiple pharmacological activities of differentiation-inducing factor-1 (DIF-1) of the cellular slime mold Dictyostelium discoideum led us to examine the use of DIF-1 as a 'drug template' to develop promising seed compounds for drug discovery. DIF-1 and its derivatives were synthesized and evaluated for their regulatory activities in innate immune responses. We found two new derivatives (4d and 5e) with highly selective inhibitory activities against production of the antimicrobial peptide attacin in Drosophila S2 cells and against production of interleukin-2 in Jurkat cells. (C) 2015 Elsevier Ltd. All rights reserved.

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Structure of Prolyl-tRNA Synthetase-Halofuginone Complex Provides Basis for Development of Drugs against Malaria and Toxoplasmosis

Vitul Jain, Manickam Yogavel, Yoshiteru Oshima, Haruhisa Kikuchi, Bastien Touquet, Mohamed-Ali Hakimi, Amit Sharma

STRUCTURE (CELL PRESS) 23 ( 5 ) 819 - 829 2015.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0969-2126

The Chinese herb Dichroa febrifuga has traditionally treated malaria-associated fever. Its active component febrifugine (FF) and derivatives such as halofuginone (HF) are potent anti-malarials. Here, we show that FF-based derivatives arrest parasite growth by direct interaction with and inhibition of the protein translation enzyme prolyl-tRNA synthetase (PRS). Dual administration of inhibitors that target different tRNA synthetases suggests high utility of these drug targets. We reveal the ternary complex structure of PRS-HF and adenosine 5'-(beta, gamma-imido) triphosphate where the latter facilitates HF integration into the PRS active site. Structural analyses also highlight spaces within the PRS architecture for HF derivatization of its quinazolinone, but not piperidine, moiety. We also show a remarkable ability of HF to kill the related human parasite Toxoplasma gondii, suggesting wider HF efficacy against parasitic PRSs. Hence, our cell-, enzyme-, and structure-based data on FF-based inhibitors strengthen the case for their inclusion in anti-malarial and anti-toxoplasmosis drug development efforts.

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Weight Loss by Ppc-1, a Novel Small Molecule Mitochondrial Uncoupler Derived from Slime Mold

Toshiyuki Suzuki, Haruhisa Kikuchi, Masato Ogura, Miwako K. Homma, Yoshiteru Oshima, Yoshimi Homma

PLOS ONE (PUBLIC LIBRARY SCIENCE) 10 ( 2 ) 2015.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 1932-6203

Mitochondria play a key role in diverse processes including ATP synthesis and apoptosis. Mitochondrial function can be studied using inhibitors of respiration, and new agents are valuable for discovering novel mechanisms involved in mitochondrial regulation. Here, we screened small molecules derived from slime molds and other microorganisms for their effects on mitochondrial oxygen consumption. We identified Ppc-1 as a novel molecule which stimulates oxygen consumption without adverse effects on ATP production. The kinetic behavior of Ppc-1 suggests its function as a mitochondrial uncoupler. Serial administration of Ppc-1 into mice suppressed weight gain with no abnormal effects on liver or kidney tissues, and no evidence of tumor formation. Serum fatty acid levels were significantly elevated in mice treated with Ppc-1, while body fat content remained low. After a single administration, Ppc-1 distributes into various tissues of individual animals at low levels. Ppc-1 stimulates adipocytes in culture to release fatty acids, which might explain the elevated serum fatty acids in Ppc-1-treated mice. The results suggest that Ppc-1 is a unique mitochondrial regulator which will be a valuable tool for mitochondrial research as well as the development of new drugs to treat obesity.

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Synthesis of prenylated quinolinecarboxylic acid derivatives and their anti-obesity activities

Haruhisa Kikuchi, Toshiyuki Suzuki, Masato Ogura, Miwako K. Homma, Yoshimi Homma, Yoshiteru Oshima

BIOORGANIC & MEDICINAL CHEMISTRY (PERGAMON-ELSEVIER SCIENCE LTD) 23 ( 1 ) 66 - 72 2015.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0968-0896

Mitochondrial uncoupling is one of the therapeutic strategies used to control energy metabolism in various metabolic diseases and in obesity. Ppc-1 (1), a prenylated quinolinecarboxylic acid isolated from cellular slime molds, shows uncoupling activity in vitro and anti-obesity activity in vivo. In this study, we synthesized Ppc-1 (1) and its derivatives, and revealed the structure-activity relationship of uncoupling activities. The triprenylated compound 18 showed mitochondrial uncoupling activity that was more potent than that of Ppc-1 (1). Compound 18 also suppressed weight gain in mice without undesired effects such as lesions on tissues. These results indicate that compound 18 could be used as a seed compound for new anti-obesity drugs. (C) 2014 Elsevier Ltd. All rights reserved.

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A Derivative of Differentiation-Inducing Factor-3 Inhibits PAK1 Activity and Breast Cancer Cell Proliferation.

Oladimeji P, Kubohara Y, Kikuchi H, Oshima Y, Rusch C, Skerl R, Diakonova M

International journal of cancer and clinical research 2 ( 3 ) 1 - 6 2015

Research paper (scientific journal), Joint Work, Accepted

Structural and functional analysis of the anti-malarial drug target prolyl-tRNA synthetase

Vitul Jain, Haruhisa Kikuchi, Yoshiteru Oshima, Amit Sharma, Manickam Yogavel

Journal of Structural and Functional Genomics (Springer Netherland) 15 ( 4 ) 181 - 190 2014.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 1570-0267

Aminoacyl-tRNA synthetases (aaRSs) drive protein translation in cells and hence these are essential enzymes across life. Inhibition of these enzymes can halt growth of an organism by stalling protein translation. Therefore, small molecule targeting of aaRS active sites is an attractive avenue from the perspective of developing anti-infectives. Febrifugine and its derivatives like halofuginone (HF) are known to inhibit prolyl-tRNA synthetase of malaria parasite Plasmodium falciparum. Here, we present functional and crystallographic data on P. falciparum prolyl-tRNA synthetase (PfPRS). Using immunofluorescence data, we show that PfPRS is exclusively resident in the parasite cytoplasm within asexual blood stage parasites. The inhibitor HF interacts strongly with PfPRS in a non-competitive binding mode in presence or absence of ATP analog. Intriguingly, the two monomers that constitute dimeric PfPRS display significantly different conformations in their active site regions. The structural analyses presented here provide a framework for development of febrifugine derivatives that can seed development of new anti-malarials.

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ZFC3H1, a Zinc Finger Protein, Modulates IL-8 Transcription by Binding with Celastramycin A, a Potential Immune Suppressor

Takeshi Tomita, Katsuaki Ieguchi, Fredric Coin, Yasuhiro Kato, Haruhisa Kikuchi, Yoshiteru Oshima, Shoichiro Kurata, Yoshiro Maru

PLOS ONE (PUBLIC LIBRARY SCIENCE) 9 ( 9 ) 2014.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 1932-6203

Celastramycin A, a small molecule that inhibits the production of antibacterial peptides in an ex vivo culture system of Drosophila, suppresses the TNFa-mediated induction of IL-8 in mammalian cells. To understand its molecular mechanism, we examined Celastramycin A binding proteins and investigated their biological functions. Our screening and subsequent pull-down assay revealed ZFC3H1 (also known as CCDC131 or CSRC2), an uncharacterized zinc finger protein, as a Celastramycin A binding protein. The knockdown of ZFC3H1 reduced IL-8 expression levels in the TNFa-stimulated lung carcinoma cell line, LU99, and UV-irradiated HeLa cells. Based on reporter assay results, we concluded that ZFC3H1 participates in the transcriptional activation of IL-8. The findings of our UV-irradiation experiments implied that ZFC3H1 may indirectly interact with ERCC1 in an activated DNA repair complex. Thus, we designated ZFC3H1 as a mammalian target of Celastramycin A (mTOC).

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Defects in the synthetic pathway prevent DIF-1 mediated stalk lineage specification cascade in the non-differentiating social amoeba, Acytostelium subglobosum

Kurato Mohri, Takashi Hata, Haruhisa Kikuchi, Yoshiteru Oshima, Hideko Urushihara

BIOLOGY OPEN (COMPANY OF BIOLOGISTS LTD) 3 ( 6 ) 553 - 560 2014.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 2046-6390

Separation of somatic cells from germ-line cells is a crucial event for multicellular organisms, but how this step was achieved during evolution remains elusive. In Dictyostelium discoideum and many other dictyostelid species, solitary amoebae gather and form a multicellular fruiting body in which germ-line spores and somatic stalk cells differentiate, whereas in Acytostelium subglobosum, acellular stalks form and all aggregated amoebae become spores. In this study, because most D. discoideum genes known to be required for stalk cell differentiation have homologs in A. subglobosum, we inferred functional variations in these genes and examined conservation of the stalk cell specification cascade of D. discoideum mediated by the polyketide differentiation-inducing factor-1 (DIF-1) in A. subglobosum. Through heterologous expression of A. subglobosum orthologs of DIF-1 biosynthesis genes in D. discoideum, we confirmed that two of the three genes were functional equivalents, while DIF-methyltransferase (As-dmtA) involved at the final step of DIF-1 synthesis was not. In fact, DIF-1 activity was undetectable in A. subglobosum lysates and amoebae of this species were not responsive to DIF-1, suggesting a lack of DIF-1 production in this species. On the other hand, the molecular function of an A. subglobosum ortholog of DIF-1 responsive transcription factor was equivalent with that of D. discoideum and inhibition of polyketide synthesis caused developmental arrest in A. subglobosum, which could not be rescued by DIF-1 addition. These results suggest that non-DIF-1 polyketide cascades involving downstream transcription factors are required for fruiting body development of A. subglobosum.

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Metarhizin A suppresses cell proliferation by inhibiting cytochrome c oxidase activity

Yasuhiro Katou, Naoya Endo, Toshiyuki Suzuki, Jiang Yu, Haruhisa Kikuchi, Yoshiteru Oshima, Yoshimi Homma

LIFE SCIENCES (PERGAMON-ELSEVIER SCIENCE LTD) 103 ( 1 ) 1 - 7 2014.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0024-3205

Aims: Metarhizin A was originally isolated from Metarhizium flavoviride as a potent inhibitor of the growth of insect and mammalian cells. In this study, we aimed to understand the molecular targets of metarhizin A involved in its anti-proliferative activity against human cells.
Main methods: Cell cycle regulators and signaling molecules were examined by immunoblotting using specific antibodies. A mitochondria-enriched fraction was prepared from mouse liver, and mitochondrial activity was monitored using an oxygen electrode. Enzyme activity was measured using purified cytochrome c oxidase and permeabilized cells.
Key findings: Metarhizin A inhibits the growth of MCF-7 cells with an IC50 value of similar to 0.2 mu M and other cells in a similar manner; a cell cycle-dependent kinase inhibitor, p21, is selectively induced. Significant amounts of reactive oxygen species (ROS) are generated and ERK1/2 is activated in cells treated with metarhizin A. Metarhizin A completely suppresses oxygen consumption by mitochondria, and potently inhibits the activity of cytochrome c oxidase. It induces cell death when MCF-7 cells are cultured under limiting conditions.
Significance: Metarhizin A is a potent inhibitor of cytochrome c oxidase and activates the MAPK pathway through the generation of ROS, which induces growth arrest of cells, and, under some conditions, enhances cell death. The cytochrome c oxidase system is a possible molecular target of metarhizin A. (C) 2014 Elsevier Inc. All rights reserved.

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Synthesis of febrifugine derivatives and development of an effective and safe tetrahydroquinazoline-type antimalarial

Haruhisa Kikuchi, Seiko Horoiwa, Ryota Kasahara, Norimitsu Hariguchi, Makoto Matsumoto, Yoshiteru Oshima

EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY (ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER) 76 10 - 19 2014.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0223-5234

Febrifugine, a quinazoline alkaloid isolated from Dichroa febrifuga roots, shows powerful antimalarial activity against Plasmodium falciparum. Although the use of ferifugine as an antimalarial drug has been precluded because of its severe side effects, its potent antimalarial activity has stimulated medicinal chemists to pursue its derivatives instead, which may provide valuable leads for novel antimalarial drugs. In the present study, we synthesized new derivatives of febrifugine and evaluated their in vitro and in vivo antimalarial activities to develop antimalarials that are more effective and safer. As a result, we proposed tetrahydroquinazoline-type derivative as a safe and effective antimalarial candidate. (C) 2014 Elsevier Masson SAS. All rights reserved.

