Development of coupled assay-based fluorescent probes to monitor post-translational modification reactions in live cells

• Project/Area Number: 17K19477
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Research Field: Pharmaceutical Sciences and related fields
• Research Institution: The University of Tokyo
• Principal Investigator: Komatsu Toru 東京大学, 大学院薬学系研究科(薬学部), 特任助教 (40599172)
• Project Period (FY): 2017-06-30 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2019: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2018: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2017: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
• Keywords: ケミカルバイオロジー / 創薬化学 / 酵素

Outline of Final Research Achievements

We have developed the methodology of "live" coupled assay that is designed to monitor the activities of enzymes in live cell context. We have successfully developed the systems below to reveal the potential of our methodology in understanding and controlling pathogenic enzymatic functions.
(1) Discovery of compounds to control pathogenic S-adenosylmethionine levels of colorectal tumor cells (JACS 2020).
(2) Development of enzymomics methodologies of monitoring and controlling altered amino acid metabolisms of tumor cells.

Academic Significance and Societal Importance of the Research Achievements

本研究課題を通じて，生きた細胞内の代謝活性を評価する新たな方法論の確立に成功し，その有用性を世界に発信することができた（JACS 2020）．
また，本研究を通じて見出された (1) 茶カテキンの細胞内メチル化状態の制御活性，(2) 既知の抗がん剤による細胞内代謝活性制御機構の発見，のいずれもが社会的にも重要なインパクトを与える研究成果であり，特に前者については，研究代表者の所属機関らによるプレスリリースがおこなわれ，医療系のニュースサイトなどで取り上げられた．

Research Products

• [Journal Article] Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples (2020)
  • Author(s): Shingo Sakamoto, Toru Komatsu, Rikiya Watanabe, Yi Zhang, Taiki Inoue, Mitsuyasu Kawaguchi, Hidehiko Nakagawa, Takaaki Ueno, Takuji Okusaka, Kazufumi Honda, Hiroyuki Noji, Yasuteru Urano
  • Journal Title: Science Advances Volume: 6 Issue: 11
  • DOI: 10.1126/sciadv.aay0888
  • Peer Reviewed / Open Access
• [Journal Article] Metabolic-Pathway-Oriented Screening Targeting S-Adenosyl-l-methionine Reveals the Epigenetic Remodeling Activities of Naturally Occurring Catechols (2019)
  • Author(s): Ogihara Shusuke、Komatsu Toru、Itoh Yukihiro、Miyake Yuka、Suzuki Takayoshi、Yanagi Kouichi、Kimura Yusuke、Ueno Tasuku、Hanaoka Kenjiro、Kojima Hirotatsu、Okabe Takayoshi、Nagano Tetsuo、Urano Yasuteru
  • Journal Title: Journal of the American Chemical Society Volume: 142 Issue: 1 Pages: 21-26
  • DOI: 10.1021/jacs.9b08698
  • Peer Reviewed / Open Access
• [Journal Article] Development of ratiometric carbohydrate sensor based on boron dipyrromethene (BODIPY) scaffold (2019)
  • Author(s): Cheruthu Nusaiba Madappuram、Komatsu Toru、Ueno Tasuku、Hanaoka Kenjiro、Urano Yasuteru
