Details of a Researcher - Hanaoka, Kenjiro

Papers - Hanaoka, Kenjiro

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Detection of NAD(P)H-dependent enzyme activity by time-domain ratiometry of terbium luminescence

Terai T., Ito H., Hanaoka K., Komatsu T., Ueno T., Nagano T., Urano Y.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters) 26 ( 9 ) 2314 - 2317 2016.05

Research paper (scientific journal), Accepted, ISSN 0960894X

NAD(P)H-dependent oxidoreductases play important roles in biology. Recently, we reported that the luminescence lifetime of some Tb3+ complexes is sensitive to NAD(P)H, and we used this phenomenon to detect activities of these enzymes. However, conventional time-resolved luminescence assays are susceptible to static quenchers such as ATP. Herein we describe a detection methodology that overcomes this issue: the intensity of the sample is measured twice with different delay times and the intensity ratio value is used as an index of NAD(P)H concentration. The method is more robust than single-point measurement, and is compatible with high-throughput assays using conventional microplate readers.

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Protein-Coupled Fluorescent Probe to Visualize Potassium Ion Transition on Cellular Membranes

Hirata T., Terai T., Yamamura H., Shimonishi M., Komatsu T., Hanaoka K., Ueno T., Imaizumi Y., Nagano T., Urano Y.

Analytical Chemistry (Analytical Chemistry) 88 ( 5 ) 2693 - 2700 2016.03

Research paper (scientific journal), Accepted, ISSN 00032700

K+ is the most abundant metal ion in cells, and changes of [K+] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K+] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K+, TLSHalo. TLSHalo is responsive to [K+] in the physiological range, with good selectivity over Na+ and retains its K+-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K+] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K+] change induced by K+ ionophores or by activation of a native Ca2+-dependent K+ channel (BK channel). Further, we show that K+ efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K+ dynamics with high spatiotemporal resolution.

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Chemical tools for the study of hydrogen sulfide (H<inf>2</inf>S) and sulfane sulfur and their applications to biological studies

Takano Y., Shimamoto K., Hanaoka K.

Journal of Clinical Biochemistry and Nutrition (Journal of Clinical Biochemistry and Nutrition) 58 ( 1 ) 7 - 15 2016.01

Research paper (scientific journal), Accepted, ISSN 09120009

Hydrogen sulfide (H2S) functions in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. On the other hand, sulfane sulfur, which is a sulfur atom with six valence electrons but no charge, has the unique ability to bind reversibly to other sulfur atoms to form hydroper sulfides (R-S-SH) and polysulfides (-S-Sn-S-). H2S and sulfane sulfur always coexist, and recent work suggests that sulfane sulfur species may be the actual signaling molecules in at least some biological phenomena. For example, one of the mechanisms of activity regulation of proteins by H2S is the S-sulfhydration of cysteine residues (protein Cys-SSH). In this review, we summarize recent progress on chemical tools for the study of H2S and sulfane sulfur, covering fluorescence probes utilizing various design strategies, H2S caged compounds, inhibitors of physiological H2S-producing enzymes (cystathionine γ-lyase, cystathionine β-synthase and 3-mercaptopyruvate sulfurtrans-ferase), and labeling reagents. Fluorescence probes offer particular advantages as chemical tools to study physiological functions of biomolecules, including ease of use and real-time, nondestructive visualization of biological processes in live cells and tissues.

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Development of silicon-substituted xanthene dyes and their application to fluorescent probes

Hanaoka K., Urano Y.

Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry (Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry) 74 ( 5 ) 512 - 520 2016

Research paper (scientific journal), Accepted, ISSN 00379980

Fluorescence imaging is one of the most powerful techniques for visualization of the temporal and spatial biological events in living cells. Many fluorescent probes have been developed so far and contributed greatly to biological and medical researches. For biological applications, xanthene dyes have many favorable characteristics, such as high water solubility and high fluorescence quantum yield, and have been utilized as the fluorescent core for a large number of fluorescent probes. In this manuscript, we introduce a novel red fluorescein analogue, TokyoMagenta (TM), in which the O atom at the 10 position of the xanthene moiety is replaced with a Si atom, and the absorption and emission wavelengths of TM are about 90 nm longer than those of fluorescein. These silicon-substituted xanthene dyes would be useful for multicolor imaging. Nowadays, we think these xanthene dyes have been becoming one of practical fluorophores for biological researches.