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Properties of a non-bioactive fluorescent derivative of differentiation-inducing factor-3, an anti-tumor agent found in Dictyostelium discoideum

Yuzuru Kubohara, Haruhisa Kikuchi, Yusuke Matsuo, Yoshiteru Oshima, Yoshimi Homma

BIOLOGY OPEN (COMPANY OF BIOLOGISTS LTD) 3 ( 4 ) 289 - 296 2014.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 2046-6390

Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives, such as butoxy-DIF-3 (Bu-DIF-3), are potent anti-tumor agents. To investigate the activity of DIF-like molecules in tumor cells, we recently synthesized a green fluorescent DIF-3 derivative, BODIPY-DIF-3G, and analyzed its bioactivity and cellular localization. In this study, we synthesized a red (orange) fluorescent DIF-3 derivative, BODIPY-DIF-3R, and compared the cellular localization and bioactivities of the two BODIPY-DIF-3s in HeLa human cervical cancer cells. Both fluorescent compounds penetrated the extracellular membrane within 0.5 h and localized mainly to the mitochondria. In formalin-fixed cells, the two BODIPY-DIF-3s also localized to the mitochondria, indicating that the BODIPY-DIF-3s were incorporated into mitochondria independently of the mitochondrial membrane potential. After treatment for 3 days, BODIPY-DIF-3G, but not BODIPY-DIF-3R, induced mitochondrial swelling and suppressed cell proliferation. Interestingly, the swollen mitochondria were stainable with BODIPY-DIF-3G but not with BODIPY-DIF-3R. When added to isolated mitochondria in vitro, BODIPY-DIF-3G increased dose-dependently the rate of O-2 consumption, but BODIPY-DIF-3R did not. These results suggest that the bioactive BODIPY-DIF-3G suppresses cell proliferation, at least in part, by altering mitochondrial activity, whereas the non-bioactive BODIPY-DIF-3R localizes to the mitochondria but does not affect mitochondrial activity or cell proliferation.

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Development of Diversity-Enhanced Extracts of Curcuma zedoaria and Their New Sesquiterpene-like Compounds

Haruhisa Kikuchi, Kazuyoshi Sakurai, Yoshiteru Oshima

ORGANIC LETTERS (AMER CHEMICAL SOC) 16 ( 7 ) 1916 - 1919 2014.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 1523-7060

Through the combination of natural products chemistry and diversity-oriented synthesis, a new approach, diversity-enhanced extracts, for increasing the diversity of natural product-like compounds is proposed. They are prepared from chemical reactions that remodel molecular scaffolds directly on extracts of natural resources. This method was applied to terpenes extracted from Curcuma zedoaria. Epoxidation and subsequent ring-opening reactions of epoxides were used to modify molecular skeletons. As a result, seven sesquiterpene-like compounds with some containing new molecular skeletons were obtained.

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Novel chlorinated dibenzofurans isolated from the cellular slime mold, Polysphondylium filamentosum, and their biological activities

Haruhisa Kikuchi, Yuzuru Kubohara, Van Hai Nguyen, Yasuhiro Katou, Yoshiteru Oshima

Bioorganic and Medicinal Chemistry 21 ( 15 ) 4628 - 4633 2013.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0968-0896

Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities
these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster. © 2013 Published by Elsevier Ltd.

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Mitochondria Are the Target Organelle of Differentiation-Inducing Factor-3, an Anti-Tumor Agent Isolated from Dictyostelium Discoideum

Yuzuru Kubohara, Haruhisa Kikuchi, Yusuke Matsuo, Yoshiteru Oshima, Yoshimi Homma

PLOS ONE (PUBLIC LIBRARY SCIENCE) 8 ( 8 ) 2013.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 1932-6203

Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3 (Bu-DIF-3) are potent anti-tumor agents. However, the precise mechanisms underlying the actions of DIF-3 remain to be elucidated. In this study, we synthesized a green fluorescent derivative of DIF-3, BODIPY-DIF-3, and a control fluorescent compound, Bu-BODIPY (butyl-BODIPY), and investigated how DIF-like molecules behave in human cervical cancer HeLa cells by using both fluorescence and electron microscopy. BODIPY-DIF-3 at 5-20 mu M suppressed cell growth in a dose-dependent manner, whereas Bu-BODIPY had minimal effect on cell growth. When cells were incubated with BODIPY-DIF-3 at 20 mu M, it penetrated cell membranes within 0.5 h and localized mainly in mitochondria, while Bu-BODIPY did not stain the cells. Exposure of cells for 1-3 days to DIF-3, Bu-DIF-3, BODIPY-DIF-3, or CCCP (a mitochondrial uncoupler) induced substantial mitochondrial swelling, suppressing cell growth. When added to isolated mitochondria, DIF-3, Bu-DIF-3, and BOIDPY-DIF-3, like CCCP, dose-dependently promoted the rate of oxygen consumption, but Bu-BODIPY did not. Our results suggest that these bioactive DIF-like molecules suppress cell growth, at least in part, by disturbing mitochondrial activity. This is the first report showing the cellular localization and behavior of DIF-like molecules in mammalian tumor cells.

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Derivatives of Dictyostelium discoideum differentiation-inducing factor-3 suppress the activities of Trypanosoma cruzi in vitro and in vivo

Junko Nakajima-Shimada, Toshimitsu Hatabu, Yukari Hosoi, Yoko Onizuka, Haruhisa Kikuchi, Yoshiteru Oshima, Yuzuru Kubohara

Biochemical Pharmacology 85 ( 11 ) 1603 - 1610 2013.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-2952

Chagas disease (human American trypanosomiasis), which is caused by the protozoan parasite Trypanosoma cruzi, is responsible for numerous deaths each year
however, established treatments for the disease are limited. Differentiation-inducing factor-1 (DIF-1) and DIF-3 are chlorinated alkylphenones originally found in the cellular slime mold Dictyostelium discoideum that have been shown to possess pharmacological activities. Here, we investigated the effects of DIF-3 derivatives on the infection rate and growth of T. cruzi by using an in vitro assay system utilizing host human fibrosarcoma HT1080 cells. Certain DIF-3 derivatives, such as butoxy-DIF-3 (Bu-DIF-3), at micro-molar levels strongly suppressed both the infection rate and growth of T. cruzi in HT1080 cells and exhibited little toxicity for HT1080 cells. For example, the IC50 of DIF-3 and Bu-DIF-3 versus the growth of T. cruzi in HT1080 cells were 3.95 and 0.72 μM, respectively, and the LD50 of the two compounds versus HT1080 cells were both greater than 100 μM. We also examined the effects of DIF-3 and Bu-DIF-3 on T. cruzi activity in C57BL/6 mice. Intraperitoneally administered Bu-DIF-3 (50 mg/kg) significantly suppressed the number of trypomastigotes in blood with no apparent adverse effects. These results strongly suggest that DIF-3 derivatives could be new lead compounds in the development of anti-trypanosomiasis drugs. © 2013 Elsevier Inc. All rights reserved.

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Terresterol, a polyoxygenated lanostanoid, isolated from the oomycete Saprolegnia terrestris, and its innate immune-promoting activity

Haruhisa Kikuchi, Yuichi Sato, Shoichiro Kurata, Yasuhiro Katou, Yoshiteru Oshima

TETRAHEDRON (PERGAMON-ELSEVIER SCIENCE LTD) 69 ( 17 ) 3536 - 3542 2013.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4020

Oomycetes are widely spread eukaryotic microorganisms. However, there have only been a few reports on the secondary metabolites produced by them. In the course of screening for innate immune regulators from natural resources, we isolated a new polyoxygenated lanostanoid, terresterol (1), from the oomycetes Saprolegnia terrestris. Compounds bearing a 21,22,24,25-tetraoxygenated side chain similar to 1 have only been rarely described, and the isolation of such compounds shows that oomycetes are promising resources for natural product chemistry. In addition, terresterol (1) has potential to be used as a lead compound for novel immunostimulating agents against bacterial infections and as probe for the innate immune system. (C) 2013 Elsevier Ltd. All rights reserved.

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The Dictyostelium prestalk inducer DIF-1 directs phosphorylation of a bZIP transcription factor

Yoko Yamada, Yuzuru Kubohara, Haruhisa Kikuchi, Yoshiteru Oshima, Hong-Yu Wang, Susan Ross, Jeffrey G. Williams

INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY (U B C PRESS) 57 ( 5 ) 375 - 381 2013

Research paper (scientific journal), Joint Work, Accepted, ISSN 0214-6282

DIF-1, a chlorinated hexaphenone produced by developing Dictyostelium cells, induces prestalk differentiation. DimB is a bZIP transcription factor that accumulates in the nucleus upon exposure to DIF-1, where it directly activates transcription of DIF-responsive genes. The signaling steps upstream of DimB and downstream of DIF-1 are entirely unknown. Analysis by mass spectrometry shows that incubation with DIF-1 rapidly stimulates phosphorylation at several sites in DimB. We characterize the most highly responsive site, S590, which is located very close to the C terminus. A point mutation in this site, S590A, does not inhibit DimB nuclear accumulation in response to DIE However, this seems likely to reflect functional redundancy with other sites; because a panel of chemical variants on the structure of DIF-1 show a correlation between their potencies as inducers of DimB nuclear accumulation and their potencies as inducers of phosphorylation at S590. Furthermore, the S590A mutant is fully active in mutant rescue of a dimB null strain, arguing against an alternative role in transcriptional activation of target genes. We conclude that i) DIF-1 directs phosphorylation at S590, ii) although it is not essential for nuclear accumulation in response to DIF-1 correlative evidence, based upon a panel of DIF-1 related molecules, suggests that this modification may play a redundant role in the process. iii) We also present evidence that the kinase activity, which phosphorylates S590, is non-nuclear and that this signalling pathway is, in part at least, independent of the DIF-regulated STATc activation pathway.

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Isolation, synthesis, and biological activity of biphenyl and m-terphenyl-type compounds from Dictyostelium cellular slime molds

Haruhisa Kikuchi, Yusuke Matsuo, Yasuhiro Katou, Yuzuru Kubohara, Yoshiteru Oshima

TETRAHEDRON (PERGAMON-ELSEVIER SCIENCE LTD) 68 ( 43 ) 8884 - 8889 2012.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4020

From the fruiting bodies of Dictyostelium polycephalum, we obtained four aromatic compounds: dictyobiphenyl A (1) and B (2) and dictyoterphenyl A (3) and B (4). The synthesis of 1-4 was performed to confirm the structures and obtain sufficient material for biological evaluation. Compound 3 was the first example of nitrogen-containing natural m-terphenyls, and the isolation of novel classes of compounds such as 3 shows that cellular slime molds are promising sources in natural product chemistry. Moreover, dictyoterphenyl A (3) showed cancer cell-selective antiproliferative activity (IC50 2.3-8.6 mu M). (C) 2012 Elsevier Ltd. All rights reserved.

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Stigmastane derivatives from the roots of Vernonia guineensis and their antimicrobial activity

Arno R. N. Donfack, Ngeh J. Toyang, Hippolyte K. Wabo, Pierre Tane, Maurice D. Awouafack, Haruhisa Kikuchi, Jean D. D. Tamokou, Jules R. Kuiate, Yoshiteru Oshima

PHYTOCHEMISTRY LETTERS (ELSEVIER SCIENCE BV) 5 ( 3 ) 596 - 599 2012.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 1874-3900

Chemical investigation of the roots of Vernonia guineensis (Asteraceae) afforded a new stigmastane derivative, vernoguinoside A (1) and the known vernoguinoside (2), stigmasterol 3-O-beta-D-glucoside (3) and sitosterol 3-O-beta-D-glucoside (4). Their structures were elucidated by spectroscopic analysis. Antimicrobial activities of 1-3 and CH2Cl2-MeOH (1:1) extract were evaluated against three bacteria species (Salmonella typhi, Staphylococcus aureus and Shigella flexneri) and three yeasts species (Candida albicans, Candida parapsilosis and Cryptococcus neoformans). Compounds 1 and 2 exhibited both antibacterial and antifungal activities that varied between the microbial species (MIC = 7.81-125 mu g/mL) while S. flexneri and C. albicans were sensitive to all the tested compounds. (C) 2012 Phytochemical Society of Europe. Published by Elsevier B. V. All rights reserved.

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New dimeric and monomeric chromanones, gonytolides D-G, isolated from the fungus Gonytrichum sp.

