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Dive into the research topics where Mitsuyasu Kawaguchi is active.

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Featured researches published by Mitsuyasu Kawaguchi.


ACS Chemical Biology | 2013

Screening and X-ray Crystal Structure-based Optimization of Autotaxin (ENPP2) Inhibitors, Using a Newly Developed Fluorescence Probe

Mitsuyasu Kawaguchi; Takayoshi Okabe; Shinichi Okudaira; Hiroshi Nishimasu; Ryuichiro Ishitani; Hirotatsu Kojima; Osamu Nureki; Junken Aoki; Tetsuo Nagano

Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), was originally identified as a tumor cell autocrine motility factor and was found to be identical to plasma lysophospholipase D, which is the predominant contributor to lysophosphatidic acid (LPA) production from lysophospholipids. ATX is therefore considered to regulate the physiological and pathological roles of LPA, including angiogenesis, lymphocyte trafficking, tissue fibrosis, and cancer cell invasion and metastasis. Thus, it is a potential therapeutic target. Here, we first developed a sensitive and specific ATX fluorescence probe, TG-mTMP, and used it to screen ATX inhibitors in a large chemical library. This probe, which is superior to previously available probes FS-3 and CPF4 in terms of sensitivity or specificity, enabled us to identify several novel ATX inhibitor scaffolds. We solved the crystal structures of ATX complexes with the hit compounds at high resolution (1.75-1.95 Å) and used this information to guide optimization of the structure of a selected inhibitor. The optimized compounds, 3BoA and its derivatives, exhibited potent ATX-inhibitory activity both in vitro and in vivo. These inhibitors are expected to be useful tools to understand the roles of ATX in vitro and in vivo and may also be candidate anti-ATX therapeutic agents.


Journal of the American Chemical Society | 2011

Fluorescence probe for lysophospholipase C/NPP6 activity and a potent NPP6 inhibitor.

Mitsuyasu Kawaguchi; Takayoshi Okabe; Shinichi Okudaira; Kenjiro Hanaoka; Yuuta Fujikawa; Takuya Terai; Toru Komatsu; Hirotatsu Kojima; Junken Aoki; Tetsuo Nagano

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC(3)C, TG-mPC(5)C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC(3)C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80,000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC(50) value of 0.21 μM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.


Bioorganic & Medicinal Chemistry Letters | 2011

Development of a highly selective fluorescence probe for alkaline phosphatase

Mitsuyasu Kawaguchi; Kenjiro Hanaoka; Toru Komatsu; Takuya Terai; Tetsuo Nagano

We have developed the first highly selective fluorescence probe for alkaline phosphatase (ALP), TG-mPhos. This probe shows selectivity for ALP over protein tyrosine phosphatase and protein serine/threonine phosphatase. Our previously developed TG-Phos, which has a phenolic phosphate linkage in place of the alcoholic phosphate linkage of TG-mPhos, lacks this selectivity. TG-mPhos should enable precise fluorescence imaging of ALP activity in biological applications.


Journal of the American Chemical Society | 2013

Diced Electrophoresis Gel Assay for Screening Enzymes with Specified Activities

Toru Komatsu; Kenjiro Hanaoka; Alexander Adibekian; Kentaro Yoshioka; Takuya Terai; Tasuku Ueno; Mitsuyasu Kawaguchi; Benjamin F. Cravatt; Tetsuo Nagano

We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants.


Bioorganic & Medicinal Chemistry Letters | 2015

Development of photo-controllable hydrogen sulfide donor applicable in live cells

Naoki Fukushima; Naoya Ieda; Mitsuyasu Kawaguchi; Kiyoshi Sasakura; Tetsuo Nagano; Kenjiro Hanaoka; Naoki Miyata; Hidehiko Nakagawa

Hydrogen sulfide (H2S) has multiple physiological roles, for example, in vasodilation and inflammation. It is a highly reactive gas under ambient conditions, so controllable H2S donors are required for studying its biological functions. Here, we describe the design, synthesis and application of a H2S donor (SPD-2) that utilizes xanthone photochemistry to control H2S release. H2S generation from SPD-2 was completely dependent on UVA-irradiation (325-385nm), as confirmed by methylene blue assay and by the use of a H2S-selective fluorescent probe. SPD-2 was confirmed to provide controlled H2S delivery in live cells, and should be suitable for various biological applications.


ACS Chemical Biology | 2016

Visible Light-Controlled Nitric Oxide Release from Hindered Nitrobenzene Derivatives for Specific Modulation of Mitochondrial Dynamics

Kai Kitamura; Mitsuyasu Kawaguchi; Naoya Ieda; Naoki Miyata; Hidehiko Nakagawa

Nitric oxide (NO) is a physiological signaling molecule, whose biological production is precisely regulated at the subcellular level. Here, we describe the design, synthesis, and evaluation of novel mitochondria-targeted NO releasers, Rol-DNB-mor and Rol-DNB-pyr, that are photocontrollable not only in the UV wavelength range but also in the biologically favorable visible wavelength range (530-590 nm). These caged NO compounds consist of a hindered nitrobenzene as the NO-releasing moiety and a rhodamine chromophore. Their NO-release properties were characterized by an electron spin resonance (ESR) spin trapping method and fluorometric analysis using NO probes, and their mitochondrial localization in live cells was confirmed by costaining. Furthermore, we demonstrated visible light control of mitochondrial fragmentation via activation of dynamin-related protein 1 (Drp1) by means of precisely controlled NO delivery into mitochondria of cultured HEK293 cells, utilizing Rol-DNB-pyr.