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Identification of lung inflammation-related elevation of acylamino acid releasing enzyme (APEH) activity using an enzymomics approach

Komatsu T., Yoshioka K., Hanaoka K., Terai T., Ueno T., Nagano T., Urano Y.

Chemical and Pharmaceutical Bulletin (Chemical and Pharmaceutical Bulletin) 64 ( 11 ) 1533 - 1538 2016

Research paper (scientific journal), Accepted, ISSN 00092363

Disease-associated alterations of enzymatic functions are potentially useful as disease biomarkers, and here we show that an enzymomics (omics of active enzymes) approach, in which enzymatic activities are screened with panels of substrates, can be an effective way to identify such alterations. In the present study, we used a panel of fluorogenic substrates to search for altered enzyme activities in bronchoalveolar lavage fluid (BALF) from a mouse model of lung inflammation. We found that acylamino acid releasing enzyme (APEH) activity was highly elevated, apparently reflecting the increased population of immune cells in the inflamed lung.

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Discovery of a pyruvylated peptide-metabolizing enzyme using a fluorescent substrate-based protein discovery technique

Yoshioka K., Komatsu T., Hanaoka K., Ueno T., Terai T., Nagano T., Urano Y.

Chemical Communications (Chemical Communications) 52 ( 23 ) 4377 - 4380 2016

Research paper (scientific journal), Accepted, ISSN 13597345

We employed a fluorescent substrate-based target discovery approach to screen the enzymome for metabolic activity towards pyruvyl-amidated peptides, and identified an acylamino acid-releasing enzyme (APEH). Cells overexpressing APEH exhibited higher metabolic activity towards the probe, N-pyruvyl-leucyl-7-amido-4-methylcoumarin (Pyr-Leu-AMC), while the selective APEH inhibitor AA74-1 blocked the reaction. Metabolism of various pyruvylated peptides in liver lysate was almost completely blocked by AA74-1. Pyruvyl peptides are synthesized in response to oxidative stress, but their biological role is poorly understood; identification of a key contributor to their metabolism should stimulate research on pathways leading from oxidative stress to protein modification and biological output.

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Development of a sensitive bioluminogenic probe for imaging highly reactive oxygen species in living rats

Kojima R., Takakura H., Kamiya M., Kobayashi E., Komatsu T., Ueno T., Terai T., Hanaoka K., Nagano T., Urano Y.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition) 54 ( 49 ) 14768 - 14771 2015.12

Research paper (scientific journal), Accepted, ISSN 14337851

A sensitive bioluminogenic probe for highly reactive oxygen species (hROS), SO3H-APL, was developed based on the concept of dual control of bioluminescence emission by means of bioluminescent enzyme-induced electron transfer (BioLeT) and modulation of cell-membrane permeability. This probe enables non-invasive visualization of physiologically relevant amounts of hROS generated deep inside the body of living rats for the first time. It is expected to serve as a practical analytical tool for investigating a wide range of biological functions of hROS in vivo. The design concept should be applicable to other in vivo bioluminogenic probes. You light up my life: A sensitive bioluminogenic probe for highly reactive oxygen species (hROS) was developed based on the concept of dual control of bioluminescence emission by means of bioluminescent enzyme-induced electron transfer (BioLeT) and modulation of cell-membrane permeability. This probe enables non-invasive visualization of physiologically relevant amounts of hROS generated deep inside the body of living rats.

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Azobenzene-caged sulforhodamine dyes: A novel class of 'turn-on' reactive probes for hypoxic tumor cell imaging

Chevalier A., Piao W., Hanaoka K., Nagano T., Renard P.Y., Romieu A.