  • Journal Title: Bioorganic & Medicinal Chemistry Letters Volume: 29 Issue: 22 Pages: 126684-126684
  • DOI: 10.1016/j.bmcl.2019.126684
  • Peer Reviewed
• [Journal Article] Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples (2019)
  • Author(s): Ichihashi Yuki、Komatsu Toru、Kyo Etsu、Matsuzaki Hiroyuki、Hata Keisuke、Watanabe Toshiaki、Ueno Tasuku、Hanaoka Kenjiro、Urano Yasuteru
  • Journal Title: Analytical Chemistry Volume: 91 Issue: 18 Pages: 11497-11501
  • DOI: 10.1021/acs.analchem.9b03016
  • Peer Reviewed
• [Journal Article] Development and validation of an improved diced electrophoresis gel assay cutter-plate system for enzymomics studies (2019)
  • Author(s): Komatsu Toru、Shimoda Masahiro、Kawamura Yukiko、Urano Yasuteru、Nagano Tetsuo
  • Journal Title: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics Volume: 1867 Issue: 1 Pages: 82-87
  • DOI: 10.1016/j.bbapap.2018.06.004
  • Peer Reviewed
• [Journal Article] Development of a Series of Practical Fluorescent Chemical Tools to Measure pH Values in Living Samples (2018)
  • Author(s): Takahashi Shodai、Kagami Yu、Hanaoka Kenjiro、Terai Takuya、Komatsu Toru、Ueno Tasuku、Uchiyama Masanobu、Koyama-Honda Ikuko、Mizushima Noboru、Taguchi Tomohiko、Arai Hiroyuki、Nagano Tetsuo、Urano Yasuteru
  • Journal Title: Journal of the American Chemical Society Volume: 140 Issue: 18 Pages: 5925
  • DOI: 10.1021/jacs.8b00277
  • Peer Reviewed
• [Journal Article] Synthesis of unsymmetrical Si-rhodamine fluorophores and application to a far-red to near-infrared fluorescence probe for hypoxia (2018)
  • Author(s): Hanaoka Kenjiro、Kagami Yu、Piao Wen、Myochin Takuya、Numasawa Koji、Kuriki Yugo、Ikeno Takayuki、Ueno Tasuku、Komatsu Toru、Terai Takuya、Nagano Tetsuo、Urano Yasuteru
  • Journal Title: Chemical Communications Volume: 54 Issue: 50 Pages: 6939-6942
  • DOI: 10.1039/c8cc02451k
  • Peer Reviewed
• [Journal Article] High-throughput single-molecule bioassay using micro-reactor arrays with a concentration gradient of target molecules (2018)
  • Author(s): Watanabe Rikiya、Komatsu Toru、Sakamoto Shingo、Urano Yasuteru、Noji Hiroyuki
  • Journal Title: Lab on a Chip Volume: 18 Issue: 18 Pages: 2849-2853
  • DOI: 10.1039/c8lc00535d
  • Peer Reviewed / Open Access
• [Journal Article] Red-Shifted Fluorogenic Substrate for Detection of lacZ-Positive Cells in Living Tissue with Single-Cell Resolution (2018)
  • Author(s): Ito Hiroki、Kawamata Yu、Kamiya Mako、Tsuda-Sakurai Kayoko、Tanaka Shinji、Ueno Tasuku、Komatsu Toru、Hanaoka Kenjiro、Okabe Shigeo、Miura Masayuki、Urano Yasuteru
  • Journal Title: Angewandte Chemie International Edition Volume: 57 Issue: 48 Pages: 15702-15706
  • DOI: 10.1002/anie.201808670
  • Peer Reviewed
• [Journal Article] Rapid detection of metastatic lymph nodes of colorectal cancer with a gamma-glutamyl transpeptidase-activatable fluorescence probe (2018)
