Published papers

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 Dictyostelium discoideum 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 μm 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.