Methods and Applications in Fluorescence (Methods and Applications in Fluorescence) 3 ( 4 ) 2015.12

Research paper (scientific journal), Accepted

New sulforhodamine-based fluorescent 'turn-on' probes have been developed for the direct imaging of cellular hypoxia. Rapid access to this novel class of water-soluble 'azobenzene-caged' fluorophores was made possible through an easily-implementable azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold (named SR101-NaphtNH2) and a tertiary aniline whose N-substituents are neutral, cationic, or zwitterionic. The detection mechanism is based on the bioreductive cleavage of the azo bond that restores strong far-red fluorescence (emission maximum at 625 nm) by regenerating the original sulforhodamine SR101-NaphtNH2. This valuable fluorogenic response was obtained for the three 'smart' probes studied in this work, as shown by an in vitro assay using rat liver microsomes placed under aerobic and then under hypoxic conditions. Most importantly, the probe namely SR101-NaphtNH2-Hyp-diMe was successfully applied for imaging the hypoxic status of tumor cells (A549 cells).

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Thiosulfate mediates cytoprotective effects of hydrogen sulfide against neuronal ischemia

Marutani E., Yamada M., Ida T., Tokuda K., Ikeda K., Kai S., Shirozu K., Hayashida K., Kosugi S., Hanaoka K., Kaneki M., Akaike T., Ichinose F.

Journal of the American Heart Association (Journal of the American Heart Association) 4 ( 11 ) 2015.11

Research paper (scientific journal), Accepted

Background-Hydrogen sulfide (H2S) exhibits protective effects in various disease models including cerebral ischemia-reperfusion (I/R) injury. Nonetheless, mechanisms and identity of molecules responsible for neuroprotective effects of H2S remain incompletely defined. In the current study, we observed that thiosulfate, an oxidation product of H2S, mediates protective effects of an H2S donor compound sodium sulfide (Na2S) against neuronal I/R injury. Methods and Results-We observed that thiosulfate in cell culture medium is not only required but also sufficient to mediate cytoprotective effects of Na2S against oxygen glucose deprivation and reoxygenation of human neuroblastoma cell line (SH-SY5Y) and murine primary cortical neurons. Systemic administration of sodium thiosulfate (STS) improved survival and neurological function of mice subjected to global cerebral I/R injury. Beneficial effects of STS, as well as Na2S, were associated with marked increase of thiosulfate, but not H2S, in plasma and brain tissues. These results suggest that thiosulfate is a circulating "carrier" molecule of beneficial effects of H2S. Protective effects of thiosulfate were associated with inhibition of caspase-3 activity by persulfidation at Cys163 in caspase-3. We discovered that an SLC13 family protein, sodium sulfate cotransporter 2 (SLC13A4, NaS-2), facilitates transport of thiosulfate, but not sulfide, across the cell membrane, regulating intracellular concentrations and thus mediating cytoprotective effects of Na2S and STS. Conclusions-The protective effects of H2S are mediated by thiosulfate that is transported across cell membrane by NaS-2 and exerts antiapoptotic effects via persulfidation of caspase-3. Given the established safety track record, thiosulfate may be therapeutic against ischemic brain injury.

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Identification of Tissue-Restricted Bioreaction Suitable for in Vivo Targeting by Fluorescent Substrate Library-Based Enzyme Discovery

Yoshioka K., Komatsu T., Nakada A., Onagi J., Kuriki Y., Kawaguchi M., Terai T., Ueno T., Hanaoka K., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society) 137 ( 38 ) 12187 - 12190 2015.09

Research paper (scientific journal), Accepted, ISSN 00027863

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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Analysis of Chemical Equilibrium of Silicon-Substituted Fluorescein and Its Application to Develop a Scaffold for Red Fluorescent Probes

Hirabayashi K., Hanaoka K., Takayanagi T., Toki Y., Egawa T., Kamiya M., Komatsu T., Ueno T., Terai T., Yoshida K., Uchiyama M., Nagano T., Urano Y.