Haruhisa Kikuchi, Masato Isobe, Shoichiro Kurata, Yasuhiro Katou, Yoshiteru Oshima

TETRAHEDRON (PERGAMON-ELSEVIER SCIENCE LTD) 68 ( 31 ) 6218 - 6223 2012.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4020

In the course of the screening of innate immune regulators from natural resources, we isolated new dimeric and monomeric chromanones, gonytolides D G (4-7), from Gonytrichum sp. along with a known innate immune promoter, gonytolide A (1). The structures of these compounds were determined by spectral analysis, chemical conversion, and biogenetic consideration. The compounds 4-7 were evaluated on the innate immune response, indicating that the 8,8'-linked bischromanone rings were crucial for innate immune-promoting activity. (C) 2012 Elsevier Ltd. All rights reserved.

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Apoptotic effects of satratoxin H is mediated through DNA double-stranded break in PC12 cells

Punnee Nusuetrong, Masaki Saito, Haruhisa Kikuchi, Yoshiteru Oshima, Takahiro Moriya, Norimichi Nakahata

JOURNAL OF TOXICOLOGICAL SCIENCES (JAPANESE SOC TOXICOLOGICAL SCIENCES) 37 ( 4 ) 803 - 812 2012.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0388-1350

Satratoxin H is an important air- and food-borne mycotoxin, which has been implicated in human health damage. Satratoxin H is known to induce apoptosis as well as genotoxicity in PC12 cells. In the present study, we further investigated the mechanism of apoptotic effects of satratoxin H with focus on caspase-3 and poly-ADP-ribose polymerase (PARP) pathway. We also examined whether it induces DNA damage in PC12 cells. In the cells treated with satratoxin H, caspase-3 was cleaved in a time-dependent manner. Furthermore, satratoxin H induced cleavage of PARP, one of the downstream molecules of caspase-3. The cleavage was inhibited by SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor. Satratoxin H, however, had no effect on expression levels of Bax and Bcl-2. Furthermore, the micronucleus assay revealed that satratoxin H induced chromosome break. Also, satratoxin H increased the level of phosphorylation of histone H2A, indicating that it caused DNA double-stranded breaks in PC12 cells. Meanwhile, no genotoxicity was detected with any of treatments carried out in the alkaline comet assay. These results imply that satratoxin H induces genotoxicity by DNA double-stranded break. Our results suggest a considerable potential for the genotoxic risk associated with the presence of satratoxin H.

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Labdane-type diterpenes and flavones from Dodonaea viscosa

Hippolyte Kamdem Wabo, Philippe Chabert, Pierre Tane, Olivier Note, Michel Feussi Tala, Jean Peluso, Christian Muller, Haruhisa Kikuchi, Yoshiteru Oshima, Annelise Lobstein

FITOTERAPIA (ELSEVIER SCIENCE BV) 83 ( 5 ) 859 - 863 2012.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0367-326X

A new labdane diterpenoid, 2,18-dihydroxylabda-7,13(E)-dien-15-oic acid (1), together with two known labdane diterpenes (3, 4), a new flavone, 5,7-dihydroxy-3,6,4'-trimethoxy-3'-(4-hydroxy-3-methyl-but-2-enyl)flavone (2) with three known flavones (5-7) were isolated from the aerial parts of Dodonaea viscosa. Their structures were determined by extensive analysis of spectroscopic data (1D and 2D NMR, MS) and by comparison with literature data. The anti-inflammatory activity of five compounds (1-5) was evaluated with a flow cytometry TNF-alpha secretion assay on human THP-1 cell line. (C) 2012 Elsevier B.V. All rights reserved.

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Br-DIF-1 accelerates dimethyl sulphoxide-induced differentiation of P19CL6 embryonic carcinoma cells into cardiomyocytes

K. Seya, K. Kanemaru, M. Matsuki, K. Hongo, H. Kitahara, H. Kikuchi, Y. Oshima, Y. Kubohara, K. Okumura, S. Motomura, K-I Furukawa

BRITISH JOURNAL OF PHARMACOLOGY (WILEY-BLACKWELL) 165 ( 4 ) 870 - 879 2012.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0007-1188

BACKGROUND AND PURPOSE
Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte-like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation-inducing factor-1 (DIF-1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells.
EXPERIMENTAL APPROACH
P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca2+ channel blocker.
KEY RESULTS
Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3-3 mu M) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca2+ channel blocker; 100 nM) and a small interfering RNA for the T-type Ca2+ channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO.
CONCLUSIONS AND IMPLICATIONS
Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca2+ channel gene.

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Phenolic Compounds and Terpenoids from Hypericum lanceolatum

Hippolyte K. Wabo, Theodora K. Kowa, Antoine Honore N. Lonfouo, Alembert T. Tchinda, Pierre Tane, Haruhisa Kikuchi, Michel Frederich, Yoshiteru Oshima

RECORDS OF NATURAL PRODUCTS (ACG PUBLICATIONS) 6 ( 2 ) 94 - 100 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 1307-6167

A benzophenone, 2,2',5,6'-tetrahydroxybenzophenone (1), and one xanthone, 5-hydroxy-3-methoxyxanthone (2), were newly described as natural products from the leaves and the stem barks of Hypericum lanceolatum, along with the known compounds friedelin (3), betulinic acid (4), allanxanthone A (5), 1,3,6-trihydroxyxanthone (6), isogarcinol (7), sitosterol 3-O-beta-D-glucopyranoside (8), 1-hydroxy-6-methoxyxanthone (9), 6,7-dihydroxy-1,3-dimethoxyxanthone (10), 3-hydroxy-5-methoxyxanthone (11), 1,7-dihydroxy-3,6-dimethoxyxanthone (12) and calophyllumin A (13). Their structures were elucidated by spectroscopic means and comparison with published data.

Coupling of Two Diazotized 3-Aminothieno[3,4-c]coumarins with Aromatic Amines

Emmanuel Sopbue Fondjo, Joseph Tsemeugne, Beibam Luc Sondengam, Thomas Oppenlaender, Hippolyte Kamdem Wabo, Pierre Tane, Joseph D. Connolly, Wim Dehaen, Taoufik Rohand, Haruhisa Kikuchi, Yoshiteru Oshima

JOURNAL OF HETEROCYCLIC CHEMISTRY (WILEY-BLACKWELL) 48 ( 6 ) 1295 - 1301 2011.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-152X

The coupling reactions of two diazotized 3-aminothieno[3,4-c]coumarins were investigated. Compounds 1a,b both react with sodium nitrite in concentrated sulphuric acid at 0-5 degrees C to give the diazotized intermediates 2 and 3, the latter resulting from the acid -catalyzed hydrolysis of the lactonic ring of 2. The in situ formed diazonium salts react with aromatic amines (4) to afford a series of arylazothiophenes dyes in the form of their ammonium sulfate salts. With diazotized aniline, besides the normally expected phenylazothiophene 10 from the reaction with compound 1a, the corresponding product of acid hydrolysis 11 was also isolated. In at least one of the cases, the thienyl diazonium salt 2 undergoes a Gomberg-Bachmann arylation reaction with p-nitroaniline to give the 2-arylthiophene 9. The direct hydrolysis of compounds 1a, b by concentrated sulphuric acid and subsequent oxidative dimerization of the primary product of acid hydrolysis led to compound 12.

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A phytoceramide analog stimulates the production of chemokines through CREB activation in human endothelial cells

Mizuki Sekiya, Kazunori Ueda, Kaori Okazaki, Jun Terashima, Yasuhiro Katou, Haruhisa Kikuchi, Shoichiro Kurata, Yoshiteru Oshima

INTERNATIONAL IMMUNOPHARMACOLOGY (ELSEVIER SCIENCE BV) 11 ( 10 ) 1497 - 1503 2011.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 1567-5769

Innate immunity is the front-line of self-defense against microbial infection. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. From a pharmaceutical point of view, innate immunity is an ideal target for the development of immunoregulators. Therefore, we aimed to isolate and characterize a novel mammalian immunoregulator isolated from the thermophilic cellulotic fungus Talaromyces sp. 2'-(R)-hydroxy-C(24) phytoceramide (C(24)(2'OH)Phy) was isolated from Talaromyces sp. using a Drosophila ex vivo culture system. C(24)(2'OH)Phy suppressed the immune deficiency (IMD) pathway-dependent expression of antibacterial peptides in Drosophila, whereas it stimulated the production of chemokines in human cells. Structure activity relationship studies of C(24)(2'OH) Phy analogs revealed that both the 2'-(R)-hydroxylignoceroyl group and phytoceramide backbone are essential for the biologic activity of C(24)(2'OH)Phy. Microarray analysis revealed that C(24)(2'OH)Phy selectively activates the transcription of inflammatory response genes, including chemokines. Furthermore, a reporter gene assay and small interfering RNA analysis demonstrated that C(24)(2'OH)Phy stimulates chemokine production through cAMP response element-binding protein activation in human cells. C(24)(2'OH)Phy may be a lead immunostimulating compound in humans. (C) 2011 Elsevier B.V. All rights reserved.

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Structures of the Dimeric and Monomeric Chromanones, Gonytolides A-C, Isolated from the Fungus Gonytrichum sp and Their Promoting Activities of Innate Immune Responses

Haruhisa Kikuchi, Masato Isobe, Mizuki Sekiya, Yuko Abe, Tsuyoshi Hoshikawa, Kazunori Ueda, Shoichiro Kurata, Yasuhiro Katou, Yoshiteru Oshima

ORGANIC LETTERS (AMER CHEMICAL SOC) 13 ( 17 ) 4624 - 4627 2011.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 1523-7060

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Antimicrobial and antioxidant properties of methanol extract, fractions and compounds from the stem bark of Entada abyssinica Stend ex A. Satabie

Gerald N. Teke, Paul K. Lunga, Hippolyte K. Wabo, Jules-Roger Kuiate, Gerard Vilarem, Geraldine Giacinti, Haruhisa Kikuchi, Yoshiteru Oshima

BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE (BIOMED CENTRAL LTD) 11 2011.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 1472-6882

Background: The aim of this study was to evaluate the antimicrobial and antioxidant activities of the methanol extract, fractions and isolated compounds from Entada abyssinica stem bark, plant used traditionally against gastrointestinal infections.
Methods: The methanol extract of E. abyssinica stem bark was pre-dissolved in a mixture of methanol and water, and then partitioned between n-hexane, ethyl acetate and n-butanol. The ethyl acetate portion was fractionated by column chromatography and the structures of isolated compounds elucidated by analysis of spectroscopic data and comparison with literature data. Antimicrobial activity was assayed by broth microdilution techniques on bacteria and yeasts. The antioxidant activity was determined by DPPH radical scavenging method.
Results: Four known compounds [(5S,6R,8aR)-5-(carboxymethyl)-3,4,4a,5,6,7,8,8a-octahydro-5,6,8a-trimethylnaphthalenecarboxylic acid (1), methyl 3,4,5-trihydroxybenzoate (2), benzene-1,2,3-triol (3) and 2,3-dihydroxypropyltriacontanoate (4)] were isolated. Compared to the methanol extract, fractionation increased the antibacterial activities of the n-hexane and ethyl acetate fractions, while the antifungal activities increased in ethyl acetate, n-butanol and aqueous residue fractions. The isolated compounds were generally more active on bacteria (9.7 to 156.2 mu g/ml) than yeasts (78.1 to 312.5 mu g/ml). Apart from compound 1, the three others displayed DPPH(center dot) scavenging activity (RSa), with RSa(50) values of 1.45 and 1.60 mu g/ml.
Conclusion: The results obtained from this study support the ethnomedicinal use of E. abyssinica in the treatment of gastrointestinal infections and the isolated compounds could be useful in the standardisation of antimicrobial phytomedicine from this plant.

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Atg1 allows second-signaled autophagic cell death in Dictyostelium

Marie-Francoise Luciani, Corinne Giusti, Birthe Harms, Yoshiteru Oshima, Haruhisa Kikuchi, Yuzuru Kubohara, Pierre Golstein

AUTOPHAGY (LANDES BIOSCIENCE) 7 ( 5 ) 501 - 508 2011.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 1554-8627

We investigated the role of Atg1 in autophagic cell death (ACD) in a Dictyostelium monolayer model. The model is especially propitious, not only because of genetic tractability and absence of apoptosis machinery, but also because induction of ACD requires two successive exogenous signals, first the combination of starvation and cAMP, second the differentiation factor DIF-1. This enables one to analyze separately first-signal-induced autophagy and subsequent second-signal-induced ACD. We used mutants of atg1, a gene that plays an essential role in the initiation of autophagy. Upon starvation/cAMP, in contrast to parental cells, atg1 mutant cells showed irreversible lesions, clearly establishing a protective role for Atg1. Upon subsequent exposure to DIF-1 or to more ACD-specific second signals, starved parental cells progressed to ACD, but starved atg1 mutant cells did not, showing that Atg1 was required for ACD. Thus, in the same cells Atg1 was required in two apparently opposite ways, upon first-signaling for cell survival and upon second-signaling for ACD. Our findings strongly suggest that Atg1, thus presumably autophagy, protects the cells from starvation-induced cell death, allowing subsequent induction of ACD by the second signal. ACD is therefore not only "with" autophagy (since it showed signs of autophagy throughout), but is also "allowed by" autophagy. This does not exclude a role for autophagy also after second signaling. These results may account for discrepancies reported in the literature, encourage searches for second signals in different developmental models of ACD, and incite caution in autophagy-related therapeutic attempts.

