Papers - Hanaoka, Kenjiro

Fluorescent Probes and Selective Inhibitors for Biological Studies of Hydrogen Sulfide-and Polysulfide-Mediated Signaling

Takano Y., Echizen H., Hanaoka K.

Antioxidants and Redox Signaling (Antioxidants and Redox Signaling)  27 ( 10 ) 669 - 683 2017.10

Research paper (scientific journal), Accepted,  ISSN  15230864

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Significance: Hydrogen sulfide (H2S) plays roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. Also, hydropersulfide (R-S-SH) and polysulfide (-S-Sn-S-) have recently been identified as reactive sulfur species (RSS) that regulate the bioactivities of multiple proteins via S-sulfhydration of cysteine residues (protein Cys-SSH) and show cytoprotection. Chemical tools such as fluorescent probes and selective inhibitors are needed to establish in detail the physiological roles of H2S and polysulfide. Recent Advances: Although many fluorescent probes for H2S are available, fluorescent probes for hydropersulfide and polysulfide have only recently been developed and used to detect these sulfur species in living cells. Critical Issues: In this review, we summarize recent progress in developing chemical tools for the study of H2S, hydropersulfide, and polysulfide, covering fluorescent probes based on various design strategies and selective inhibitors of H2S- and polysulfide-producing enzymes (cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase), and we summarize their applications in biological studies. Future Directions: Despite recent progress, the precise biological functions of H2S, hydropersulfide, and polysulfide remain to be fully established. Fluorescent probes and selective inhibitors are effective chemical tools to study the physiological roles of these sulfur molecules in living cells and tissues. Therefore, further development of a broad range of practical fluorescent probes and selective inhibitors as tools for studies of RSS biology is currently attracting great interest.

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An Activatable Photosensitizer Targeted to γ-Glutamyltranspeptidase

Chiba M., Ichikawa Y., Kamiya M., Komatsu T., Ueno T., Hanaoka K., Nagano T., Lange N., Urano Y.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  56 ( 35 ) 10418 - 10422 2017.08

Research paper (scientific journal),  ISSN  14337851

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We adopted a spirocyclization-based strategy to design γ-glutamyl hydroxymethyl selenorhodamine green (gGlu-HMSeR) as a photo-inactive compound that would be specifically cleaved by the tumor-associated enzyme γ-glutamyltranspeptidase (GGT) to generate the potent photosensitizer HMSeR. gGlu-HMSeR has a spirocyclic structure and is colorless and does not show marked phototoxicity toward low-GGT-expressing cells or normal tissues upon irradiation with visible light. In contrast, HMSeR predominantly takes an open structure, is colored, and generates reactive oxygen species upon irradiation. The γ-glutamyl group thus serves as a tumor-targeting moiety for photodynamic therapy (PDT), switching on tumor-cell-specific phototoxicity. To validate this system, we employed chick chorioallantoic membrane (CAM), a widely used model for preliminary evaluation of drug toxicity. Photoirradiation after gGlu-HMSeR treatment resulted in selective ablation of implanted tumor spheroids without damage to healthy tissue.

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Silicon-substituted Xanthene Dyes and Their Unique Photophysical Properties for Fluorescent Probes

Ikeno T., Nagano T., Hanaoka K.

Chemistry - An Asian Journal (Chemistry - An Asian Journal)  12 ( 13 ) 1435 - 1446 2017.07

Research paper (scientific journal), Accepted,  ISSN  18614728

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Polysulfides (H<inf>2</inf>S<inf>n</inf>) produced from the interaction of hydrogen sulfide (H<inf>2</inf>S) and nitric oxide (NO) activate TRPA1 channels

Miyamoto R., Koike S., Takano Y., Shibuya N., Kimura Y., Hanaoka K., Urano Y., Ogasawara Y., Kimura H.

Scientific Reports (Scientific Reports)  7 2017.04

Research paper (scientific journal), Accepted

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Hydrogen sulfide (H2S) exerts synergistic effects with another gaseous signaling molecule nitric oxide (NO) on ion channels and vasculature. However, the mechanism of the synergy is not well understood. Here, we show that the interaction between H2S and NO generates polysulfides (H2Sn), which activate transient receptor potential ankyrin 1 (TRPA1) channels. High performance liquid chromatography with tandem mass spectrometry analysis, along with the imaging of intracellular Ca2+ and H2Sn, showed that H2Sn and their effects were abolished by cyanolysis and by reducing substances such as dithiothreitol (DTT), cysteine, and glutathione (GSH). However, the effects of nitroxyl or nitrosopersulfide, other potential products of H2S and NO interaction, are not affected by cyanolysis or reducing substances. This study demonstrates that H2Sn are products of synergy between H2S and NO and provides a new insight into the signaling mechanisms.

