Details of a Researcher - Kikuchi, Haruhisa

Papers - Kikuchi, Haruhisa

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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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