論文 - 花岡　健二郎

Alkyl gallates inhibit serine O-acetyltransferase in bacteria and enhance susceptibility of drug-resistant Gram-negative bacteria to antibiotics.

Toyomoto T, Ono K, Shiba T, Momitani K, Zhang T, Tsutsuki H, Ishikawa T, Hoso K, Hamada K, Rahman A, Wen L, Maeda Y, Yamamoto K, Matsuoka M, Hanaoka K, Niidome T, Akaike T, Sawa T

Frontiers in microbiology 14   1276447 2023年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1664-302X

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Folate receptor-targeted near-infrared photodynamic therapy for folate receptor-overexpressing tumors.

Aung W, Tsuji AB, Hanaoka K, Higashi T

World journal of clinical oncology 13 （ 11 ） 880 - 895 2022年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2218-4333

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General Design Strategy to Precisely Control the Emission of Fluorophores via a Twisted Intramolecular Charge Transfer (TICT) Process

Hanaoka Kenjiro, Iwaki Shimpei, Yagi Kiyoshi, Myochin Takuya, Ikeno Takayuki, Ohno Hisashi, Sasaki Eita, Komatsu Toru, Ueno Tasuku, Uchigashima Motokazu, Mikuni Takayasu, Tainaka Kazuki, Tahara Shinya, Takeuchi Satoshi, Tahara Tahei, Uchiyama Masanobu, Nagano Tetsuo, Urano Yasuteru

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 144 （ 43 ） 19778 - 19790 2022年10月

研究論文（学術雑誌）, 共著, 筆頭著者, 責任著者, 査読有り,  ISSN  0002-7863

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RELATIONSHIP BETWEEN MOUSE WOUND HEALING AND KERATIN 17

Ito M., Imbe Y., Wang Q., Aramaki N., Okabe K., Sakai S., Takaya K., Kanazawa H., Hanaoka K., Kishi K.

WOUND REPAIR AND REGENERATION 30 （ 4 ） A9 - A9 2022年07月

ISSN  1067-1927

SEARCH FOR THE FORMATION MECHANISM OF ACTIN CABLE FOR COMPLETE SKIN REGENERATION

Imbe Y., Ito M., Wang Q., Aramaki N., Okabe K., Sakai S., Takaya K., Kanazawa H., Hanaoka K., Kishi K.

WOUND REPAIR AND REGENERATION 30 （ 4 ） A9 - A9 2022年07月

ISSN  1067-1927

Development of a fluorescent probe library enabling efficient screening of tumour-imaging probes based on discovery of biomarker enzymatic activities.

Kuriki Y, Yoshioka T, Kamiya M, Komatsu T, Takamaru H, Fujita K, Iwaki H, Nanjo A, Akagi Y, Takeshita K, Hino H, Hino R, Kojima R, Ueno T, Hanaoka K, Abe S, Saito Y, Nakajima J, Urano Y

Chemical science 13 （ 16 ） 4474 - 4481 2022年04月

研究論文（学術雑誌）, 査読有り,  ISSN  2041-6520

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Molecular design of near-infrared (NIR) fluorescent probes targeting exopeptidase and application for detection of dipeptidyl peptidase 4 (DPP-4) activity

Hoshino Yuki, Hanaoka Kenjiro, Sakamoto Kei, Yasunaga Masahiro, Kojima Takashi, Kotani Daisuke, Nomoto Ayumu, Sasaki Eita, Komatsu Toru, Ueno Tasuku, Takamaru Hiroyuki, Saito Yutaka, Seto Yasuyuki, Urano Yasuteru

RSC CHEMICAL BIOLOGY 3 （ 7 ） 859 - 867 2022年03月

研究論文（学術雑誌）, 筆頭著者, 責任著者, 査読有り,  ISSN  2633-0679

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Rapid Visualization of Deeply Located Tumors In Vivo by Intravenous Administration of a gamma-Glutamyltranspeptidase-Activated Fluorescent Probe