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Synthesis and innate immunosuppressive effect of 1,2-cyclopentanediol derivatives

Haruhisa Kikuchi, Kaori Okazaki, Mizuki Sekiya, Yasuyuki Uryu, Kazunori Ueda, Yasuhiro Katou, Shoichiro Kurata, Yoshiteru Oshima

EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY (ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER) 46 ( 4 ) 1263 - 1273 2011.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0223-5234

Innate immunity is the front line of self-defense against infectious microorganisms. In mammals, innate immunity interacts with adaptive immunity and plays a key role in regulating the immune response. Therefore, innate immunity is a good pharmaceutical target for the development of immune regulators. After searching for natural substances that regulate innate immunity using an ex vivo Drosophila culture system, we identified a cyclopentanediol-type compound 1 as an immunosuppressor. In this study, we synthesized and evaluated 1 and its derivatives. Several methylamide- or phenylamide-containing derivatives showed effects that were 20-25 times more potent than those of 1. (c) 2011 Elsevier Masson SAS. All rights reserved.

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Derivatives of Dictyostelium differentiation-inducing factors promote mitogen-activated IL-2 production via AP-1 in Jurkat cells

Katsunori Takahashi, Masami Murakami, Haruhisa Kikuchi, Yoshiteru Oshima, Yuzuru Kubohara

LIFE SCIENCES (PERGAMON-ELSEVIER SCIENCE LTD) 88 ( 11-12 ) 480 - 485 2011.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 0024-3205

Aims: Differentiation-inducing factors (DIFs) are chlorinated alkylphenones found in the cellular slime mold Dictyostelium discoideum. DIF derivatives exhibit antiproliferative activities and promote glucose consumption in mammalian cells in vitro. Here, we assessed the ability of DIFs to regulate the immune system in a mammalian cell-line and investigated their mechanisms of action.
Main methods: We examined the effects of 30 DIF derivatives on concanavalin A-induced interleukin-2 (IL-2) production (CIIP) in Jurkat T-cells. We also examined the effects of these DIF derivatives on the activity of three transcription factors required for CIIP: namely, activator protein-1 (AP-1), nuclear factor of activated T-cells (NFAT), and nuclear factor kappa B (NF kappa B).
Key findings: A reporter gene assay suggested that 2 DIF derivatives, termed DIF-1(+ 1) and DIF-3(3 M), significantly promoted CIIP in Jurkat cells, at least in part, by enhancing the activity of AP-1. These 2 DIF derivatives had no significant effect on concanavalin A-induced interferon-gamma production.
Significance: The results suggest that DIF derivatives could be developed as novel drugs for the activation of IL-2 production and resultant stimulation of the immune system. (C) 2011 Elsevier Inc. All rights reserved.

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Artificial Compounds Differentially Control Dictyostelium Chemotaxis and Cell Differentiation

Hidekazu Kuwayama, Haruhisa Kikuchi, Yoshiteru Oshima, Yuzuru Kubohara

CELL STRUCTURE AND FUNCTION (JAPAN SOC CELL BIOLOGY) 36 ( 1 ) 21 - 26 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 0386-7196

Differentiation- inducing factor-1 and -2 (DIF-1 and DIF-2) are small lipophilic signal molecules that control both cell differentiation and chemotaxis in the cellular slime mold Dictyostelium discoideum. In this study, we examined the effects of four amide derivatives of DIF-1 on stalk cell differentiation and chemotaxis. The DIF derivatives differentially affected cell differentiation and chemotaxis, suggesting the possible existence of at least three receptors for DIFs: one receptor responsible for stalk cell induction, and two receptors responsible for chemotaxis modulation. Furthermore, our results indicate that DIF derivatives can be utilized to analyze the DIF-signaling pathways.

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Inhibitory effect of hericenone B from Hericium erinaceus on collagen-induced platelet aggregation

Koichiro Mori, Haruhisa Kikuchi, Yutaro Obara, Masaya Iwashita, Yoshihito Azumi, Satomi Kinugasa, Satoshi Inatomi, Yoshiteru Oshima, Norimichi Nakahata

PHYTOMEDICINE (ELSEVIER GMBH, URBAN & FISCHER VERLAG) 17 ( 14 ) 1082 - 1085 2010.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0944-7113

Platelet aggregation in the blood vessel causes thrombosis. Therefore, inhibitors of platelet aggregation promise to be preventive or therapeutic agents of various vascular diseases, including myocardial infarction and stroke. In the present study, we found that hericenone B had a strong anti-platelet activity and it might be a novel compound for antithrombotic therapy possessing a novel mechanism. Prior to this study, we examined anti-platelet aggregation activity of ethanol extracts of several species of mushrooms, and found that extract of Hericium erinaceus potently inhibited platelet aggregation induced by collagen. Therefore, we first fractionated the ethanol extract of H. erinaceus to identify the active substances. The anti-platelet activity of each fraction was determined using washed rabbit platelets. As a result, an active component was isolated and identified as hericenone B. Hericenone B selectively inhibited collagen-induced platelet aggregation, but it did not suppress the aggregation induced by U46619 (TXA(2) analogue), ADP, thrombin, or adrenaline. Furthermore, hericenone B did not inhibit arachidonic acid- or convulxin (GPVI agonist)-induced platelet aggregation. Therefore, hericenone B was considered to block collagen signaling from integrin alpha 2/beta 1 to arachidonic acid release. Moreover, we found that collagen-induced aggregation was inhibited by hericenone B in human platelets, similar to in rabbit platelets. (C) 2010 Elsevier GmbH. All rights reserved.

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Exploitation of new biological resources, entomopathogenic fungi and oomycetes

Kikuchi Haruhisa, Sato Yuichi, Hoshi Tomoko, Kitayama Minoru, Sekiya Mizuki, Isobe Masato, Mihara Ken, Katou Yasuhiro, Kurata Shoichiro, Oshima Yoshiteru, Kubohara Yuzuru, Sato Hiroki, Shimazu Mitsuaki

Symposium on the Chemistry of Natural Products, symposium papers (Symposium on the chemistry of natural products) ( 52 ) 703 - 708 2010.09

Research paper (scientific journal), Joint Work, Accepted

Entomopathogenic fungi infect insects through the cuticle, grow as hyphal bodies or hyphae in the hemocoel, and cause host death by nutritional destruction of tissues, and producint toxic metabolites and pathogenic enzymes. To date, entomopathogenic fungi have not been extensively studied by natural product chemists. We focused on entomopathogenic fungi as new resources for biologically active compounds, and studied the diversity of their secondary metabolites. In this study, we show the isolation and structure elucidation of novel pyrone diterpene-type compounds, metarhizin A-H (1-8) from Metarhizium flavoviride, and their antiproliferative activities on human cancer cell lines. Additionally, we synthesized many metarhizin derivatives, evaluated their antiproliferative activities, and discuss the structural requirements for this activity. A novel pyrone diterpene-type compound, Nf-1 (9), was also isolated from Nectria flammea. Oomycetes from fungus-like mycelium and were calssified as fungi in the past. However, the cell walls of them are c omposed of cellulose rather than chitin, as in the fungi. In the present, the ultrastructure, biochemistry and molecular sequences of them indicate that the oomycetes belong to the Kingdom Chromista, not the Kingdom Fungi. We also focused on oomycetes as new resources for natural product chemistry. A new oxidized lanostane-type compound, St-1 (23), was isolated from the mycelium of oomycete Saprolegnia terrestris. The structure of 23 was determined by spectral means including EIMS, and ^1H and ^<13>C NMR.

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Novel prenylated and geranylated aromatic compounds isolated from Polysphondylium cellular slime molds

Haruhisa Kikuchi, Shinya Ishiko, Koji Nakamura, Yuzuru Kubohara, Yoshiteru Oshima

TETRAHEDRON (PERGAMON-ELSEVIER SCIENCE LTD) 66 ( 32 ) 6000 - 6007 2010.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4020

We have studied the diversity of secondary metabolites of cellular slime molds to utilize them as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium tenuissimum, we obtained five prenylated and geranylated aromatic compounds, Pt-1-5 (1-5). An additional aromatic compound, Ppc-1 (6), was isolated from Polysphondylium pseudo-candidum. The structures of these compounds were determined by spectral analysis, and synthetic routes to 4, 5, and 6 were developed. Compound 5 showed the glucose consumption-promotive activity on 3T3-L1 cells. (c) 2010 Elsevier Ltd. All rights reserved.

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Xanthones and a benzophenone from the roots of Pentadesma butyracea and their antiproliferative activity

Hippolyte K. Wabo, Haruhisa Kikuchi, Yasuhiro Katou, Pierre Tane, Yoshiteru Oshima

PHYTOCHEMISTRY LETTERS (ELSEVIER SCIENCE BV) 3 ( 2 ) 104 - 107 2010.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 1874-3900

A new xanthone, 3,4-dihydro-8,10,12-trihydroxy-2,2-dimethylpyrano[2,3-b]xanthen-11(2H)-one or butyraxanthone E (1), along with the known compounds 30-epi-cambogin (2), 1,7-dihydroxyxanthone (3) and 1,5-dihydroxyxanthone (4) were isolated from the roots of Pentadesma butyracea. Their structures were elucidated by spectroscopic means and comparison with published data. Their antiproliferative activities were evaluated against Drosophila S2 cells and two human cancer cell lines, THP-1 (leukemia) and HCT116 (colon cancer). Compounds 1 and 2 showed moderate antiproliferative activity against Drosophila S2 cells and the HCT116 cell line, respectively. Compound 2 was active against Drosophila S2 cells. (C) 2010 Phytochemical Society of Europe. Published by Elsevier B. V. All rights reserved.

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Preparation of an antibody that recognizes and neutralizes Dictyostelium differentiation-inducing factor-1

Yuzuru Kubohara, Haruhisa Kikuchi, Koji Nakamura, Yusuke Matsuo, Yoshiteru Oshima

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (ACADEMIC PRESS INC ELSEVIER SCIENCE) 396 ( 2 ) 364 - 369 2010.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

In the development of the cellular slime mold Dictyostelium discoideum the differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) plays an important role in the regulation of cell differentiation and chemotaxis; however, the cellular signaling systems involving DIF-1 remain to be elucidated. To obtain a probe for DIF-1, we synthesized a DIF derivative (DIF-1-NH(2); 6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), and prepared an anti-DIF-1 antibody using a DIF-1-NH(2)-conjugated macromolecule as the immunogen. A 100-fold dilution of the antibody bound to DIF-1-NH(2)-conjugated resin, and this binding was inhibited by co-addition of 20 mu M DIF-1 or DIF-1-NH(2). In a monolayer culture of HM44 cells, a DIF-deficient D. discoideum strain, 0.5 nM exogenous DIF-1 induced stalk cell formation in similar to 60% of the cells; this induction was dose-dependently inhibited by the antibody (diluted 12.5- or 25-fold). Furthermore, this inhibition by the antibody was recovered by co-addition of 2.5 or10 nM DIF-1. The results indicate that the anti-DIF-1 antibody recognizes DIF-1 and neutralizes its function. (C) 2010 Elsevier Inc. All rights reserved.

