Yasushi Takemoto

Gliotoxin analogues from a marine-derived fungus, Penicillium sp., and their cytotoxic and histone methyltransferase inhibitory activities.

Seven gliotoxin-related compounds were isolated from the fungus Penicillium sp. strain JMF034, obtained from deep sea sediments of Suruga Bay, Japan. These included two new metabolites, bis(dethio)-10a-methylthio-3a-deoxy-3,3a-didehydrogliotoxin (1) and 6-deoxy-5a,6-didehydrogliotoxin (2), and five known metabolites (3-7). The structures of the new compounds were elucidated by analysis of spectroscopic data and the application of the modified Moshers analysis. All of the compounds exhibited cytotoxic activity, whereas compounds containing a disulfide bond showed potent inhibitory activity against histone methyltransferase (HMT) G9a. None of them inhibited HMT SET7/9.

Involvement of 14-3-3 proteins in the second epidermal growth factor-induced wave of Rac1 activation in the process of cell migration.

Background: The spatiotemporal regulation of Rac1 controls cell migration. Results: EGF induced two waves of Rac1 activation in the process of cell migration. Conclusion: 14-3-3 proteins regulate the second EGF-induced wave of Rac1 activation by interacting with RacGEF. Significance: The second wave of Rac1 activation might be required for EGF-induced cell migration. Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ζ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ζ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ζ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1.

A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor.

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

5‐Lipoxygenase and cysteinyl leukotriene receptor 1 regulate epidermal growth factor‐induced cell migration through Tiam1 upregulation and Rac1 activation

Cell migration is an essential step for tumor metastasis. The small GTPase Rac1 plays an important role in cell migration. Previously, we reported that epidermal growth factor (EGF) induced two waves of Rac1 activation; namely, at 5 min and 12 h after stimulation. A second wave of EGF‐induced Rac1 activation was required for EGF‐induced cell migration, however, the spatiotemporal regulation of the second wave of EGF‐induced Rac1 activation remains largely unclear. In this study, we found that 5‐lipoxygenase (5‐LOX) is activated in the process of EGF‐induced cell migration, and that leukotriene C4 (LTC4) produced by 5‐LOX mediated the second wave of Rac1 activation, as well as cell migration. Furthermore, these effects caused by LTC4 were found to be blocked in the presence of the antagonist of cysteinyl leukotriene receptor 1 (CysLT1). This blockage indicates that LTC4‐mediated CysLT1 signaling regulates the second EGF‐induced wave of Rac1 activation. We also found that 5‐LOX inhibitors, CysLT1 antagonists and the knockdown of CysLT1 inhibited EGF‐induced T cell lymphoma invasion and metastasis‐inducing protein 1 (Tiam1) expression. Tiam1 expression is required for the second wave of EGF‐induced Rac1 activation in A431 cells. Therefore, our results indicate that the 5‐LOX/LTC4/CysLT1 signaling pathway regulates EGF‐induced cell migration by increasing Tiam1 expression, leading to a second wave of Rac1 activation. Thus, CysLT1 may serve as a new molecular target for antimetastatic therapy. In addition, the CysLT1 antagonist, montelukast, which is used clinically for allergy treatment, might have great potential as a novel type of antimetastatic agent.

Synthesis and anti-migrative evaluation of moverastin derivatives

Cell migration of tumor cells is essential for invasion of the extracellular matrix and for cell dissemination. Inhibition of the cell migration involved in the invasion process represents a potential therapeutic approach to the treatment of tumor metastasis; therefore, a novel series of derivatives of moverastins (moverastins A and B), an inhibitor of tumor cell migration, was designed and chemically synthesized. Among these moverastin derivatives, several compounds showed stronger cell migration inhibitory activity than parental moverastins, and UTKO1 was found to have the most potent inhibitory activity against the migration of human esophageal tumor EC17 cells in a chemotaxis cell chamber assay. Interestingly, although moverastins are considered to inhibit tumor cell migration by inhibiting farnesyltransferase (FTase), UTKO1 did not inhibit FTase, indicating that UTKO1 inhibited tumor cell migration by a mechanism other than the inhibition of FTase.

Identification of Cyproheptadine as an Inhibitor of SET Domain Containing Lysine Methyltransferase 7/9 (Set7/9) That Regulates Estrogen-Dependent Transcription

SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization.

Suppression of multidrug resistance by migrastatin.

Migrastatin (MGS) is a Streptomyces metabolite that inhibits cancer cell migration. In this study, we found that MGS also enhanced the cytotoxicity of vinblastine, vincristine, and taxol in P-glycoprotein-overexpressing VJ-300 cells and P388/VCR cells. Furthermore, MGS increased the intracellular concentration of labeled vinblastine, vincristine, and taxol in both VJ-300 cells and P388/VCR cells. P-glycoprotein was photolabeled with [3H]azidopine, but this photolabeling was significantly inhibited in the presence of MGS. These results indicated that MGS directly interacts with and inhibits P-glycoprotein, thereby sensitizing drug-resistant cells to anticancer drugs.

Epiblastin A Induces Reprogramming of Epiblast Stem Cells Into Embryonic Stem Cells by Inhibition of Casein Kinase 1.

The discovery of novel small molecules that induce stem cell reprogramming and give efficient access to pluripotent stem cells is of major importance for potential therapeutic applications and may reveal novel insights into the factors controlling pluripotency. Chemical reprogramming of mouse epiblast stem cells (EpiSCs) into cells corresponding to embryonic stem cells (cESCs) is an inefficient process. In order to identify small molecules that promote this cellular transition, we analyzed the LOPAC library in a phenotypic screen monitoring Oct4-GFP expression and identified triamterene (TR) as initial hit. Synthesis of a TR-derived compound collection and investigation for reprogramming of EpiSCs into cESCs identified casein kinases 1 (CK1) α/δ/ɛ as responsible cellular targets of TR and unraveled the structural parameters that determine reprogramming. Delineation of a structure-activity relationship led to the development of Epiblastin A, which engages CK1 isoenzymes in cell lysate and induces efficient conversion of EpiSCs into cESCs.

Design and synthesis of peptide-MCA substrates for a novel assay of histone methyltransferases and their inhibitors.

Histone methyltransferases (HMTs) play an important role in controlling gene expression through site-specific methylation of lysines in core and linker histones within chromatin. As the typical HMTs, G9a and Set7/9 have been intensively studied that G9a is specific to the methylation at H3K9 and H3K27 and represses transcription, while Set7/9 methylates at H3K4. In this report we prepared various peptide-MCAs (4-methylcoumaryl-7-amides) related to histone tail and protein-substrates such as p53 and estrogen receptor-α. The fluorogenic substrates are applied for the assay of HMTs and an inhibitor, for example. The most sensitive and specific MCA-substrates to G9a and Set7/9 are discovered. The peptide-MCAs corresponding to the methylation sequences are promising for screening of HMT inhibitors.