Nakada Akihiro, Maruyama Takuma, Kamiya Mako, Hanaoka Kenjiro, Urano Yasuteru

BIOCONJUGATE CHEMISTRY 33 （ 3 ） 523 - 529 2022年03月

研究論文（学術雑誌）, 査読有り,  ISSN  1043-1802

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Sulfide catabolism ameliorates hypoxic brain injury

Marutani E., Morita M., Hirai S., Kai S., Grange R.M.H., Miyazaki Y., Nagashima F., Traeger L., Magliocca A., Ida T., Matsunaga T., Flicker D.R., Corman B., Mori N., Yamazaki Y., Batten A., Li R., Tanaka T., Ikeda T., Nakagawa A., Atochin D.N., Ihara H., Olenchock B.A., Shen X., Nishida M., Hanaoka K., Kevil C.G., Xian M., Bloch D.B., Akaike T., Hindle A.G., Motohashi H., Ichinose F.

Nature Communications （Nature Communications）  12 （ 1 ） 3108 2021年12月

研究論文（学術雑誌）, 共著, 査読有り

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The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain’s sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

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Discovery of an F-actin-binding small molecule serving as a fluorescent probe and a scaffold for functional probes.

Takagi T, Ueno T, Ikawa K, Asanuma D, Nomura Y, Uno SN, Komatsu T, Kamiya M, Hanaoka K, Okimura C, Iwadate Y, Hirose K, Nagano T, Sugimura K, Urano Y

Science advances （Science Advances）  7 （ 47 ） eabg8585 2021年11月

研究論文（学術雑誌）, 査読有り,  ISSN  2375-2548

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Actin is a ubiquitous cytoskeletal protein, forming a dynamic network that generates mechanical forces in the cell. There is a growing demand for practical and accessible tools for dissecting the role of the actin cytoskeleton in cellular function, and the discovery of a new actin-binding small molecule is an important advance in the field, offering the opportunity to design and synthesize of new class of functional molecules. Here, we found an F-actin-binding small molecule and introduced two powerful tools based on a new class of actin-binding small molecule: One enables visualization of the actin cytoskeleton, including super-resolution imaging, and the other enables highly specific green light-controlled fragmentation of actin filaments, affording unprecedented control of the actin cytoskeleton and its force network in living cells.

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Recent advances in detection, isolation, and imaging techniques for sulfane sulfur-containing biomolecules

Echizen H., Sasaki E., Hanaoka K.

Biomolecules （Biomolecules）  11 （ 11 ）  2021年11月

研究論文（学術雑誌）, 査読有り

　概要を見る

Hydrogen sulfide and its oxidation products are involved in many biological processes, and sulfane sulfur compounds, which contain sulfur atoms bonded to other sulfur atom(s), as found in hydropersulfides (R-S-SH), polysulfides (R-S-Sn-S-R), hydrogen polysulfides (H2Sn), etc., have at-tracted increasing interest. To characterize their physiological and pathophysiological roles, selective detection techniques are required. Classically, sulfane sulfur compounds can be detected by cyanolysis, involving nucleophilic attack by cyanide ion to cleave the sulfur–sulfur bonds. The generated thiocyanate reacts with ferric ion, and the resulting ferric thiocyanate complex can be easily detected by absorption spectroscopy. Recent exploration of the properties of sulfane sulfur compounds as both nucleophiles and electrophiles has led to the development of various chemical techniques for detection, isolation, and bioimaging of sulfane sulfur compounds in biological samples. These include tag-switch techniques, LC-MS/MS, Raman spectroscopy, and fluorescent probes. Herein, we present an overview of the techniques available for specific detection of sulfane sulfur species in biological contexts.

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A Sulfonyl Azide-Based Sulfide Scavenger Rescues Mice from Lethal Hydrogen Sulfide Intoxication

Miyazaki Y., Marutani E., Ikeda T., Ni X., Hanaoka K., Xian M., Ichinose F.