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Antimycobacterial Flavonoids from the Leaf Extract of Galenia africana

Sannah P. N. Mativandlela, Tshilidzi Muthivhi, Haruhisa Kikuchi, Yoshiteru Oshima, Chris Hamilton, Ahmed A. Hussein, Martha L. van der Walt, Peter J. Houghton, Namrita Lall

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 72 ( 12 ) 2169 - 2171 2009.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0163-3864

The bioassay-guided fractionation of the EtOH extract of the leaves of Galenia africana led to the isolation of three known flavonoids, (2S)-5,7,2'-trihydroxyflavanone (1), (E)-3,2',4'-trihydroxychalcone (2), and (E)-2',4'-dihydroxychalcone (3), and the new (E)-3,2',4'-trihydroxy-3'-methoxychalcone (4). Compounds 1 and 3 exhibited moderate antituberculosis activity. During synergistic studies, a combination of compound 4 and an existing antituberculosis drug, isoniazid, reduced their original MICs 4-fold, resulting in a fractional inhibitory concentration of 0.50. The most pronounced effect was demonstrated by compound I and isoniazid reducing their MICs 16-fold and resulting in an FIC of 0.12. Both EtOH extract and isolated compounds failed to exhibit any NADPH oxidase activity at 800.0 mu M concentrations, indicating that mycothiol disulfide reductase is not the target for their antituberculosis activity.

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Exploration of Innate Immune Regulators from Natural Resources

Kikuchi Haruhisa, Sekiya Mizuki, Isobe Masato, Kabeya Takahiro, Nakamura Tetsuya, Ueda Kazunori, Katou Yasuhiro, Kurata Shoichiro, Oshima Yoshiteru

Symposium on the Chemistry of Natural Products, symposium papers (Symposium on the chemistry of natural products) ( 51 ) 545 - 550 2009.09

Research paper (scientific journal), Joint Work, Accepted

Innate immunity is the first line of defense against infectious microorganisms, and the basic mechanisms of this process, including pathogen recognition and immune response activation, are evolutionarily conserved. In mammals, the innate immunity has an instructive role in the adaptive immune response specific to antigen by inducing co-stimulatory molecules and cytokines, indicating that a concerted and interactive innate and adaptive immune reaction is key for the regulated immune response. Therefore, from a pharmaceutical point of view, innate immunity is a good target for the development of immune regulators to suppress unwanted immune responses, such as septic shock, inflammatory diseases and autoimmunity, and to stimulate protective immune responses to some of the disease that largely elude the immune system, such as infectious diseases and cancer. To screen pharmaceuticals that target innate immunity, we established an ex vivo culture system based on the innate immune response of Drosophila, which is highly useful for idintifying immune regulators that act on human innate immunity. We used this system to search for natural substances that regulate innate immunity, and identified celastramycin A (1) and TP-554 (2) as a potent suppressor and an activator, respectively. The previously reported structure 3 for celastramycin A was corrected to 1 by synthesizing both compounds. In addition, we synthesized some derivatives of celastramycin A (1) to evaluate structure-activity relationship of these compounds.

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Regulation of IL-2 production in Jurkat cells by Dictyostelium-derived factors

Katsunori Takahashi, Masami Murakami, Kohei Hosaka, Haruhisa Kikuchi, Yoshiteru Oshima, Yuzuru Kubohara

LIFE SCIENCES (PERGAMON-ELSEVIER SCIENCE LTD) 85 ( 11-12 ) 438 - 443 2009.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0024-3205

Aims: Differentiation-inducing factors (DIFs) are chlorinated alkylphenones found in the cellular slime mold Dictyostelium discoideum. DIF derivatives exhibit anti proliferative activities and promote glucose consumption in mammalian cells in vitro. In this study, we assessed the ability of DIFs to regulate the immune system and investigated their mechanisms of action.
Main methods: We examined the effects of 30 DIF derivatives on concanavalin A-induced IL-2 production (CIIP) in Jurkat T-cells. We also examined the effects of some DIF derivatives on the activity of AP-1 (activator protein-1), NFAT (nuclear factor of activated T-cells), and NF kappa B (nuclear factor kappa B), which are transcription factors required for CIIP.
Key findings: Of the derivatives tested, some compounds suppressed CIIP as well as the known immunosuppressants cyclosporine A and FK506. A reporter gene assay revealed that 4 DIF derivatives tested suppressed CHIP, at least in part, by inhibiting the activity of AP-1, NFAT, and/or NF kappa B. Unlike cyclosporine A and FK506, the DIF derivatives had little effect on concanavalin A-induced interferon-gamma production in Jurkat cells.
Significance: The present results suggest that DIF derivatives could be developed as novel immunosuppressive drugs. (C) 2009 Elsevier Inc. All rights reserved.

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A reduction of epidermal growth factor receptor is involved in brefelamide-induced inhibition of phosphorylation of ERK in human astrocytoma cells

Shigeyoshi Honma, Masaki Saito, Haruhisa Kikuchi, Yoshinori Saito, Yoshiteru Oshima, Norimichi Nakahata, Makoto Yoshida

EUROPEAN JOURNAL OF PHARMACOLOGY (ELSEVIER SCIENCE BV) 616 ( 1-3 ) 38 - 42 2009.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0014-2999

Brefelamide is an aromatic amide isolated from Dictyostelium cellular slime molds. We found that brefelamide has a potent inhibitory growth effect measured by MTT assay in 1321N1 human astrocytoma cells. The inhibition was associated with reduced phosphorylation of extracellular signal-regulated kinase (ERK). Brefelamide inhibited epidermal growth factor (EGF)-induced phosphorylation of ERK in a concentration-dependent manner. Furthermore, brefelamide diminished EGF-induced phosphorylation of EGF receptor at Tyr(1068), a Grb2 binding site that leads to an activation of the Ras/Raf/ERK system. Brefelamide also reduced the expression level of the EGF receptor. These results suggest that one of the mechanisms of action of brefelamide is assumed to be inhibition of phosphorylation of ERK through a reduction of EGF receptor activity in 1321 NI human astrocytoma cells. (C) 2009 Elsevier B.V. All rights reserved.

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Revised Structure and Synthesis of Celastramycin A, A Potent Innate Immune Suppressor

Haruhisa Kikuchi, Mizuki Sekiya, Yasuhiro Katou, Kazunori Ueda, Takahiro Kabeya, Shoichiro Kurata, Yoshiteru Oshima

ORGANIC LETTERS (AMER CHEMICAL SOC) 11 ( 8 ) 1693 - 1695 2009.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 1523-7060

After searching for natural substances that regulate innate immunity using the ex vivo Drosophila culture system, a benzoyl pyrrole-type compound, celastramycin A, was identified and isolated as a potent suppressor. By synthesizing the previously reported structure 1 and another benzoyl pyrrole-type compound 2 reported in a Japanese patent, the correct structure of celastramycin A was confirmed to be 2. Compound 2 suppressed the production of IL-8 (IC(50) 0.06 mu g/mL) in human umbilical vein endothelial cells (HUVECs).

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Autophagic or necrotic cell death triggered by distinct motifs of the differentiation factor DIF-1

M. F. Luciani, Y. Kubohara, H. Kikuchi, Y. Oshima, P. Golstein

CELL DEATH AND DIFFERENTIATION (NATURE PUBLISHING GROUP) 16 ( 4 ) 564 - 570 2009.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 1350-9047

Autophagic or necrotic cell death (ACD and NCD, respectively), studied in the model organism Dictyostelium which offers unique advantages, require triggering by the same differentiation-inducing factor DIF-1. To initiate these two types of cell death, does DIF-1 act through only one or through two distinct recognition structures? Such distinct structures may recognize distinct motifs of DIF-1. To test this albeit indirectly, DIF-1 was modified at one or two of several positions, and the corresponding derivatives were tested for their abilities to induce ACD or NCD. The results strongly indicated that distinct biochemical motifs of DIF-1 were required to trigger ACD or NCD, and that these motifs were separately recognized at the onset of ACD or NCD. In addition, both ACD and NCD were induced more efficiently by DIF-1 than by either its precursors or its immediate catabolite. These results showed an unexpected relation between a differentiation factor, the cellular structures that recognize it, the cell death types it can trigger and the metabolic state of the cell. The latter seems to guide the choice of the signaling pathway to cell death, which in turn imposes the cell death type and the recognition pattern of the differentiation factor.

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Peltophorum Africanum, a Traditional South African Medicinal Plant, Contains an Anti HIV-1 Constituent, Betulinic Acid

Andros Theo, Tracy Masebe, Yasuhiro Sijzuki, Haruhisa Kikuchi, Shoko Wada, Chikwelu Larry Obi, Pascal Obong Bessong, Motoki Usuzawa, Yoshiteru Oshima, Toshio Hattori

TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE (TOHOKU UNIV MEDICAL PRESS) 217 ( 2 ) 93 - 99 2009.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-8727

The biodiversity of medicinal plants in South Africa makes them rich sources of leading compounds for the development of novel drugs. Peltophorum africanum (Fabaceae) is a deciduous tree widespread in South Africa. The stem bark has been traditionally employed to treat diarrhoea, dysentery, sore throat, wounds, human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS), venereal diseases and infertility. To evaluate these ethnobotanical clues and isolate lead compounds, butanol and ethyl acetate extracts of the stem bark were screened for their inhibitory activities against HIV-1 using MAGI CCR5+ cells, which are derived from HeLa cervical cancer cells and express HIV receptor CD4, a chemokine receptor CCR5 and HIV-LTR-/3- galactosidase. Bioassay-guided fractionation using silica gel chromatography was also conducted. The ethyl acetate and butanol extracts of the stem bark of Peltophorum africanum showed inhibitory activity against HIV-1, CXCR4 (X4) and CCR5 (R5) tropic viruses. The ethyl acetate and butanol extracts yielded previously reported anti-HIV compounds, (+)-catechin, a flavonoid, and bergenin, a C-galloylglycoside, respectively. Furthermore, we identified betulinic acid from the ethyl acetate fraction for the first time. The fractions, which contained betulinic acid, showed the highest selective index. We therefore describe the presence of betulinic acid, a not well-known anti-HIV compound, in an African medicinal herb, which has been used for therapy, and claim that betulinic acid is the predominant anti-HIV-1 constituent of Peltophorum africanum. These data suggest that betulinic acid and its analogues could be used as potential therapeutics for HIV-1 infection.

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New diterpene pyrone-type compounds, metarhizins A and B, isolated from entomopathogenic fungus, Metarhizium flavoviride and their inhibitory effects on cellular proliferation

Haruhisa Kikuchi, Tomoko Hoshi, Minoru Kitayama, Mizuki Sekiya, Yasuhiro Katou, Kazunori Ueda, Yuzuru Kubohara, Hiroki Sato, Mitsuaki Shimazu, Shoichiro Kurata, Yoshiteru Oshima

TETRAHEDRON (PERGAMON-ELSEVIER SCIENCE LTD) 65 ( 2 ) 469 - 477 2009.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4020

To date, entomopathogenic fungi have not been extensively examined by natural product chemists. In this study, we isolated novel pyrone diterpene-type compounds, metarhizins A (1) and B (2), from methanol extracts of entomopathogenic fungus, Metarhizium flavoviride. They showed potent and selective antiproliferative activity against both insect and human cancer cell lines. These results indicate that metarhizins A (1) and B (2) can be used as novel lead compounds for anti-cancer agents and probes for cell cycle regulation. To further investigate the structural requirements for this inhibitory activity, we synthesized many metarhizin derivatives and evaluated their antiproliferative activities. (C) 2008 Elsevier Ltd. All rights reserved.

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Biological activities of novel derivatives of DIF-1 isolated from Dictyostelium

Haruhisa Kikuchi, Shinya Ishiko, Yoshiteru Oshima, Naomi Gokan, Kohei Hosaka, Yuzuru Kubohara

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (ACADEMIC PRESS INC ELSEVIER SCIENCE) 377 ( 3 ) 1012 - 1017 2008.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-291X

The differentiation-inducing factor-1 (DIF-1) is a lipophilic signal molecule (chlorinated alkylphenone) that induces stalk cell differentiation in the cellular slime mold Dictyostelium discoideum. in addition, DIF-1 and its derivatives have been shown to possess anti-leukemic activity and glucose Consumption-promoting activity in vitro in mammalian cells. In this study. to assess the chemical structure-effect relationship of DIF-1, we synthesized eight derivatives of DIF-1 and investigated their stalk cell-inducing activity in Dictyostelium cells and pharmacological activities in mammalian cells. Of the derivatives, two amide derivatives of DIF-1, whose hydrophobic indexes are close to that of DIF-1. induced stalk cell differentiation as strongly as DIF-1 in Dictyostelium cells. It Was also found that some derivatives Suppressed cell growth in human K562 leukemia cells and promoted glucose consumption in mouse 3T3-L1 cells. These results give LIS valuable information as to the chemical structure-effect relationship of DIF-1. (C) 2008 Elsevier Inc. All rights reserved.