Analytical Chemistry (Analytical Chemistry) 87 ( 17 ) 9061 - 9069 2015.09

Research paper (scientific journal), Accepted, ISSN 00032700

Fluorescein is a representative green fluorophore that has been widely used as a scaffold of practically useful green fluorescent probes. Here, we report synthesis and characterization of a silicon-substituted fluorescein, i.e., 2-COOH TokyoMagenta (2-COOH TM), which is a fluorescein analogue in which the O atom at the 10′ position of the xanthene moiety of fluorescein is replaced with a Si atom. This fluorescein analogue forms a spirolactone ring via intramolecular nucleophilic attack of the carboxylic group in a pH-dependent manner. Consequently, 2-COOH TM exhibits characteristic large pH-dependent absorption and fluorescence spectral changes: (1) 2-COOH TM is colorless at acidic pH, whereas fluorescein retains observable absorption and fluorescence even at acidic pH, and the absorption maximum is also shifted; (2) the absorption spectral change occurs above pH 7.0 for 2-COOH TM and below pH 7.0 for fluorescein; (3) 2-COOH TM shows a much sharper pH response than fluorescein because of its pK<inf>a</inf> inversion, i.e., pK<inf>a1</inf> > pK<inf>a2</inf>. These features are also different from those of a compound without the carboxylic group, 2-Me TokyoMagenta (2-Me TM). Analysis of the chemical equilibrium between pH 3.0 and 11.0 disclosed that 2-COOH TM favors the colorless and nonfluorescent lactone form, compared with fluorescein. Substitution of Cl atoms at the 4′ and 5′ positions of the xanthene moiety of 2-COOH TM to obtain 2-COOH DCTM shifted the equilibrium so that the new derivative exists predominantly in the strongly fluorescent open form at physiological pH (pH 7.4). To demonstrate the practical utility of 2-COOH DCTM as a novel scaffold for red fluorescent probes, we employed it to develop a probe for β-galactosidase. (Chemical Equation Presented).

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Artificial Ligands of Streptavidin (ALiS): Discovery, Characterization, and Application for Reversible Control of Intracellular Protein Transport

Terai T., Kohno M., Boncompain G., Sugiyama S., Saito N., Fujikake R., Ueno T., Komatsu T., Hanaoka K., Okabe T., Urano Y., Perez F., Nagano T.

Journal of the American Chemical Society (Journal of the American Chemical Society) 137 ( 33 ) 10464 - 10467 2015.08

Research paper (scientific journal), Accepted, ISSN 00027863

Artificial ligands of streptavidin (ALiS) with association constants of 106 M-1 were discovered by high-throughput screening of our chemical library, and their binding characteristics, including X-ray crystal structure of the streptavidin complex, were determined. Unlike biotin and its derivatives, ALiS exhibits fast dissociation kinetics and excellent cell permeability. The streptavidin-ALiS system provides a novel, practical compound-dependent methodology for repeated reversible cycling of protein localization between intracellular organella.

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Detection of NAD(P)H-dependent enzyme activity with dynamic luminescence quenching of terbium complexes

Ito H., Terai T., Hanaoka K., Ueno T., Komatsu T., Nagano T., Urano Y.

Chemical Communications (Chemical Communications) 51 ( 39 ) 8319 - 8322 2015.05

Research paper (scientific journal), Accepted, ISSN 13597345

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Fluorescent probes for hydrogen sulfide (H<inf>2</inf>S) and sulfane sulfur and their applications to biological studies

Shimamoto K., Hanaoka K.

Nitric Oxide - Biology and Chemistry (Nitric Oxide - Biology and Chemistry) 46 72 - 79 2015.04

Research paper (scientific journal), Accepted, ISSN 10898603

Hydrogen sulfide (H2S), a toxic gas with the smell of rotten eggs, plays key roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. The most commonly used methods or detecting H2S are the methylene blue method and the electrode method, but these methods require destructive sampling, e.g., homogenization of biological samples. On the other hand, the fluorescence detection method has been widely used in biological studies to study the physiological roles of H2S, because this technology provides real-time, easy-to-use, nondestructive detection in live cells or tissues. Many selective fluorescent probes for H2S have been reported. Sulfane sulfur compounds contain divalent sulfur atoms bonded to other sulfur atom(s), as in persulfides (RSSH) and polysulfides (RSSnSR). They are currently attracting increasing interest because one of the mechanisms of activity regulation of proteins by H2S is sulfhydration of cysteine residues (RSH→RSSH). Since H2S and sulfane sulfur are redox partners, they are very likely to coexist in biological systems, and from a reactivity point-of-view, sulfane sulfur seems likely to be much more effective than H2S in S-sulfhydration. Therefore, sulfane sulfur may be involved in mediating at least some of the biological activities of H2S. In this review, we summarize recent work on fluorescent probes selective for H2S and/or sulfane sulfur, and we briefly review their applications to biological studies.