Toxicological Sciences （Toxicological Sciences）  183 （ 2 ） 393 - 403 2021年10月

研究論文（学術雑誌）, 査読有り,  ISSN  10966080

　概要を見る

Exposure to hydrogen sulfide (H2S) can cause neurotoxicity and cardiopulmonary arrest. Resuscitating victims of sulfide intoxication is extremely difficult, and survivors often exhibit persistent neurological deficits. However, no specific antidote is available for sulfide intoxication. The objective of this study was to examine whether administration of a sulfonyl azide-based sulfide-specific scavenger, SS20, would rescue mice in models of H2S intoxication: Ongoing exposure and post-cardiopulmonary arrest. In the ongoing exposure model, SS20 (1250 μmol/kg) or vehicle was administered to awake CD-1 mice intraperitoneally at 10 min after breathing 790 ppm of H2S followed by another 30 min of H2S inhalation. Effects of SS20 on survival were assessed. In the post-cardiopulmonary arrest model, cardiopulmonary arrest was induced by an intraperitoneal administration of sodium sulfide nonahydrate (125 mg/kg) in anesthetized mice. After 1 min of cardiopulmonary arrest, mice were resuscitated with intravenous administration of SS20 (250 μmol/kg) or vehicle. Effects of SS20 on survival, neurological outcomes, and plasma H2S levels were evaluated. Administration of SS20 during ongoing H2S inhalation improved 24-h survival (6/6 [100%] in SS20 vs 1/6 [17%] in vehicle; p =. 0043). Post-arrest administration of SS20 improved 7-day survival (4/10 [40%] in SS20 vs 0/10 [0%] in vehicle; p =. 0038) and neurological outcomes after resuscitation. SS20 decreased plasma H2S levels to pre-arrest baseline immediately after reperfusion and shortened the time to return of spontaneous circulation and respiration. These results suggest that SS20 is an effective antidote against lethal H2S intoxication, even when administered after cardiopulmonary arrest.

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Matrix metalloprotease–14 is a target enzyme for detecting peritoneal metastasis in gastric cancer

Ogawa S., Kubo H., Murayama Y., Kubota T., Yubakami M., Matsumoto T., Ohashi T., Okamoto K., Kuriki Y., Hanaoka K., Urano Y., Otsuji E.

Photodiagnosis and Photodynamic Therapy （Photodiagnosis and Photodynamic Therapy）  35   102420 2021年09月

研究論文（学術雑誌）, 査読有り,  ISSN  15721000

　概要を見る

Background: Accurate diagnosis of peritoneal metastasis in gastric cancer (GC) is important to determine the appropriate treatment. This study aimed to examine whether matrix metalloprotease–14 (MMP–14) was a candidate enzyme in fluorescence imaging for the diagnosis of peritoneal metastasis in GC. Methods: GC and normal peritoneal (NP) tissues from 96 and 20 patients, respectively were evaluated for MMP–14 expression. Live cell imaging of GC cell lines (NUGC4, MKN45, MKN74, HGC-27, and Kato-III) was performed using the MMP–14-activatable fluorescence probe; BODIPY-MMP. Furthermore, the overall survival (OS) was calculated in all patients (n = 96). Results: MMP–14 expression was significantly higher in GC tissues (median: 3.57 ng/mg protein; range:0.64–24.4 ng/mg protein) than in NP tissues (median: 1.34 ng/mg protein; median: 0.53–3.09 ng/mg protein) (P < 0.01). Receiver operating characteristic curves showed that the area under the curve, sensitivity, and specificity were 0.907, 84.4%, and 90.0%, respectively. In live cell imaging using the BODIPY-MMP, fluorescence was observed in five GC cell lines. In the analysis of OS, the high expression of the MMP–14 group had a significantly poorer OS rate than the low expression of the MMP–14 group (P = 0.02). In the multivariate analyses, MMP-14 expression was an independent risk factor for OS (hazard ratio: 2.33; 95 % confidence interval: 1.05–5.45; P = 0.04). Conclusion: MMP–14 is a promising enzyme in intraoperative fluorescence imaging for peritoneal metastasis in GC, especially in patients with poor prognosis.