  • Author(s): Kubo Hidemasa、Hanaoka Kenjiro、Kuriki Yugo、Komatsu Toru、Ueno Tasuku、Kojima Ryosuke、Kamiya Mako、Murayama Yasutoshi、Otsuji Eigo、Urano Yasuteru
  • Journal Title: Scientific Reports Volume: 8 Issue: 1 Pages: 17781-6942
  • DOI: 10.1038/s41598-018-36062-3
  • Peer Reviewed / Open Access
• [Journal Article] Establishment of Molecular Design Strategy To Obtain Activatable Fluorescent Probes for Carboxypeptidases (2018)
  • Author(s): Kuriki Yugo、Kamiya Mako、Kubo Hidemasa、Komatsu Toru、Ueno Tasuku、Tachibana Ryo、Hayashi Kento、Hanaoka Kenjiro、Yamashita Suguru、Ishizawa Takeaki、Kokudo Norihiro、Urano Yasuteru
  • Journal Title: Journal of the American Chemical Society Volume: 140 Issue: 5 Pages: 1767-1773
  • DOI: 10.1021/jacs.7b11014
  • Peer Reviewed
• [Journal Article] Fluorescence detection of serum albumin with a turnover-based sensor utilizing Kemp elimination reaction (2017)
  • Author(s): Sakamoto Shingo、Komatsu Toru、Ueno Tasuku、Hanaoka Kenjiro、Urano Yasuteru
  • Journal Title: Bioorganic & Medicinal Chemistry Letters Volume: 27 Issue: 15 Pages: 3464-3467
  • DOI: 10.1016/j.bmcl.2017.05.076
  • Peer Reviewed
• [Journal Article] Development of an Azo-Based Photosensitizer Activated under Mild Hypoxia for Photodynamic Therapy (2017)
  • Author(s): Piao Wen、Hanaoka Kenjiro、Fujisawa Tomotsumi、Takeuchi Satoshi、Komatsu Toru、Ueno Tasuku、Terai Takuya、Tahara Tahei、Nagano Tetsuo、Urano Yasuteru
  • Journal Title: J. Am. Chem. Soc. Volume: 139 Issue: 39 Pages: 13713-13719
  • DOI: 10.1021/jacs.7b05019
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] An Activatable Photosensitizer Targeted to γ-Glutamyltranspeptidase (2017)
  • Author(s): Chiba Mayumi、Ichikawa Yuki、Kamiya Mako、Komatsu Toru、Ueno Tasuku、Hanaoka Kenjiro、Nagano Tetsuo、Lange Norbert、Urano Yasuteru
  • Journal Title: Angewandte Chemie International Edition Volume: 56 Issue: 35 Pages: 10418-10422
  • DOI: 10.1002/anie.201704793
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Potential of Enzymomics Methodologies to Characterize Disease-Related Protein Functions (2017)
  • Author(s): Toru Komatsu
  • Journal Title: Chemical and Pharmaceutical Bulletin Volume: 65 Issue: 7 Pages: 605-610
  • DOI: 10.1248/cpb.c17-00144
  • NAID: 130006772511
  • ISSN: 0009-2363, 1347-5223
  • Peer Reviewed
• [Presentation] 物質代謝の局所性の制御と理解を目指すケミカルバイオロジー研究 (2019)
  • Author(s): 小松徹
  • Organizer: 第19回日本蛋白質科学会年会・第71回日本細胞生物学会大会 合同年次大会
  • Invited
• [Presentation] Diced electrophoresis gel 法を用いたがんにおけるペプチド代謝活性異常の網羅的解析 (2019)
  • Author(s): 小松徹
  • Organizer: 日本プロテオーム学会2019年大会／第70回日本電気泳動学会総会
  • Invited
• [Presentation] 緑茶カテキンによる細胞内「メチル化」状態の制御と健康寿命 (2019)
  • Author(s): 小松徹
  • Organizer: 科学的根拠に基づく健康寿命を延ばす会 第15回講演・研究会
  • Invited