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Exploitation of the derivatives of Dictyostelium differentiation-inducing factor-1, which promote glucose consumption in mammalian cells

Yuzuru Kubohara, Haruhisa Kikuchi, Yoshiteru Oshima

LIFE SCIENCES (PERGAMON-ELSEVIER SCIENCE LTD) 83 ( 17-18 ) 608 - 612 2008.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0024-3205

Aims: The differentiation-inducing factor-1 (DIF-1) is a signal molecule that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. DIF-1 has also been shown to possess pharmacological activities, such as the suppression of tumor cell growth and the promotion of glucose uptake in non-transformed mammalian cells. In this study, we tried to develop compounds that possess weaker anti-tumor activity and stronger glucose uptake-promoting activity than DIF-1.
Main methods: We investigated the in Vitro effects of 12 derivatives of DIF-1 on glucose consumption in mouse 3T3-L1 cells and on cell growth in K562 human leukemia cells. We also examined the effect of a good compound oil the blood glucose concentration in KK-Ay diabetic mice.
Key findings: We found that some derivatives at 20 mu M promoted glucose consumption more than twice as fast as the control. Of the derivatives, a compound named DIF-1(3M), which has a weaker anti-leukemic effect than DIF-1, promoted glucose consumption as strongly as DIF-1 in confluent 3T3-L1 cells. While DIF-1 at 20 mu M was inhibitory to the cell growth of 3T3-L1, DIF-1(3M) at 20 mu M exhibited no inhibitory effect on the growing cells. We also found that DIF-1(3M) injected (10-12.5 mg/kg body weight) intraperitoneally in mice tended to lower the blood glucose concentration.
Significance: The present results open the possibility for the development of new agents that possess strong glucose-Uptake-promoting activity but little anti-tumor activity and may have therapeutic potential for the treatment of diabetes and/or obesity. (C) 2008 Elsevier Inc. All rights reserved.

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Satratoxin H generates reactive oxygen species and lipid peroxides in PC12 cells

Punnee Nusuetrong, Thitima Pengsuparp, Duangdeun Meksuriyen, Masaaki Tanitsu, Haruhisa Kikuchi, Michinao Mizugaki, Ken-ichi Shimazu, Yoshiteru Oshima, Norimichi Nakahata, Makoto Yoshida

BIOLOGICAL & PHARMACEUTICAL BULLETIN (PHARMACEUTICAL SOC JAPAN) 31 ( 6 ) 1115 - 1120 2008.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0918-6158

Satratoxin H, a mycotoxin, is thought to induce apoptosis of PC12 cells through the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in a glutathione (GSH)-sensitive manner. The present study was undertaken to further elucidate the mechanism by which satratoxin H induces cell death in PC12 cells. Satratoxin H caused apoptosis of PC12 cells within 24-h, as determined by DNA fragmentation and flow cytometric analysis. Satratoxin H increased reactive oxygen species (ROS) production and lipid peroxidation, as determined by malondialdehyde formation. These effects were attenuated by incubation of cells with GSH, suggesting that satratoxin H-induced increase in apoptosis of serum-deprived PC12 cells may be partially mediated through the generation of ROS.

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A cyclopentanediol analogue selectively suppresses the conserved innate immunity pathways, Drosophila IMD and TNF-alpha pathways

Mizuki Sekiya, Kazunori Ueda, Kaori Okazaki, Haruhisa Kikuchi, Shoichiro Kurata, Yoshiteru Oshima

BIOCHEMICAL PHARMACOLOGY (PERGAMON-ELSEVIER SCIENCE LTD) 75 ( 11 ) 2165 - 2174 2008.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-2952

Innate immunity comprises evolutionarily conserved self-defense mechanisms against microbial infections. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Using Drosophila ex vivo culture systems, we isolated a cyclopentanediol analogue from Aspergillus sp. as an immunosuppressive substance. This compound selectively suppressed activation of the IMD pathway in Drosophila in vivo and the target molecules of the compound lie between the Imd adaptor protein and dTAK1 kinase in the IMD pathway. In human cells, the compound suppressed TNF-alpha, but not IL-1 beta, stimulation-induced activation of NF-kappa B, suggesting that its target molecules are upstream of TAK1 in mammalian innate immunity. (C) 2008 Elsevier Inc. All rights reserved.

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5,19-cyclo-9 beta,10 xi-androstane-3,17-dione promotes neurotrophic factor biosynthesis in 1321N1 human astrocytorna cells and improves passive avoidance learning impairment

Yutaro Obara, Asami Haganuma, Shinsuke Murakami, Toshiki Chiba, Koichiro Mori, Osamu Nakagawasai, Takeshi Tadano, Haruhisa Kikuchi, Yoshiteru Oshima, Norimichi Nakahata

BRAIN RESEARCH (ELSEVIER SCIENCE BV) 1184 ( 1 ) 57 - 64 2007.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-8993

Since neurotrophic factors are essential for neurons to form neuronal networks and maintain neuronal functions, neurotrophic factor-like substances or inducers of neurotrophic factors can be useful for the treatment of serious neuronal diseases such as Alzheimer's and Parkinson's diseases. In the present study, we examined an effect of 5,19-cyclo-9 beta,10 xi-androstane-3,17-dione (CAD) on neurotrophic factor synthesis in glial cells and scopolamine-induced impairment of learning in mice. 1321N1 human astrocytoma cells promoted secretion of certain neurotrophic factors in response to CAD with no cytotoxicity, which caused dramatic neurite outgrowth in rat pheochromocytoma (PC12) cells. In fact, CAD significantly enhanced nerve growth factor (NGF) secretion and its gene expression in 1321N1 cells, in a time and concentration-dependent manner. Because second messengers such as cAMP, inositol 1,4,5-trisphosphates and Ca2+ induce NGF gene expression, we measured activities of adenylyl cyclase and phospholipase C and intracellular Ca2+ concentration in 1321N1 cells. However, CAD changed neither second messenger levels. CAD enhanced the gene expression of proto-oncogene, c-fos that is one of the components of transcription factor (AP-1). In addition to those above, the in vivo effects of CAD were also examined. Although injection of muscarinic receptor antagonist scopolamine impaired passive avoidance learning in mice, pretreatment with CAD significantly reversed the adverse effect in a dose-dependent manner. Taking these results together, CAD has enormous therapeutic potential for serious neuronal diseases. (C) 2007 Elsevier B.V. All rights reserved.

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Dihydrodictyopyrone A and C: new members of dictyopyrone family isolated from Dictyostelium cellular slime molds

Haruhisa Kikuchi, Koji Nakamura, Yuzuru Kubohara, Naomi Gokan, Kohel Hosaka, Yasuo Maeda, Yoshiteru Oshima

TETRAHEDRON LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 48 ( 33 ) 5905 - 5909 2007.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4039

We have explored the diversity of secondary metabolites produced by cellular slime molds to evaluate if they are valuable resources for biologically potential substances. From the methanol extract of fruiting bodies of Dicillosteliumfirinibasis, we obtained new alpha-pyranoids, dihydrodictyopyrone A (1) and C (2). Their structures including absolute configurations were determined by spectyal means and asymmetric total synthesis. Compounds I and 2 are new members of the dictyopyrone family, which are characteristic secondary metabolites of various species of Dictyostelillin cellular slime molds. (c) 2007 Elsevier Ltd. All rights reserved.

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Dictyostelium differentiation-inducing factor-1 induces glucose transporter 1 translocation and promotes glucose uptake in mammalian cells

Waka Omata, Hiroshi Shibata, Masahiro Nagasawa, Itaru Kojima, Haruhisa Kikuchi, Yoshiteru Oshima, Kohei Hosaka, Yuzuru Kubohara

FEBS JOURNAL (BLACKWELL PUBLISHING) 274 ( 13 ) 3392 - 3404 2007.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 1742-464X

The differentiation-inducing factor-1 (DIF-1) is a signal molecule that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum, while DIF-1 and its analogs have been shown to possess antiproliferative activity in vitro in mammalian tumor cells. In the present study, we investigated the effects of DIF-1 and its analogs on normal (nontransformed) mammalian cells. Without affecting the cell morphology and cell number, DIF-1 at micromolar levels dose-dependently promoted the glucose uptake in confluent 3T3-L1 fibroblasts, which was not inhibited with wortmannin or LY294002 (inhibitors for phosphatidylinositol 3-kinase). DIF-1 affected neither the expression level of glucose transporter 1 nor the activities of four key enzymes involved in glucose metabolism, such as hexokinase, fluctose 6-phosphate kinase, pyruvate kinase, and glucose 6-phosphate dehydrogenase. Most importantly, stimulation with DIF-1 was found to induce the translocation of glucose transporter 1 from intracellular vesicles to the plasma membranes in the cells. In differentiated 3T3-L1 adipocytes, DIF-1 induced the translocation of glucose trasporter 1 (but not of glucose transporter 4) and promoted glucose uptake, which was not inhibited with wortmannin. These results indicate that DIF-1 induces glucose transporter 1 translocation and thereby promotes glucose uptake, at least in part, via a inhibitors for phosphatidylinositol 3-kinase/Akt-independent pathway in mammalian cells. Furthermore, analogs of DIF-1 that possess stronger antitumor activity than DIF-1 were less effective in promoting glucose consumption, suggesting that the mechanism of the action of DIF-1 for stimulating glucose uptake should be different from that for suppressing tumor cell growth.

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MalariaCount: An image analysis-based program for the accurate determination of parasitemia

Selena W. S. Sio, Weiling Sun, Saravana Kumar, Wong Zeng Bin, Soon Shan Tan, Sim Heng Ong, Haruhisa Kikuchi, Yoshiteru Oshima, Kevin S. W. Tan

JOURNAL OF MICROBIOLOGICAL METHODS (ELSEVIER SCIENCE BV) 68 ( 1 ) 11 - 18 2007.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0167-7012

Malaria is a serious global health problem and rapid, precise determination of parasitemia is necessary for malaria research and in clinical settings. Manual counting by light microscopy is the most widely used technique for parasitemia determination but it is a time-consuming and laborious process. The aim of our study was to develop an automated image analysis-based system for the rapid and accurate determination of parasitemia. We have developed, for the first time, a software, MalariaCount, that automatically generates parasitemias from images of Giemsa-stained blood smears. The potential application and robustness of MalariaCount was tested in normal and drug-treated in vitro cultures of Plasmodium falciparum. The results showed a tight correlation between MalariaCount and manual count parasitemia values. These findings suggest that MalariaCount can potentially be used as a tool to provide rapid and accurate determination of parasitemia in research laboratories where frequent, large-scale, efficient determination of parasitemia is required. (c) 2006 Elsevier B.V. All rights reserved.

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Anti-leukemic activities of Dictyostelium secondary metabolites: A novel aromatic metabolite, 4-methyl-5-n-pentylbenzene-1,3-diol, isolated from Dictyostelium mucoroides suppresses cell growth in human leukemia K562 and HL-60 cells

Haruhisa Kikuchi, Yoshiteru Oshima, Aya Ichimura, Naomi Gokan, Aiko Hasegawa, Kohei Hosaka, Yuzuru Kubohara

LIFE SCIENCES (PERGAMON-ELSEVIER SCIENCE LTD) 80 ( 2 ) 160 - 165 2006.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0024-3205

It has previously been shown that DIF-1, a differentiation-inducing factor of the cellular slime mold Dictyostelium discoideum, possesses antitumor activities in mammalian tumor cells and that neuronal differentiation of PC12 cells can be induced with furanodictines (FDs), aminosugar analogs found in D. discoideum, or dictyoglucosamines (DGs), N-acetyl glucosamine derivatives (DG-A from D. purpureum and DG-B from D. discoideum). Thus, cellular slime molds are attractive natural resources that may provide valuable lead compounds to be utilized in the field of pharmacology and medicine. In this study, we have isolated a novel aromatic compound, 4-methyl-5-n-pentylbenzene-1,3-diol (MPBD), from fruiting bodies of the cellular slime mold D. mucoroides and assessed the in vitro antiproliferative activities of MPBD, FDs, and DGs in human leukemia K562 and HL-60 cells. MPBD at 20-80 mu M dose-dependently suppressed cell growth in both K562 and HL-60 cells. While FDs at 10-80 mu M did not affect cell growth, DGs at 10-40 mu M dose-dependently suppressed cell growth in the cells. Although we failed to find the roles of FDs and DGs in the original organisms, MPBD at 5-20 mu M was found to promote stalk cell formation in D. discoideum. The present results indicate that MPBD, DGs or their derivatives may have therapeutic potential in the treatment of cancer and confirm our expectations regarding cellular slime molds as drug resources. (c) 2006 Elsevier Inc. All rights reserved.

