Yoshiteru Oshima

Revised structure and synthesis of celastramycin a, a potent innate immune suppressor.

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 microg/mL) in human umbilical vein endothelial cells (HUVECs).

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

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.

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

AIMSnDifferentiation-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. In this study, we assessed the ability of DIFs to regulate the immune system and investigated their mechanisms of action.nnnMAIN METHODSnWe 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 NFkappaB (nuclear factor kappa B), which are transcription factors required for CIIP.nnnKEY FINDINGSnOf 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 CIIP, at least in part, by inhibiting the activity of AP-1, NFAT, and/or NFkappaB. Unlike cyclosporine A and FK506, the DIF derivatives had little effect on concanavalin A-induced interferon-gamma production in Jurkat cells.nnnSIGNIFICANCEnThe present results suggest that DIF derivatives could be developed as novel immunosuppressive drugs.

Antimycobacterial flavonoids from the leaf extract of Galenia africana.

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 1 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 muM concentrations, indicating that mycothiol disulfide reductase is not the target for their antituberculosis activity.

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

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-1beta, stimulation-induced activation of NF-kappaB, suggesting that its target molecules are upstream of TAK1 in mammalian innate immunity.

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

AIMSnThe 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.nnnMAIN METHODSnWe 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 on the blood glucose concentration in KK-Ay diabetic mice.nnnKEY FINDINGSnWe found that some derivatives at 20 microM 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 microM was inhibitory to the cell growth of 3T3-L1, DIF-1(3M) at 20 microM 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.nnnSIGNIFICANCEnThe 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.

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

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 us valuable information as to the chemical structure-effect relationship of DIF-1.

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

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 1321N1 human astrocytoma cells.

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

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 Alzheimers and Parkinsons diseases. In the present study, we examined an effect of 5,19-cyclo-9beta,10xi-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 Ca(2+) induce NGF gene expression, we measured activities of adenylyl cyclase and phospholipase C and intracellular Ca(2+) 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.