Bioorganic & Medicinal Chemistry Letters | 2008

Development of a novel fluorescent probe for fluorescence correlation spectroscopic detection of kinase inhibitors

Mitsuyasu Kawaguchi; Takuya Terai; Rei Utata; Miki Kato; Keiko Tsuganezawa; Akiko Tanaka; Hirotatsu Kojima; Takayoshi Okabe; Tetsuo Nagano

We have developed a fluorescently labeled probe for high-throughput screening of kinase inhibitors using fluorescence correlation spectroscopy. With this probe, we have successfully evaluated the inhibitory activities of known inhibitors of a model kinase, ASK1. Because the probe contains a general kinase inhibitor, staurosporine, we believe that this homogeneous, high-throughput, and simple method can be applied to the inhibitor screening of other kinases as well.


The EMBO Journal | 2018

PlexinD1 signaling controls morphological changes and migration termination in newborn neurons

Masato Sawada; Nobuhiko Ohno; Mitsuyasu Kawaguchi; Shihhui Huang; Takao Hikita; Youmei Sakurai; Huy Nguyen; Truc Quynh Thai; Yuri Ishido; Yutaka Yoshida; Hidehiko Nakagawa; Akiyoshi Uemura; Kazunobu Sawamoto

Newborn neurons maintain a very simple, bipolar shape, while they migrate from their birthplace toward their destinations in the brain, where they differentiate into mature neurons with complex dendritic morphologies. Here, we report a mechanism by which the termination of neuronal migration is maintained in the postnatal olfactory bulb (OB). During neuronal deceleration in the OB, newborn neurons transiently extend a protrusion from the proximal part of their leading process in the resting phase, which we refer to as a filopodium‐like lateral protrusion (FLP). The FLP formation is induced by PlexinD1 downregulation and local Rac1 activation, which coincide with microtubule reorganization and the pausing of somal translocation. The somal translocation of resting neurons is suppressed by microtubule polymerization within the FLP. The timing of neuronal migration termination, controlled by Sema3E‐PlexinD1‐Rac1 signaling, influences the final positioning, dendritic patterns, and functions of the neurons in the OB. These results suggest that PlexinD1 signaling controls FLP formation and the termination of neuronal migration through a precise control of microtubule dynamics.


Bioorganic & Medicinal Chemistry | 2016

(7-Diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid as a caged inhibitor for photocontrol of histone deacetylase activity

Naoya Ieda; Sota Yamada; Mitsuyasu Kawaguchi; Naoki Miyata; Hidehiko Nakagawa

Histone deacetylases (HDACs) are involved in epigenetic control of the expression of various genes by catalyzing deacetylation of ε-acetylated lysine residues. Here, we report the design, synthesis and evaluation of the (7-diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid (AC-SAHA) as a caged HDAC inhibitor, which releases the known pan-HDAC inhibitor SAHA upon cleavage of the photolabile (7-diethylaminocoumarin-4-yl)methyl protecting group in response to photoirradiation. A key advantage of AC-SAHA is that the caged derivative itself shows essentially no HDAC-inhibitory activity. Upon photoirradiation, AC-SAHA decomposes to SAHA and a 7-diethylaminocoumarin derivative, together with some minor products. We confirmed that AC-SAHA inhibits HDAC in response to photoirradiation in vitro by means of chemiluminescence assay. AC-SAHA also showed photoinduced inhibition of proliferation of human colon cancer cell line HCT116, as determined by MTT assay. Thus, AC-SAHA should be a useful tool for spatiotemporally controlled inhibition of HDAC activity, as well as a candidate chemotherapeutic reagent for human colon cancer.


Journal of the American Chemical Society | 2015

Identification of tissue-restricted bioreaction suitable for in vivo targeting by fluorescent substrate library-based enzyme discovery.

Kentaro Yoshioka; Toru Komatsu; Akihiro Nakada; Jun Onagi; Yugo Kuriki; Mitsuyasu Kawaguchi; Takuya Terai; Tasuku Ueno; Kenjiro Hanaoka; Tetsuo Nagano; Yasuteru Urano

Tissue-restricted bioreactions can be utilized to design chemical-biological tools and prodrugs. We have developed a fluorescent-substrate-library-based enzyme discovery approach to screen tissue extracts for enzymatic activities of interest. Assay-positive candidate proteins were identified by diced electrophoresis gel assay followed by peptide mass fingerprinting. We discovered that pyruvyl anilide is specifically hydrolyzed by carboxylesterase 2 (CES2), which is predominantly localized in the liver and kidney. We show that the pyruvyl targeting group/CES2 enzyme pair can be used to deliver the 7-amino-4-methylcoumarin fluorophore specifically to the liver and kidney in vivo. Our screening approach should be useful to find other masking group/enzyme pairs suitable for development of fluorescent substrates and prodrugs.

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Naoya Ieda

Nagoya City University

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