Hiroki Shimogawa

Chromatin Regulator PRC2 Is a Key Regulator of Epigenetic Plasticity in Glioblastoma

Tumor cell plasticity contributes to functional and morphologic heterogeneity. To uncover the underlying mechanisms of this plasticity, we examined glioma stem-like cells (GSC) where we found that the biologic interconversion between GSCs and differentiated non-GSCs is functionally plastic and accompanied by gain or loss of polycomb repressive complex 2 (PRC2), a complex that modifies chromatin structure. PRC2 mediates lysine 27 trimethylation on histone H3 and in GSC it affected pluripotency or development-associated genes (e.g., Nanog, Wnt1, and BMP5) together with alterations in the subcellular localization of EZH2, a catalytic component of PRC2. Intriguingly, exogenous expression of EZH2-dNLS, which lacks nuclear localization sequence, impaired the repression of Nanog expression under differentiation conditions. RNA interference (RNAi)-mediated attenuation or pharmacologic inhibition of EZH2 had little to no effect on apoptosis or bromodeoxyuridine incorporation in GSCs, but it disrupted morphologic interconversion and impaired GSC integration into the brain tissue, thereby improving survival of GSC-bearing mice. Pathologic analysis of human glioma specimens revealed that the number of tumor cells with nuclear EZH2 is larger around tumor vessels and the invasive front, suggesting that nuclear EZH2 may help reprogram tumor cells in close proximity to this microenvironment. Our results indicate that epigenetic regulation by PRC2 is a key mediator of tumor cell plasticity, which is required for the adaptation of glioblastoma cells to their microenvironment. Thus, PRC2-targeted therapy may reduce tumor cell plasticity and tumor heterogeneity, offering a new paradigm for glioma treatment.

Cell-morphology profiling of a natural product library identifies bisebromoamide and miuraenamide A as actin filament stabilizers

Natural products provide a rich source of biological tools, but elucidating their molecular targets remains challenging. Here we report a cell morphological profiling of a natural product library, which permitted the identification of bisebromoamide and miuraenamide A as actin filament stabilizers. Automated high-content image analysis showed that these two structurally distinct marine natural products induce morphological changes in HeLa cells similar to those induced by known actin-stabilizing compounds. Bisebromoamide and miuraenamide A stabilized actin filaments in vitro, and fluorescein-conjugated bisebromoamide localized specifically to actin filaments in cells. Cell morphological profiling was also used to identify actin-stabilizing or -destabilizing natural products from marine sponge extracts, leading to the isolation of pectenotoxin-2 and lyngbyabellin C. Overall, the results demonstrate that high-content imaging of nuclei and cell shapes offers a sensitive and convenient method for detecting and isolating molecules that target actin.

A Mitochondrial Surface‐Specific Fluorescent Probe Activated by Bioconversion

Kawazoe, Y.; Shimogawa, H.; Sato, A.; Uesugi, M., Mitochondrial Surface-specific Fluorescent Probe Activated by Bioconversion, Angew. Chem. Int. Ed., 50(24), 5478-5481 (2011). Sumiya, E.; Shimogawa, H.; Sasaki, H.; Tsutsumi, M.; Yoshita, K.; Ojika, M.; Suenaga, K.; Uesugi, M., Cell-morphology Profiling of a Natural Product Library Identifies Bisebromoamide and Miuraenamide A as Actin-filament Stabilizers, ACS Chem. Biol., 6(5), 425-431 (2011). Shirakawa, T.; Kawazoe, Y.; Tsujikawa, T.; Jung, D.; Sato, S.; Uesugi, M., Deactivation of STAT6 through Serine 707 Phosphorylation by JNK, J. Biol. Chem., 286, 4003-4010 (2011). Sato, S; Murata, A.; Orihara, T.; Shirakawa, T.; Suenaga, K.; Kigoshi, H.; Uesugi, M., Marine Natural Product Aurilide Activates the OPA1mediated Apoptosis by Binding to Prohibitin, Chem. Biol., 18 (1), 131-139 (2011). Kamisuki, S.; Shirakawa, T.; Kugimiya, A.; Abu-Elheiga, L.; Choo, H. Y.; Yamada, K.; Shimogawa, H.; Wakil, S. J.; Uesugi, M., Synthesis and Evaluation of Diarylthiazole Derivatives that Inhibit Activation of Sterol Regulatory Element-binding Proteins, J. Med. Chem., 54(13), 4923-4927(2011). Murata, A.; Sato, S.; Kawazoe, Y.; Uesugi, M., Small-molecule Fuorescent Probes for Specific RNA Targets, Chem. Comm., 47, 4712-4714 (2011). Proj Res**** WATANABE, Mizuki (D Pharm Sc) Proj Res** JIN, Guihua (D Med Sc) Proj Res**,***** KUO, Ting-Fang (D Sc) Guest Scholar*** SHEN, Yan (D Med Sc)