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Development of a Series of Near-Infrared Dark Quenchers Based on Si-rhodamines and Their Application to Fluorescent Probes

Myochin T., Hanaoka K., Iwaki S., Ueno T., Komatsu T., Terai T., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society) 137 ( 14 ) 4759 - 4765 2015.04

Research paper (scientific journal), Accepted, ISSN 00027863

Near-infrared (NIR) fluorescent probes based on the Förster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers, which are nonfluorescent dyes, as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). Here, we describe Si-rhodamine-based NIR dark quenchers (SiNQs), which show broad absorption covering this region. They are nonfluorescent independently of solvent polarity and pH, probably due to free rotation of the bond between the N atom and the xanthene moiety. SiNQs can easily be structurally modified to tune their water-solubility and absorption spectra, enabling flexible design of appropriate FRET pair for various NIR fluorescent dyes. To demonstrate the usefulness of SiNQs, we designed and synthesized a NIR fluorescent probe for matrix metalloproteinase (MMP) activity using SiNQ780. This probe 1 could detect MMP activity in vitro, in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized, by NIR fluorescence. We believe SiNQs will be useful for the development of a wide range of practical NIR fluorescent probes. (Chemical Equation Presented).

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New class of bioluminogenic probe based on bioluminescent enzyme-induced electron transfer: BioLeT

Takakura H., Kojima R., Kamiya M., Kobayashi E., Komatsu T., Ueno T., Terai T., Hanaoka K., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society) 137 ( 12 ) 4010 - 4013 2015.04

Research paper (scientific journal), Accepted, ISSN 00027863

Bioluminescence imaging (BLI) has advantages for investigating biological phenomena in deep tissues of living animals, but few design strategies are available for functional bioluminescent substrates. We propose a new design strategy (designated as bioluminescent enzyme-induced electron transfer: BioLeT) for luciferin-based bioluminescence probes. Luminescence measurements of a series of aminoluciferin derivatives confirmed that bioluminescence can be controlled by means of BioLeT. Based on this concept, we developed bioluminescence probes for nitric oxide that enabled quantitative and sensitive detection even in vivo. Our design strategy should be applicable to develop a wide range of practically useful bioluminogenic probes.

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Gliotoxin suppresses NF-κB activation by selectively inhibiting linear ubiquitin chain assembly complex (LUBAC)

Sakamoto H., Egashira S., Saito N., Kirisako T., Miller S., Sasaki Y., Matsumoto T., Shimonishi M., Komatsu T., Terai T., Ueno T., Hanaoka K., Kojima H., Okabe T., Wakatsuki S., Iwai K., Nagano T.

ACS Chemical Biology (ACS Chemical Biology) 10 ( 3 ) 675 - 681 2015.03

Research paper (scientific journal), Accepted, ISSN 15548929

A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb3+-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.

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

Fukushima N., Ieda N., Kawaguchi M., Sasakura K., Nagano T., Hanaoka K., Miyata N., Nakagawa H.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters) 25 ( 2 ) 175 - 178 2015.01

Research paper (scientific journal), Accepted, ISSN 0960894X

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 (325-385 nm), 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.

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Synthesis of a photocontrollable hydrogen sulfide donor using ketoprofenate photocages

Fukushima N., Ieda N., Sasakura K., Nagano T., Hanaoka K., Suzuki T., Miyata N., Nakagawa H.

Chemical Communications (Chemical Communications) 50 ( 5 ) 587 - 589 2014.12

Research paper (scientific journal), Accepted, ISSN 13597345

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Sodium thiosulfate attenuates acute lung injury in Mice

Sakaguchi M., Marutani E., Shin H.S., Chen W., Hanaoka K., Xian M., Ichinose F.

Anesthesiology (Anesthesiology) 121 ( 6 ) 1248 - 1257 2014.12

Research paper (scientific journal), Accepted, ISSN 00033022

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