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Establishment of live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells

Yanagi K., Komatsu T., Ogihara S., Okabe T., Kojima H., Nagano T., Ueno T., Hanaoka K., Urano Y.

Cell Reports （Cell Reports）  36 （ 1 ） 109311 2021年07月

研究論文（学術雑誌）, 査読有り,  ISSN  2211-1247

　概要を見る

In this study, we present a live-cell-based fluorometric coupled assay system to identify the compounds that can regulate the targeted metabolic pathways in live cells. The assay is established through targeting specific metabolic pathways and using “input” and “output” metabolite pairs. The changes in the extracellular output that are generated and released into the extracellular media from the input are assessed as the activity of the pathway. The screening for the glycolytic pathway and amino acid metabolism reveals the activities of the present drugs, 6-BIO and regorafenib, that regulate the metabolic fate of tumor cells.

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Design, synthesis and evaluation of enzyme-responsive fluorogenic probes based on pyridine-flanked diketopyrrolopyrrole dyes

Jenni S., Ponsot F., Baroux P., Collard L., Ikeno T., Hanaoka K., Quesneau V., Renault K., Romieu A.

Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy （Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy）  248   119179 2021年03月

研究論文（学術雑誌）, 査読有り,  ISSN  13861425

　概要を見る

The ever-growing demand for fluorogenic dyes usable in the rapid construction of analyte-responsive fluorescent probes, has recently contributed to a revival of interest in the chemistry of diketopyrrolopyrrole (DPP) pigments. In this context, we have explored the potential of symmetrical and unsymmetrical DPP derivatives bearing two or one 4-pyridyl substituents acting as optically tunable group(s). The unique fluorogenic behavior of these molecules, closely linked to N-substitution/charge state of their pyridine unit (i.e., neutral pyridine or cationic pyridinium), has been used to design DPP-based fluorescent probes for detection of hypoxia-related redox enzymes and penicillin G acylase (PGA). In this paper, we describe synthesis, spectral characterization and bioanalytical validations of these probes. Dramatic differences in terms of aqueous stability and enzymatic fluorescence activation were observed. This systematic study enables to delineate the scope of application of pyridine-flanked DPP fluorophores in the field of enzyme biosensing.

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Recent advances in probe design to detect reactive sulfur species and in the chemical reactions employed for fluorescence switching

Echizen H., Hanaoka K.

Journal of Clinical Biochemistry and Nutrition （Journal of Clinical Biochemistry and Nutrition）  68 （ 1 ） 9 - 17 2021年01月

研究論文（学術雑誌）, 査読有り,  ISSN  09120009

　概要を見る

Reactive sulfur species, including hydrogen sulfide, hydropersulfide, and polysulfide, have many roles in biological systems. For example, hydrogen sulfide is involved in the relaxation of vascular smooth muscles and mediation of neurotransmission, while sulfane sulfur, which exists in cysteine persulfide/polysulfide, and glutathione persulfide/polysulfide, is involved in physiological antioxidation and cytoprotection mechanisms. Fluorescence imaging is well suited for real-time monitoring of reactive sulfur species in living cells, and many fluorescent probes for reactive sulfur species have been reported. In such probes, the choice of detection chemistry is extremely important, not only to achieve effective fluorescence switching and high selectivity, but also because the reactions may be applicable to develop other chemical tools, such as reactive sulfur species donors/scavengers. Here, we present an overview of both widely used and recently developed fluorescent probes for reactive sulfur species, focusing especially on the chemical reactions employed in them for fluorescence switching. We also briefly introduce some applications of fluorescent probes for hydrogen sulfide and sulfane sulfur.

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Fluorescent probes for detection of supersulfides

Sasaki E., Hanaoka K.