Synthesis and Evaluation of Diarylthiazole Derivatives That Inhibit Activation of Sterol Regulatory Element-Binding Proteins

Fatostatin, a recently discovered small molecule that inhibits activation of sterol regulatory element-binding protein (SREBP), blocks biosynthesis and accumulation of fat in obese mice. We synthesized and evaluated a series of fatostatin derivatives. Our structure-activity relationships led to the identification of N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide (24, FGH10019) as the most potent druglike molecule among the analogues tested. Compound 24 has high aqueous solubility and membrane permeability and may serve as a seed molecule for further development.

A Dumbbell-Shaped Small Molecule that Promotes Cell Adhesion and Growth

During an image-based phenotype screening of our chemical library, we noted a small molecule that boosts the adhesion and growth of human cells. Chemical and cell biological experiments suggest that the diaryldispirotripiperazine derivative (adhesamine) targets selective cell-surface glycosaminoglycans, especially heparan sulfate, for increasing cell adhesion and growth. The addition of adhesamine to the culture medium enables the adhesion of even floating lymphocytes to cell culture plates and the microinjection into them. Unlike poly-L-lysine, adhesamine induces apparently normal cell adhesion accompanied by organized actin structures and activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Adhesamine may be useful as a cell-attaching reagent for cell engineering and basic cell biology.

Wrenchnolol derivative optimized for gene activation in cells.

Naturally occurring transcription factors usually have two independent domains, a DNA-binding domain and an activation domain. In designing a synthetic small molecule that mimics a transcription factor, each of the two domains needs to be replaced by small-molecule counterparts. Results of the present study show that derivatives of wrenchnolol, a synthetic molecule that interacts with Sur-2 coactivator, serve as activation modules and stimulate gene transcription in vitro and in cells when tethered to a DNA-binding molecule. Thirteen derivatives of wrenchnolol were chemically synthesized and tested for their ability to activate transcription in vitro and in cells. When tethered to the GAL4 DNA-binding domain, one derivative increased transcription of a GAL4-responsive reporter gene in cells 9-fold. This optimized derivative also induced up to 45% myogenesis of C2C12 cells when tethered to the DNA-binding domain of myogenic transcription factor MyoD. This optimized derivative may serve as a starting point for designing biological tools or components of fully synthetic transcription factors that permit selective up-regulation of genes.

Catharsitoxins from the Chinese remedy qiung laug

Abstract Novel imidazole compounds, catharsitoxins A, B, C, D, E and F, were isolated from the Chinese remedy qiung laug. Their structures were determined by 2D NMR. To confirm their structures and to ensure an adequate supply for further biological studies, catharsitoxins A and D were synthesized.

Chemical Library Screening Identifies a Small Molecule That Downregulates SOD1 Transcription for Drugs to Treat Amyotrophic Lateral Sclerosis

Familial amyotrophic lateral sclerosis (fALS) accounts for 10% of ALS cases, and about 25% of fALS cases are due to mutations in superoxide dismutase 1 (SOD1). Mutant SOD1-mediated ALS is caused by a gain of toxic function of the mutant protein, and the SOD1 level in nonneuronal neighbors, including astrocytes, determines the progression of ALS (non-cell-autonomous toxicity). Therefore, the authors hypothesized that small molecules that reduce SOD1 protein levels in astrocytes might slow the progression of mutant SOD1-mediated ALS. They developed and optimized a cell-based, high-throughput assay to identify low molecular weight compounds that decrease SOD1 expression transcriptionally in human astrocyte-derived cells. Screening of a chemical library of 9600 compounds with the assay identified two hit compounds that selectively and partially downregulate SOD1 expression in a dose-dependent manner, without any detectable cellular toxicity. Western blot analysis showed that one hit compound significantly decreased the level of endogenous SOD1 protein in H4 cells, with no reduction in expression of β-actin. The assay developed here provides a powerful strategy for discovering novel lead molecules for treating familial SOD1-mediated ALS.