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Discovery of Cell-Type-Specific and Disease-Related Enzymatic Activity Changes via Global Evaluation of Peptide Metabolism

Onagi J., Komatsu T., Ichihashi Y., Kuriki Y., Kamiya M., Terai T., Ueno T., Hanaoka K., Matsuzaki H., Hata K., Watanabe T., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society)  139 ( 9 ) 3465 - 3472 2017.03

Research paper (scientific journal),  ISSN  00027863

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Cellular homeostasis is maintained by a complex network of reactions catalyzed by enormous numbers of enzymatic activities (the enzymome), which serve to determine the phenotypes of cells. Here, we focused on the enzymomics of proteases and peptidases because these enzymes are an important class of disease-related proteins. We describe a system that (A) simultaneously evaluates metabolic activities of peptides using a series of exogenous peptide substrates and (B) identifies the enzymes that metabolize the specified peptide substrate with high throughput. We confirmed that the developed system was able to discover cell-type-specific and disease-related exo- and endopeptidase activities and identify the responsible enzymes. For example, we found that the activity of the endopeptidase neurolysin is highly elevated in human colorectal tumor tissue samples. This simple but powerful enzymomics platform should be widely applicable to uncover cell-type-specific reactions and altered enzymatic functions with potential value as biomarkers or drug targets in various disease states and to investigate the mechanisms of the underlying pathologies.

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Development of an Azoreductase-based Reporter System with Synthetic Fluorogenic Substrates

Shin N., Hanaoka K., Piao W., Miyakawa T., Fujisawa T., Takeuchi S., Takahashi S., Komatsu T., Ueno T., Terai T., Tahara T., Tanokura M., Nagano T., Urano Y.

ACS Chemical Biology (ACS Chemical Biology)  12 ( 2 ) 558 - 563 2017.02

Research paper (scientific journal), Accepted,  ISSN  15548929

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Enzyme/substrate pairs, such as β-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biological events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, we focused on azoreductase (AzoR). We designed and synthesized a library of azo-rhodamine derivatives as candidate fluorogenic substrates. These derivatives were nonfluorescent, probably due to ultrafast conformational change around the N=N bond after photoexcitation. We found that AzoR-mediated reduction of the azo bond of derivatives bearing an electron-donating group on the azobenzene moiety was followed by nonenzymatic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. We show that the AzoR/compound 9 reporter system can detect azoreductase-expressing live cells at the single cell level.

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Discovery and mechanistic characterization of selective inhibitors of H<inf>2</inf>S-producing Enzyme: 3-Mercaptopyruvate

Hanaoka K., Sasakura K., Suwanai Y., Toma-Fukai S., Shimamoto K., Takano Y., Shibuya N., Terai T., Komatsu T., Ueno T., Ogasawara Y., Tsuchiya Y., Watanabe Y., Kimura H., Wang C., Uchiyama M., Kojima H., Okabe T., Urano Y., Shimizu T., Nagano T.

Scientific Reports (Scientific Reports)  7 2017.01

Research paper (scientific journal), Accepted

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Very recent studies indicate that sulfur atoms with oxidation state 0 or -1, called sulfane sulfurs, are the actual mediators of some physiological processes previously considered to be regulated by hydrogen sulfide (H2S). 3-Mercaptopyruvate sulfurtransferase (3MST), one of three H2S-producing enzymes, was also recently shown to produce sulfane sulfur (H2S n). Here, we report the discovery of several potent 3MST inhibitors by means of high-throughput screening (HTS) of a large chemical library (174,118 compounds) with our H2S-selective fluorescent probe, HSip-1. Most of the identified inhibitors had similar aromatic ring-carbonyl-S-pyrimidone structures. Among them, compound 3 showed very high selectivity for 3MST over other H2S/sulfane sulfur-producing enzymes and rhodanese. The X-ray crystal structures of 3MST complexes with two of the inhibitors revealed that their target is a persulfurated cysteine residue located in the active site of 3MST. Precise theoretical calculations indicated the presence of a strong long-range electrostatic interaction between the persulfur anion of the persulfurated cysteine residue and the positively charged carbonyl carbon of the pyrimidone moiety of the inhibitor. Our results also provide the experimental support for the idea that the 3MST-catalyzed reaction with 3-mercaptopyruvate proceeds via a ping-pong mechanism.