• [Presentation] 酵素活性の網羅的探索（enzymomics）に基づく疾患関連タンパク質の探索 (2019)
  • Author(s): 小松徹
  • Organizer: 第92回日本生化学会大会
  • Invited
• [Presentation] 疾患と関わるエピジェネティック代謝異常の天然物カテコールによる制御 (2019)
  • Author(s): 小松徹
  • Organizer: 2019年度さきがけ疾患代謝領域研究会
  • Invited
• [Presentation] 酵素活性の網羅的解析（enzymomics）による疾患関連タンパク質の探索 (2018)
  • Author(s): 小松徹
  • Organizer: レドックス研究会 2018
  • Invited
• [Presentation] Development of enzymomics approach to search for disease-related alternation of enzymatic functions (2018)
  • Author(s): Toru Komatsu
  • Organizer: Mass Spectrometry and Proteomics (MSP) 2018
  • Int'l Joint Research / Invited
• [Presentation] ペプチド代謝活性の網羅的解析（enzymomics）による新規疾患関連タンパク質の探索 (2018)
  • Author(s): 小松徹
  • Organizer: 新学術領域研究「化学コミュニケーションのフロンティア」第1回若手シンポジウム
  • Invited
• [Presentation] ペプチド代謝活性の網羅的解析（enzymomics）による疾患関連タンパク質の探索 (2018)
  • Author(s): 小松徹
  • Organizer: 第69回日本電気泳動学会総会シンポジウム
  • Invited
• [Presentation] 酵素活性の網羅的解析（Enzymomics）法による疾患関連タンパク質の探索 (2018)
  • Author(s): 小松徹
  • Organizer: 第56回生物物理学会年会
  • Invited
• [Presentation] Development of enzymomics approach to search for disease-related alternation of enzymatic functions (2018)
  • Author(s): Toru Komatsu
  • Organizer: ICPAC Langkawi 2018
  • Int'l Joint Research / Invited
• [Presentation] 酵素活性の網羅的評価（enzymomics）による新規疾患関連タンパク質の探索 (2018)
  • Author(s): 小松徹
  • Organizer: 第41回日本分子生物学会年会
  • Invited
• [Presentation] Diced Electrophoresis Gel (DEG) 法を用いた癌細胞における活性異常酵素の探索 (2018)
  • Author(s): 小松徹
  • Organizer: 第38回 キャピラリー電気泳動シンポジウム (SCE2018)
  • Invited
• [Presentation] 分子夾雑の理解に基づく癌関連タンパク質のオルガネラ膜局時局在機構の解明と制御 (2018)
  • Author(s): 小松徹
  • Organizer: 新学術領域研究「分子夾雑の生命化学」関東地区シンポジウム
  • Invited
• [Presentation] 酵素のはたらきを「視る」手法を開発して新たな疾患関連タンパク質を「探す」 (2018)
  • Author(s): 小松徹
  • Organizer: ITbM/IGER Chemistry Workshop 2018 - Exploring the Science with Molecules
  • Invited
• [Presentation] Discovery of Disease-related Alternation of Enzymatic Functions with Enzymomics Approach (2018)
  • Author(s): Toru Komatsu, Jun Onagi, Yuki Ichikawa, Yasuteru Urano
  • Organizer: 日本化学会第98年春季大会
  • Invited
• [Presentation] Pathway-oriented screening法による解糖系酵素阻害剤の探索 (2018)
  • Author(s): 柳 光一 、小松徹、小島宏建、岡部隆義、長野哲雄、浦野泰照
  • Organizer: 日本薬学会第138年会
• [Presentation] マイクロデバイスを用いた単一酵素活性検出による病態診断法の開発 (2018)
  • Author(s): 坂本眞伍、小松徹、渡邉力也、張翼、野地博行、浦野泰照
  • Organizer: 日本薬学会第138年会
• [Presentation] 荻原州介、小松徹、浦野泰照 (2018)
  • Author(s): 有機小分子蛍光プローブを用いたSAM高感度検出系の開発と応用
  • Organizer: 日本薬学会第138年会
• [Presentation] Development of enzymomics approach to search for disease-related alternation of enzymatic functions (2017)
  • Author(s): Toru Komatsu, Jun Onagi, Yuki Ichihashi, Tetsuo Nagano and Yasuteru Urano
  • Organizer: International Chemical Biology Society 2017
  • Int'l Joint Research
