Details of a Researcher - Hanaoka, Kenjiro

Papers - Hanaoka, Kenjiro

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Development of a Series of Practical Fluorescent Chemical Tools to Measure pH Values in Living Samples

Takahashi S., Kagami Y., Hanaoka K., Terai T., Komatsu T., Ueno T., Uchiyama M., Koyama-Honda I., Mizushima N., Taguchi T., Arai H., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society) 140 ( 18 ) 5925 - 5933 2018.05

Research paper (scientific journal), Accepted, ISSN 00027863

In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate pKa for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) SiRpH5 has suitable pKa and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with SiRpH5, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) Me-pEPPR is a near-infrared (NIR) probe; by using dextrin tagged with Me-pEPPR, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging.

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N-Acetyl Cysteine Functions as a Fast-Acting Antioxidant by Triggering Intracellular H <inf>2</inf> S and Sulfane Sulfur Production

Ezeriņa D., Takano Y., Hanaoka K., Urano Y., Dick T.P.

Cell Chemical Biology (Cell Chemical Biology) 25 ( 4 ) 447 - 459.e4 2018.04

Research paper (scientific journal), Accepted, ISSN 24519456

The cysteine prodrug N-acetyl cysteine (NAC) is widely used as a pharmacological antioxidant and cytoprotectant. It has been reported to lower endogenous oxidant levels and to protect cells against a wide range of pro-oxidative insults. As NAC itself is a poor scavenger of oxidants, the molecular mechanisms behind the antioxidative effects of NAC have remained uncertain. Here we show that NAC-derived cysteine is desulfurated to generate hydrogen sulfide, which in turn is oxidized to sulfane sulfur species, predominantly within mitochondria. We provide evidence suggesting the possibility that sulfane sulfur species produced by 3-mercaptopyruvate sulfurtransferase and sulfide:quinone oxidoreductase are the actual mediators of the immediate antioxidative and cytoprotective effects provided by NAC. N-Acetyl cysteine (NAC), by itself a poor scavenger of oxidants, is converted inside cells to yield sulfane sulfur species, which are very potent scavengers of oxidants. This conversion may account for many of the antioxidative effects provided by NAC which have hitherto remained unexplained.

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Establishment of Molecular Design Strategy to Obtain Activatable Fluorescent Probes for Carboxypeptidases

Kuriki Y., Kamiya M., Kubo H., Komatsu T., Ueno T., Tachibana R., Hayashi K., Hanaoka K., Yamashita S., Ishizawa T., Kokudo N., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society) 140 ( 5 ) 1767 - 1773 2018.02

Research paper (scientific journal), ISSN 00027863

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins. However, methodology to monitor the activities of CPs is poorly developed. Here, we present the first versatile design strategy to obtain activatable fluorescent probes for CPs by utilizing intramolecular spirocyclization of rhodamine to translate the "aliphatic carboxamide to aliphatic carboxylate" structural conversion catalyzed by CPs into dynamic fluorescence activation. Based on this novel strategy, we developed probes for carboxypeptidases A and B. One of these probes was able to detect pancreatic juice leakage in mice ex vivo, suggesting that its suitability for intraoperative diagnosis of pancreatic fistula. This design strategy should be broadly applicable to CPs, as well as other previously untargetable enzymes, enabling development of fluorescent probes to study various pathological and biological processes.

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Development of a novel inhibitor for reactive sulfur species-producing enzymes

Echizen H., Hanaoka K.

Seikagaku (Seikagaku) 90 ( 3 ) 388 - 393 2018

Research paper (scientific journal), Accepted, ISSN 00371017

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Synthesis of unsymmetrical Si-rhodamine fluorophores and application to a far-red to near-infrared fluorescence probe for hypoxia

Hanaoka K., Kagami Y., Piao W., Myochin T., Numasawa K., Kuriki Y., Ikeno T., Ueno T., Komatsu T., Terai T., Nagano T., Urano Y.

Chemical Communications (Chemical Communications) 54 ( 50 ) 6939 - 6942 2018

Research paper (scientific journal), Accepted, ISSN 13597345

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Molecular imaging of aberrant crypt foci in the human colon targeting glutathione S-transferase P1-1

Muguruma N., Okamoto K., Nakagawa T., Sannomiya K., Fujimoto S., Mitsui Y., Kimura T., Miyamoto H., Higashijima J., Shimada M., Horino Y., Matsumoto S., Hanaoka K., Nagano T., Shibutani M., Takayama T.

Scientific Reports (Scientific Reports) 7 ( 1 ) 2017.12

Research paper (scientific journal)

Aberrant crypt foci (ACF), the earliest precursor lesion of colorectal cancers (CRCs), are a good surrogate marker for CRC risk stratification and chemoprevention. However, the conventional ACF detection method with dye-spraying by magnifying colonoscopy is labor- A nd skill-intensive. We sought to identify rat and human ACF using a fluorescent imaging technique that targets a molecule specific for ACF. We found that glutathione S-transferase (GST) P1-1 was overexpressed in ACF tissues in a screening experiment. We then synthesized the fluorogenic probe, DNAT-Me, which is fluorescently quenched but is activated by GSTP1-1. A CRC cell line incubated with DNAT-Me showed strong fluorescence in the cytosol. Fluorescence intensities correlated significantly with GST activities in cancer cell lines. When we sprayed DNAT-Me onto colorectal mucosa excised from azoxymethane-treated rats and surgically resected from CRC patients, ACF with strong fluorescent signals were clearly observed. The ACF number determined by postoperative DNAT-Me imaging was almost identical to that determined by preoperative methylene blue staining. The signal-to-noise ratio for ACF in DNAT-Me images was significantly higher than that in methylene blue staining. Thus, we sensitively visualized ACF on rat and human colorectal mucosa by using a GST-activated fluorogenic probe without dye-spraying and magnifying colonoscopy.

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Development of an azo-based photosensitizer activated under mild hypoxia for photodynamic therapy

Piao W., Hanaoka K., Fujisawa T., Takeuchi S., Komatsu T., Ueno T., Terai T., Tahara T., Nagano T., Urano Y.

Journal of the American Chemical Society (Journal of the American Chemical Society) 139 ( 39 ) 13713 - 13719 2017.10

Research paper (scientific journal), Accepted, ISSN 00027863

Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (1O2), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate 1O2 only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to 1O2 generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of 1O2 to occur. In PDT in vitro, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O2), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azobased strategy should be widely applicable to design a range of activatable photosensitizers.

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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

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

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

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

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

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

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

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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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

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

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

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

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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