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Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

Kimura Y., Komatsu T., Yanagi K., Hanaoka K., Ueno T., Terai T., Kojima H., Okabe T., Nagano T., Urano Y.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  56 ( 1 ) 153 - 157 2017.01

Research paper (scientific journal), Accepted,  ISSN  14337851

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We have established a coupled assay system targeting protein l-isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1′ position of the caspase-3 recognition sequence. Following PIMT-catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase-3 to give fluorescence activation. High-throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.

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Development of a reversible fluorescent probe for reactive sulfur species, sulfane sulfur, and its biological application

Takano Y., Hanaoka K., Shimamoto K., Miyamoto R., Komatsu T., Ueno T., Terai T., Kimura H., Nagano T., Urano Y.

Chemical Communications (Chemical Communications)  53 ( 6 ) 1064 - 1067 2017

Research paper (scientific journal), Accepted,  ISSN  13597345

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We report a reversible off/on fluorescent probe for monitoring concentration changes of sulfane sulfur by utilizing the unique ability of sulfane sulfur to bind reversibly to other sulfur atoms and the intramolecular spirocyclization reaction of xanthene dyes. It reversibly visualized sulfane sulfur in living A549 cells and primary-cultured hippocampal astrocytes.

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Fluorescence detection of serum albumin with a turnover-based sensor utilizing Kemp elimination reaction

Sakamoto S., Komatsu T., Ueno T., Hanaoka K., Urano Y.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  27 ( 15 ) 3464 - 3467 2017

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

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The Kemp elimination reaction is a well-known chemical reaction that is facilitated on a protein surface microenvironment, and in particular is highly accelerated in a unique binding pocket of serum albumin. We have designed and synthesized a fluorescently activatable coumarin derivative with a benzisoxazole scaffold to enable monitoring of the Kemp elimination reaction in terms of fluorescence change for the first time. We show that this fluorescent sensor can sensitively and selectively quantitate serum albumin in blood samples. It also works in a dry-chemistry format.

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Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability

Hirabayashi K., Hanaoka K., Egawa T., Kobayashi C., Takahashi S., Komatsu T., Ueno T., Terai T., Ikegaya Y., Nagano T., Urano Y.

Cell Calcium (Cell Calcium)  60 ( 4 ) 256 - 265 2016.10

Research paper (scientific journal), Accepted,  ISSN  01434160

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Fluorescence imaging of calcium ions (Ca2+) has become an essential technique for investigation of signaling pathways involving Ca2+ as a second messenger. But, Ca2+ signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca2+ and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca2+ that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca2+ concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca2+ red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms.

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A Single Fluorescent Probe to Visualize Hydrogen Sulfide and Hydrogen Polysulfides with Different Fluorescence Signals

Chen W., Pacheco A., Takano Y., Day J.J., Hanaoka K., Xian M.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  55 ( 34 ) 9993 - 9996 2016.08

Research paper (scientific journal), Accepted,  ISSN  14337851

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Hydrogen sulfide (H2S) and hydrogen polysulfides (H2Sn, n>1) are endogenous regulators of many physiological processes. In order to better understand the symbiotic relationship and cellular cross-talk between H2S and H2Sn, it is highly desirable to develop single fluorescent probes which enable dual-channel discrimination between H2S and H2Sn. Herein, we report the rational design, synthesis, and evaluation of the first dual-detection fluorescent probe DDP-1 that can visualize H2S and H2Snwith different fluorescence signals. The probe showed high selectivity and sensitivity to H2S and H2Snin aqueous media and in cells.

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Unexpected Photo-instability of 2,6-Sulfonamide-Substituted BODIPYs and Its Application to Caged GABA

Takeda A., Komatsu T., Nomura H., Naka M., Matsuki N., Ikegaya Y., Terai T., Ueno T., Hanaoka K., Nagano T., Urano Y.

ChemBioChem (ChemBioChem)     1233 - 1240 2016.07

Research paper (scientific journal), Accepted,  ISSN  14394227

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Investigation of the unexpected photo-instability of 2,6-sulfonamide-substituted derivatives of the boron dipyrromethene (BODIPY) fluorophore led to the discovery of a photoreaction accompanied by multiple bond scissions. We characterized the photoproducts and utilized the photoreaction to design a caged γ-aminobutyric acid (GABA) derivative that can release GABA upon irradiation in the visible range (>450 nm). This allowed us to stimulate neural cells in mouse brain slices.

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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

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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

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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

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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

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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

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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

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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

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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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