• [Presentation] Development of enzymomicsapproach to search for disease-related alternation of enzymatic functions (2017)
  • Author(s): Toru Komatsu, Jun Onagi, Yuki Ichihashi, Tetsuo Nagano and Yasuteru Urano
  • Organizer: The Second International Symposium on Biofunctional Chemistry (ISBC 2017)
  • Int'l Joint Research
• [Presentation] Development of activatable fluorescent probes for carboxypeptidase and their application (2017)
  • Author(s): Yugo Kuriki, Mako Kamiya, Hidemasa Kubo, Toru Komatsu, Tasuku Ueno, Suguru Yamashita, Norihiro Kokudo and Yasuteru Urano
  • Organizer: The Second International Symposium on Biofunctional Chemistry (ISBC 2017)
  • Int'l Joint Research
• [Presentation] 新規Activatable型Carboxypeptidase活性検出蛍光プローブの開発と応用 (2017)
  • Author(s): 栗木優五、神谷真子、小松徹、上野匡、山下俊、國土典宏、浦野泰照
  • Organizer: 日本ケミカルバイオロジー学会 第12年会
• [Presentation] Pathway-oriented screening法による解糖系酵素阻害剤の探索 (2017)
  • Author(s): 柳光一、小松徹、小島宏建、岡部隆義、長野哲雄、浦野泰照
  • Organizer: 日本ケミカルバイオロジー学会 第12年会
• [Presentation] ペプチドライブラリーを用いた酵素活性の網羅的解析法の確立と発展 (2017)
  • Author(s): 市橋裕樹、小名木淳、小松徹、神谷真子、松崎裕幸、畑啓介、渡邉聡明、浦野泰照
  • Organizer: 日本ケミカルバイオロジー学会 第12年会
• [Presentation] マイクロデバイス中の単一酵素活性検出による病態診断法の開発 (2017)
  • Author(s): 坂本眞伍、小松徹、渡邉力也、張翼、野地博行、浦野泰照
  • Organizer: 日本ケミカルバイオロジー学会 第12年会
• [Presentation] 蛍光物質Boron dipyrromethene (BODIPY) 2,6位化学修飾新規蛍光群開発 (2017)
  • Author(s): 小松徹、Cherythu Nusaiba MADAPPURAM、浦野泰照
  • Organizer: 生体機能関連化学部会若手の会第29回サマースクール
• [Presentation] Diced Electrophoresis Gel (DICE) 法を用いた癌細胞における機能異常酵素の探索 (2017)
  • Author(s): 小松徹、小名木淳、市橋裕樹、浦野泰照
  • Organizer: 日本プロテオーム学会2017年大会
• [Presentation] ペプチドの「代謝」を視る技術を開発し新たな疾患関連タンパク質の機能を解明する (2017)
  • Author(s): 小松徹
  • Organizer: 第49回若手ペプチド夏の勉強会
  • Invited
• [Presentation] 酵素の動的機能の理解による疾患関連タンパク質の探索 (2017)
  • Author(s): 小松徹
  • Organizer: 第30回バイオメディカル分析科学シンポジウム(BMAS2017)
  • Invited
• [Presentation] Pathway-oriented screening法による解糖系酵素阻害剤の探索 (2017)
  • Author(s): 柳光一、小松徹、小島宏建、岡部隆義、長野哲雄、浦野泰照
  • Organizer: 第11回バイオ関連化学シンポジウム
• [Presentation] S-adenosyl-L-methionine(SAM)の高感度検出に基づく細胞内メチオニン代謝活性のハイスループット評価系の開発 (2017)
  • Author(s): 第35回メディシナルケミストリーシンポジウム
  • Organizer: 小松徹、荻原洲介、木村勇亮、浦野泰照
• [Presentation] ペプチドライブラリーを用いたenzymomicsプラットフォームの構築 (2017)
  • Author(s): 小松徹、小名木淳、市橋裕樹、浦野泰照
  • Organizer: 第68回日本電気泳動学会総会
• [Presentation] Pathway-oriented screening法による癌細胞の代謝制御化合物の探索 (2017)
  • Author(s): 小松徹、柳光一、荻原洲介、小島宏建、岡部隆義、長野哲雄、浦野泰照
  • Organizer: ConBio2017(生命科学系合同年次大会) （第40回日本分子生物学会年会、第90回日本生化学大会）
  • Invited
• [Presentation] Pathway-oriented screening法による解糖系酵素阻害剤の探索 (2017)
  • Author(s): 柳 光一, 小松 徹, 小島 宏建, 岡部 隆義, 長野 哲雄, 浦野 泰照
  • Organizer: ConBio2017(生命科学系合同年次大会) （第40回日本分子生物学会年会、第90回日本生化学大会）
• [Presentation] ペプチドライブラリーを用いた酵素活性の網羅的解析法の確立と発展 (2017)
  • Author(s): 市橋裕樹、小名木淳、小松徹、神谷真子、松崎裕幸、畑啓介、渡邉聡明、浦野泰照
  • Organizer: ConBio2017(生命科学系合同年次大会) （第40回日本分子生物学会年会、第90回日本生化学大会）