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Establishment of ex vivo systems to identify compounds acting on innate immune responses and to determine their target molecules using transgenic Drosophila

Mizuki Sekiya, Kazunori Ueda, Tomomitsu Fujita, Minoru Kitayama, Haruhisa Kikuchi, Yoshiteru Oshima, Shoichiro Kurata

LIFE SCIENCES (PERGAMON-ELSEVIER SCIENCE LTD) 80 ( 2 ) 113 - 119 2006.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0024-3205

Innate immunity is an evolutionarily conserved self-defense mechanism against microbial infections. In Drosophila, induction of antimicrobial peptides is a major immune response that is regulated by two distinct signaling pathways called the IMD pathway and the Toll pathway, similar to the tumor necrosis factor-alpha signaling and Toll-like receptor/interleukin-1 signaling pathways, respectively, in mammals. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Previously, based on the striking conservation between the mechanisms that regulate Drosophila immunity and human innate immunity, we established an ex vivo culture in which compounds acting on innate immunity can be evaluated using a reporter gene that reflects activation of the IMD pathway [Yajima et al. [Yajima, M., Takada, M., Takahashi, N., Kikuchi, H., Natori, S., Oshima, Y., Kurata, S., 2003. A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity. The Biochemical Journal 37 1 (Pt 1), 205-210] Biochem J 371, 205-210]. Here, we combined the ex vivo culture with a reporter gene that reflects the heat shock response and demonstrated that the resulting systems are useful for screening compounds that act specifically on innate immunity, including mammalian innate immune responses. Identification of target molecules is essential for the development of more potent medicines with fewer side effects. In this study, we also established ex vivo systems capable of identifying target molecules of the identified compounds using targeted activation of the IMD pathway. (c) 2006 Elsevier Inc. All rights reserved.

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Isolation of Cholest-5-en-3-ol formate from the red alga Grateloupia turuturu Yamada and its chemotaxonomic significance

Erwan Plouguerne, Haruhisa Kikuchi, Yoshiteru Oshima, Eric Deslandes, Valerie Stiger-Pouvreau

BIOCHEMICAL SYSTEMATICS AND ECOLOGY (PERGAMON-ELSEVIER SCIENCE LTD) 34 ( 9 ) 714 - 717 2006.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0305-1978

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Exploration of a new type of antimalarial compounds based on febrifugine

Haruhisa Kikuchi, Keisuke Yamamoto, Seiko Horoiwa, Shingo Hirai, Ryota Kasahara, Norimitsu Hariguchi, Makoto Matsumoto, Yoshiteru Oshima

JOURNAL OF MEDICINAL CHEMISTRY (AMER CHEMICAL SOC) 49 ( 15 ) 4698 - 4706 2006.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-2623

Febrifugine (1), a quinazoline alkaloid, isolated from Dichroa febrifuga roots, shows powerful antimalarial activity against Plasmodium falciparum. The use of 1 as an antimalarial drug has been precluded because of side effects, such as diarrhea, vomiting, and liver toxicity. However, the potent antimalarial activity of 1 has stimulated medicinal chemists to pursue compounds derived from 1, which may be valuable leads for novel drugs. In this study, we synthesized a new series of febrifugine derivatives formed by structural modifications at (i) the quinazoline ring, (ii) the linker, or (iii) the piperidine ring. Then, we evaluated their antimalarial activities. Thienopyrimidine analogue 15 exhibited a potent antimalarial activity and a high therapeutic selectivity both in vitro and in vivo, suggesting that 15 is a good antimalarial candidate.

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Isolation and synthesis of a new aromatic compound, brefelamide, from Dictyostelium cellular slime molds and its inhibitory effect on the proliferation of astrocytoma cells

H Kikuchi, Y Saito, J Sekiya, Y Okano, M Saito, N Nakahata, Y Kubohara, Y Oshima

JOURNAL OF ORGANIC CHEMISTRY (AMER CHEMICAL SOC) 70 ( 22 ) 8854 - 8858 2005.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-3263

We have explored the diversity of secondary metabolites produced by cellular slime molds to examine the possible use of such cellular slime molds as a resource for novel drug development. A new aromatic amide, brefelamide (1), was isolated from methanol extracts of the fruiting bodies of Dictyostelium brefeldianum and D. giganteum. The structure of 1 was determined by spectral means including EIMS and H-1 and C-13 NMR. The total synthesis of 1 was carried out to confirm the structure and obtain sufficient samples for performing biological evaluation. Interestingly, compound 1 inhibited the cellular proliferation of 1321N1 human astrocytoma cells.

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Structural requirements of Dictyostelium differentiation-inducing factors for their stalk-cell-inducing activity in Dictyostelium cells and anti-proliferative activity in K562 human leukemic cells

N Gokan, H Kikuchi, K Nakamura, Y Oshima, K Hosaka, Y Kubohara

BIOCHEMICAL PHARMACOLOGY (PERGAMON-ELSEVIER SCIENCE LTD) 70 ( 5 ) 676 - 685 2005.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006-2952

The differentiation-inducing factor-1 (DIF-1) is a lipophilic signal molecule (chlorinated alkylphenone) that induces stalk-cell differentiation in the cellular slime mould Dictyostelium discoideum. It has also been shown that DIF-1 and its derivative (DIF-3) suppress cell growth in mammalian tumor cells. In the present study, in order to assess the chemical structure-effect relationship of DIF derivatives and to develop useful agents for the study of both Dictyostelium development and cancer biology, we synthesized 28 analogues of DIF-1 and DIF-3 and investigated their stalk-cell-inducing activity in Dictyostelium HM44 cells (mutant strain) and anti-proliferative activity in human leukemia K562 cells. HM44 cells are defective in endogenous DIF-1 production and should be suitable for the assay for stalk-cell-inducing activity of DIF analogues. DIF-1 and some of its derivatives at nanomolar levels were good stalk-cell inducers in HM44 cells, whereas DIF-3 and some DIF-3 derivatives at micromolar levels were potent anti-proliferative agents in K562 cells. We also tried to search for antagonistic molecules against DIF-1 and DIF-3 but failed to find such molecules from the analogues used here. The present findings would give us hints for identifying the target molecule(s) of DIFs and also for developing novel anti-cancer drugs. (c) 2005 Elsevier Inc. All rights reserved.

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Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

A Arai, Y Goto, A Hasegawa, K Hosaka, H Kikuchi, Y Oshima, S Tanaka, Y Kubohara

DIFFERENTIATION (BLACKWELL PUBLISHING) 73 ( 7 ) 377 - 384 2005.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0301-4681

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 mu M promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 mu M suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 mu M dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 mu M slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.

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Involvement of reactive oxygen species and stress-activated MAPKs in satratoxin H-induced apoptosis

P Nusuetrong, M Yoshida, MA Tanitsu, H Kikuchi, M Mizugaki, K Shimazu, T Pengsuparp, D Meksuniyen, Y Oshima, N Nakahata

EUROPEAN JOURNAL OF PHARMACOLOGY (ELSEVIER SCIENCE BV) 507 ( 1-3 ) 239 - 246 2005.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0014-2999

Satratoxins, members of the trichothecene mycotoxin family, have been known to be harmful to health. However, the mechanisms underlying the toxicity still remain unclear. The present study is undertaken to elucidate the mechanisms of the satratoxin H-induced cytotoxicity in PC12 cells. Satratoxin H caused cytotoxicity, which was reflected from apoptosis determined by chromatin staining and flow cytometry. Satratoxin H stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Pre-incubation with SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor, but not PD98059, an ERK inhibitor, reduced satratoxin-induced cytotoxicity. Co-incubation of cells with slutatbione. N-acetyl-L-cysteine or glutathione reductase inhibited cytotoxicity and the phosphorylation of p38 MAPK induced by satratoxin H. Our data suggest that satratoxin H-induced apoptosis in PC12 cells is dependent on the activation of p38 MAPK/JNK and the increase in reactive oxygen species. (C) 2004 Elsevier B.V. All rights reserved.

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A novel carbon skeletal trichothecane, tenuipesine A, isolated from an entomopathogenic fungus, Paecilomyces tenuipes

H Kikuchi, Y Miyagawa, K Nakamura, Y Sahashi, S Inatomi, Y Oshima

ORGANIC LETTERS (AMER CHEMICAL SOC) 6 ( 24 ) 4531 - 4533 2004.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 1523-7060

Tenuipesine A (1), a novel trichothecane with an unprecedented carbon-migrated skeleton that embodies of a cyclopropane ring, was isolated from cultivated fruiting bodies of Paecilomyces tenuipes (Isaria japonica), a popular e ntomo pathogenic fungi employed in folk medicine and health foods in China, Korea, and Japan. The structure was determined on the basis of two-dimensional NMR data. Its stereochemistry was elucidated by spectroscopic data and the chemical transformation of the coexisting trichothecene, 4beta-acetoxy-12,13-epoxytrichothec-g-ene3cc,15-diol (2).

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Novel trichothecanes, paecilomycine A, B, and C, isolated from entomopathogenic fungus, Paecilomyces tenuipes

H Kikuchi, Y Miyagawa, Y Sahashi, S Inatomi, A Haganuma, N Nakahata, Y Oshima

TETRAHEDRON LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 45 ( 33 ) 6225 - 6228 2004.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4039

Paecilomycine A (1), B (2), and C (3) were isolated from cultivated fruiting bodies of Paecilomyces tenuipes (Isaria japonica), which is a popular entomopathogenic fungus used in folk medicine and health foods in China, Korea, and Japan. The structures of 1 3 were deduced from their spectroscopic data and their absolute configurations were elucidated by preparing their MPA esters. Compound I showed activity in the neurotrophic factor biosynthesis in glial cells. (C) 2004 Elsevier Ltd. All rights reserved.

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Structural requirements of dictyopyrones isolated from Dictyostelium spp. in the regulation of Dictyostelium development and in anti-leukemic activity

H Kikuchi, K Sasaki, J Sekiya, Y Maeda, A Amagai, Y Kubohara, Y Oshima

BIOORGANIC & MEDICINAL CHEMISTRY (PERGAMON-ELSEVIER SCIENCE LTD) 12 ( 12 ) 3203 - 3214 2004.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0968-0896

Cellular slime molds are fascinating to the field of developmental biology, and have long been used as excellent model organisms for the study of various aspects of multicellular development. We have recently isolated alpha-pyronoids, named dictyopyrones A-D (1-4), from various species of Dictyostelium cellular slime molds, and it was shown that compound 3 may regulate Dictyostelium development. In this study, we synthesized dictyopyrones A-D (1-4) and their analogues, investigated the physiological role of the molecules in cell growth and morphogenesis in D. discoideum, and further verified their effects on human leukemia K562 cells. Nitrogen-containing compounds 22 and 37 strongly inhibited cell growth in K562 leukemia cells, indicating that these compounds may be utilized as novel lead compounds for anti-leukemic agents. (C) 2004 Elsevier Ltd. All rights reserved.

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Novel spirocyclic trichothecanes, spirotenuipesine A and B, isolated from entomopathogenic fungus, Paecilomyces tenuipes

H Kikuchi, Y Miyagawa, Y Sahashi, S Inatomi, A Haganuma, N Nakahata, Y Oshima

JOURNAL OF ORGANIC CHEMISTRY (AMER CHEMICAL SOC) 69 ( 2 ) 352 - 356 2004.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-3263

Entomopathogenic fungi forming fruiting bodies have been employed as tonics and antitussives from ancient times. Paecilomyces tenuipes, which is also called Isaria japonica, is a very popular entomopathogenic fungus and is often considered a health food in northeast Asian countries such as China, Korea, and Japan. We cultivated the fruiting bodies of Paecilomyces tenuipes. Among the large-scale cultivations, fruiting body grown in barley grain contained two novel spirocyclic trichothecane derivatives, spirotenuipesine A (1) and B (2), and known trichothecane mycotoxins. Compounds 1 and 2 showed potent activity in neurotrophic factor biosynthesis in glial cells. The isolation of these compounds indicated that P. tenuipes is a promising source for producing various biologically active substances including trichothecanes. It is noteworthy that trichothecane mycotoxins are present in Paecilomyces tenuipes, which is typically used in medicinal health food.