Seikagaku （Seikagaku）  93 （ 5 ） 604 - 612 2021年

ISSN  00371017

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A cytosolically localized far-red to near-infrared rhodamine-based fluorescent probe for calcium ions

Numasawa K., Hanaoka K., Ikeno T., Echizen H., Ishikawa T., Morimoto M., Komatsu T., Ueno T., Ikegaya Y., Nagano T., Urano Y.

Analyst （Analyst）  145 （ 23 ） 7736 - 7740 2020年12月

研究論文（学術雑誌）, 査読有り,  ISSN  00032654

　概要を見る

Ca2+ is one of the most important second messengers in cells. A far-red to near-infrared (NIR) Ca2+ fluorescent probe is useful for multi-color imaging in GFP or YFP-expressing biosamples. Here we developed a cytosolically localized far-red to NIR rhodamine-based fluorescent probe for Ca2+, CaSiR-2 AM, while rhodamine dyes are basically localized to mitochondria or lysosomes in cells. This journal is

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Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling

Okada K., Mori D., Makii Y., Nakamoto H., Murahashi Y., Yano F., Chang S.H., Taniguchi Y., Kobayashi H., Semba H., Takeda N., Piao W., Hanaoka K., Nagano T., Tanaka S., Saito T.

Scientific Reports （Scientific Reports）  10 （ 1 ） 5425 2020年12月

研究論文（学術雑誌）, 査読有り

　概要を見る

HIF-1α, an essential transcription factor under hypoxic condition, is indispensable for chondrocytes during skeletal development but its expression and roles in articular chondrocytes are yet to be revealed. We examined HIF-1α protein expression and the hypoxic condition during mouse osteoarthritis (OA) development using state of the art hypoxic probes and found that its expression decreased as OA progressed, coinciding with the change in hypoxic conditions in articular cartilage. Gain- and loss-of-function of HIF-1α in cell culture experiments showed that HIF-1α suppressed catabolic genes such as Mmp13 and Hif2a. We confirmed these anticatabolic effects by measuring glycosaminoglycan release from wild type and conditional knock-out mice femoral heads cultured ex vivo. We went on to surgically induce OA in mice with chondrocyte-specific deletion of Hif1a and found that the development of OA was exacerbated. Increased expression of catabolic factors and activation of NF-κB signalling was clearly evident in the knock-out mice. By microarray analysis, C1qtnf3 was identified as a downstream molecule of HIF-1α, and experiments showed it exerted anti-catabolic effects through suppression of NF-κB. We conclude that HIF-1α has an anti-catabolic function in the maintenance of articular cartilage through suppression of NF-κB signalling.

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Calciprotein particle-induced cytotoxicity via lysosomal dysfunction and altered cholesterol distribution in renal epithelial HK-2 cells

Kunishige R., Mizoguchi M., Tsubouchi A., Hanaoka K., Miura Y., Kurosu H., Urano Y., Kuro-o M., Murata M.

Scientific Reports （Scientific Reports）  10 （ 1 ） 20125 2020年12月

研究論文（学術雑誌）, 査読有り

　概要を見る

Dietary phosphate overload induces chronic kidney disease (CKD), and calciprotein particles (CPPs), a form of nanoparticle comprising calcium phosphate and serum proteins, has been proposed to cause renal toxicity. However, the mechanism of CPP cytotoxicity in renal tubular cells is unknown. Here we show that in renal proximal tubular epithelial HK-2 cells, endocytosed CPPs accumulate in late endosomes/lysosomes (LELs) and increase their luminal pH by ~ 1.0 unit. This results in a decrease in lysosomal hydrolase activity and autophagic flux blockage without lysosomal rupture and reactive oxygen species generation. CPP treatment led to vulnerability to H2O2-induced oxidative stress and plasma membrane injury, probably because of autophagic flux blockage and decreased plasma membrane cholesterol, respectively. CPP-induced disruption of lysosomal homeostasis, autophagy flux and plasma membrane integrity might trigger a vicious cycle, leading to progressive nephron loss.

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