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Novel aromatics bearing 4-O-methylglucose unit isolated from the oriental crude drug Bombyx Batryticatus

H Kikuchi, N Takahashi, Y Oshima

TETRAHEDRON LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 45 ( 2 ) 367 - 370 2004.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4039

Oriental crude drug, Bombyx Batryticatus, is dried silkworm larva, Bombyx mori L., which are dead and stiffened due to a Beauveria bassiana infection. In traditional Japanese, Korean, and Chinese medicine, it is employed as analgesic and anticonvulsant. We investigated the constituents of Bombyx Batryticatus and isolated four novel aromatics bearing 4-O-methylglucose moiety, BB-1, 2, 3, and 4 (1-4). It is speculated that these compounds are produced by an interaction between plants, insects, and microorganisms. (C) 2003 Elsevier Ltd. All rights reserved.

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Adenosine as an active constituent of Auricularia auricula for the inhibition of rabbit platelet aggregation

Murata A, Oka N, Ohkubo S, Sato Y, Funamori M, Kikuchi H, Tanitsu M.-A, Kogi K, Sato S, Tadano T, Oshima Y, Nakahata N

Pharmacometrics 67 ( 3-4 ) 359 - 364 2004

Research paper (scientific journal), Joint Work, Accepted

Metabolites of febrifugine and its synthetic analogue by mouse liver S9 and their antimalarial activity against Plasmodium malaria parasite

S Hirai, H Kikuchi, HS Kim, K Begum, Y Wataya, H Tasaka, Y Miyazawa, K Yamamoto, Y Oshima

JOURNAL OF MEDICINAL CHEMISTRY (AMER CHEMICAL SOC) 46 ( 20 ) 4351 - 4359 2003.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-2623

Quinazolinone type alkaloids, febrifugine (1) and isofebrifugine (2), isolated from Dichroa febrifuga roots, show powerful antimalarial activity against Plasmodium falciparum. Unfortunately, their emetic effect and other undesirable side effects have precluded their clinical use for malaria. Because of their antimalarial potency, analogues were searched for, with the goal of preserving the strong antimalarial activity, while dramatically reducing side effects. We expected that compounds useful in drug development would exist in metabolites derived from I and Df-1 (3), the condensation product of 1 with acetone, by mouse liver S9. Feb-A and -B (4 and 5) were isolated as the major metabolites of 1. In addition to 4 and 5, feb-C and -D (6 and 7) were also purified from the metabolic mixture of 3. Compounds 4 and 5 were compounds oxidized at C-6 and C-2 of the quinazolinone ring of 1, respectively. Compounds 6 and 7, derived from 3, also bear febrifugine type structures in which the 4"- and 6"-positions of the piperidine ring of 1 were oxidized. In vitro antimalarial. and cytotoxic tests using synthetically obtained racemic 4-6 and enantiomerically pure 7 demonstrated that 4 and 6 had antimalarial activity against P. falciparum, of similar potency to that of 1, with high selectivity. The antimalarial. activity of 5 and 7, however, was dramatically decreased in the test. The in vitro antimalarial activity of analogues 22 and 43, which are stereoisomers of 4 and 6, was also evaluated, showing that 22 is active. The results suggest that basicity of both the 1- and the 1"-nitrogen atoms of 1 is crucial in conferring powerful antimalarial activity. Racemic 4 and 6 exhibited powerful in vivo antimalarial activity against mouse malaria P. berghei, and especially, no serious side effects were observed with 4. Thus, the metabolite 4 appears to be a promising lead compound for the development of new types of antimalarial drugs.

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A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A(2)-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity

M Yajima, M Takada, N Takahashi, H Kikuchi, S Natori, Y Oshima, S Kurata

BIOCHEMICAL JOURNAL (PORTLAND PRESS) 371 ( 1 ) 205 - 210 2003.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0264-6021

Innate immunity is the first line of defence against infectious micro-organisms, and the basic mechanisms of pathogen recognition and response activation are evolutionarily conserved. In mammals, the innate immune response in combination with antigen-specific recognition is required for the activation of adaptive immunity. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Here, for the purpose of pharmaceutical screening, we established an in vitro culture based on the innate immune response of Drosophila. The in vitro system is capable of measuring lipopolysaccharide (LPS)-dependent activation of the immune deficiency (imd) pathway, which is similar to the tumour necrosis factor signalling pathway in mammals. Screening revealed that well-known inhibitors of phospholipase A(2) (PLA(2)), dexamethasone (Dex) and p-bromophenacyl bromide (BPB) inhibit LPS-dependent activation of the imd pathway. The inhibitory effects of Dex and BPB were suppressed by the addition of an excess of three (arachidonic acid, eicosapentaenoic acid and gamma-linolenic acid) of the fatty acids so far tested. Arachidonic acid, however, did not activate the imd pathway when used as the sole agonist. These findings indicate that PLA, participates in LPS-dependent activation of the imd pathway via the generation of arachidonic acid and other mediators, but requires additional signalling from LPS stimulation. Moreover, PLA(2) was activated in response to bacterial infection in Sareophaga. These results suggest a functional link between the PLA(2)-generated fatty acid cascade and the LPS-stimulated imd pathway in insect immunity.

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Multiple activities of a novel substance, dictyopyrone C isolated from Dictyostelium discoideum, in cellular growth and differentiation

Y Maeda, H Kikuchi, K Sasaki, A Amagai, J Sekiya, Y Takaya, Y Oshima

PROTOPLASMA (SPRINGER-VERLAG WIEN) 221 ( 3-4 ) 185 - 192 2003

Research paper (scientific journal), Joint Work, Accepted, ISSN 0033-183X

We report that a novel substance named dictyopyrone C (DPC) has remarkable effects on growth and differentiation of Dictyostelium discoideum Ax-2 cells, in a dose-dependent manner. In the presence of 3-15 muM DPC, differentiation of starving Ax-2 (clone MS) cells was greatly enhanced in submerged culture, when vegetative MS cells were harvested at the mid-late-exponential growth phase (>3 x 10(6) cells per ml) and starved. In contrast, DPC above 30 muM markedly impaired the progression of differentiation including cell aggregation, most of starved cells being round after 3-4 h of DPC application and then lysed during further incubation. In the presence of 30 muM DPC however, MS cells that had been harvested at the early exponential growth phase (<5 x 10(5) cells per ml) and starved became neither round nor lysed and exhibited rather enhanced differentiation. Essentially the same results were obtained in cultures of starved cells on nonnutrient agar. With respect to the DPC effect on MS cells growing in axenic medium, cell lysis and growth inhibition by DPC at concentrations higher than 15 muM were realized in the mid-late-exponential-growth-phase cells (>3 x 10(6) cells per ml) but not in the early-exponential-growth-phase cells (<5 x 10(5) cells per ml). Moreover, analysis using synchronized MS cells has demonstrated that the DPC effect changes in a cell-cycle-dependent manner. In contrast to such unique DPC actions, the pyrone ring of DPC had no effects on growth and differentiation within the range of 3-120 muM tested. These findings strongly suggested the importance of the combined structure of the pyrone ring and the linear carbon chain in revelation of the DPC activities.

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Potent antimalarial febrifugine analogues against the Plasmodium malaria parasite

H Kikuchi, H Tasaka, S Hirai, Y Takaya, Y Iwabuchi, H Ooi, S Hatakeyama, HS Kim, Y Wataya, Y Oshima

JOURNAL OF MEDICINAL CHEMISTRY (AMER CHEMICAL SOC) 45 ( 12 ) 2563 - 2570 2002.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-2623

Although febrifugine (1) and isofebrifugine (2), alkaloids isolated from roots of the Dichroa febrifuga plant, show powerful antimalarial activity against Plasmodium falciparum, strong side effects such as the emetic effect have precluded their clinical use against malaria. However, their antimalarial potency makes them attractive substances as leads for developing new types of chemotherapeutic antimalarial drugs. Thus, we have evaluated the in vitro antimalarial activity of the analogues of febrifugine (1) and isofebrifugine (2). The activities of the analogues derived from Df-1 (3) and Df-2 (4), condensation products of 1 and 2 with acetone, respectively, were also obtained. The 3"-keto derivative (7, EC50 = 2.0 x 10(-8) M) of 1 was found to exhibit potential antimalarial activity with high selectivity against P. falciparum in vitro. The in vitro activities of the reduction product (8, EC50 = 2.0 x 10-8 M) of 1 at C-2' and its cyclic derivatives 9 and 10 (EC50 = 3.7 x 10(-9) and 8.6 x 10(-9) M, respectively) were found to be strongly active and selective. Additionally, the Dess-Martin oxidation product of 3 was found to be strongly active with high selectivity against P. falciparum. A structure-activity relationship study (SAR) demonstrates that the essential role played by the 4-quinazolinone ring in the appearance of activity and the presence of a 1"-amino group and C-2', C-3" O-functionalities are crucial in the activity of 1. For 7, 8, and 9, prepared as racemic forms, an in vivo study has also been conducted.

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The isolation and synthesis of two novel N-acetyl glucosamine derivatives from Dictyostelium cellular slime molds which exhibit neurite outgrowth activity

H Kikuchi, J Komiya, Y Saito, JI Sekiya, S Honma, N Nakahata, Y Oshima

TETRAHEDRON LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 43 ( 8 ) 1477 - 1480 2002.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4039

To clarify the diversity of secondary metabolites of Dictyostelium cellular slime molds and explore any biologically active substances which they may hold, constituents of two species were investigated. From a methanol extract or their fruit bodies, two novel acylated amino sugar were isolated, dictyoglucosamine A (1) from D. purpureum and dictyoglucosamine B (2) from D. discoideum. For further analysis, these compounds were then synthesized from N-aceyl-D-glucosamine. Biological evaluation of 1 and 2 shocked the induce neuronal differentiation of rat pheochromocytoma (PC-12) cells. (C) Elsevier Science Ltd. All rights reserved.

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Furanodictine A and B: Amino sugar analogues produced by cellular slime mold Dictyostelium discoideum showing neuronal differentiation activity

H Kikuchi, Y Saito, J Komiya, Y Takaya, S Honma, N Nakahata, A Ito, Y Oshima

JOURNAL OF ORGANIC CHEMISTRY (AMER CHEMICAL SOC) 66 ( 21 ) 6982 - 6987 2001.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-3263

We investigated the constituents of Dictyostelium discoideum to clarify the diversity of secondary metabolites of Dictyostelium cellular slime molds and to explore biologically active substances that could be useful in the development of novel drugs. From a methanol extract of the multicellular fruit body of D. discoideum, we isolated two novel amino sugar analogues, furanodictine A (1) and B (2). They are the first 3,6-anhydrosugars to be isolated from natural sources. Their relative structures were elucidated by spectral means, and the absolute configurations were confirmed by asymmetric syntheses of 1 and 2. These furanodictines potently induce neuronal differentiation of rat pheochromocytoma (PC-12) cells.

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Novel aromatic substances, dictyomedin A and B, from Dictyostelium cellular slime molds and their inhibitory effects on Dictyostelium development

Y Takaya, H Kikuchi, Y Terui, J Komiya, Y Maeda, A Ito, Y Oshima

TETRAHEDRON LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 42 ( 1 ) 61 - 63 2001.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0040-4039

To elucidate the diversity of secondary metabolites of Dictyostelium cellular slime molds and search for physiologically active substances affecting their development, we investigated the constituents of D, medium. From the methanol extract of its fruit body, we obtained two novel aromatic compounds named dictyomedin A (1) and B (2), and their structures were elucidated by means of physicochemical data. Biological evaluation of the compounds showed that they delayed the differentiation of D. discoideum cells. (C) 2000 Elsevier Science Ltd. All rights reserved.

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Novel acyl alpha-pyronoids, dictyopyrone A, B, and C, from Dictyostelium cellular slime molds

Y Takaya, H Kikuchi, Y Terui, J Komiya, KI Furukawa, K Seya, S Motomura, A Ito, Y Oshima

JOURNAL OF ORGANIC CHEMISTRY (AMER CHEMICAL SOC) 65 ( 4 ) 985 - 989 2000.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-3263

For the elucidation of the diversity of secondary metabolites of Dictyostelium cellular slime molds, we investigate the constituent of three species of slime molds. From the methanol extract of their fruit bodies, we obtained three novel compounds, dictyopyrone A (1) and B (2) from D. discoideum and D, rhizoposium and dictyopyrone C (3) from D, longosporum. They possess a unique cr-pyrone moiety with a side chain at the C-3 position. Their relative structures were elucidated by spectral means, and the absolute configuration was confirmed by asymmetric synthesis of 1. Since these compounds were obtained from different species of Dictyostelium slime molds, they may be a type of compound common to this genus.

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