Screening of post-translational modified molecules by using functional zinc complexes

• Project/Area Number: 20K06988
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 47020:Pharmaceutical analytical chemistry and physicochemistry-related
• Research Institution: Hiroshima University
• Principal Investigator: Koike Tohru 広島大学, 医系科学研究科(薬), 教授 (90186586)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2022: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2021: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2020: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: リン酸化タンパク質 / フォスタグ技術 / チオタグ技術 / 翻訳後修飾 / マイクロビーズ法 / 蛍光分析法 / リン酸化生体分子 / タンパク質の翻訳後修飾 / プロテオミクス / フォスタグ / チオタグ / リン酸化生体ぶんし / タンパク質の翻訳語修飾 / Phos-tag / Thio-tag

Outline of Research at the Start

本研究では，これまでに開発した蛍光発色基（TAMRA, Cy3, Cy5など）を有するフォスタグおよびチオタグ誘導体を当初使用するが，必要に応じてより水溶性の高い蛍光発色基（Sulfo-Cy3など）を有する新規蛍光性誘導体なども合成する。それら亜鉛錯体化合物を用いた翻訳後修飾調節分子の迅速スクリーニング法を確立するため，アセチル化酵素反応の蛍光検出法，マルチリン酸化反応の蛍光検出法，阻害剤プロファイリング実施例の作成，それら蛍光分析法を用いた迅速スクリーニング法を開発し，本法の創薬分野における医薬品シーズ探索研究法としての有用性を実証する。

Outline of Final Research Achievements

By using original functional zinc(II) complexes, phosphate- and thiol-capturing molecules (Phos-tag and Thio-tag), we have developed reliable analytical probes for the screening of post-translational modified molecules. In this study, we synthesized several original zinc(II) complex compounds (fluorescent Phos-tag, microbead-bound Phos-tag and Thio-tag), and used them to collect the basic chemical data necessary for the development of a rapid screening method for post-translational modification regulatory molecules. Based on the data, we developed a method for phosphorylation profiling of human proteins involved in kinase reactions and a method for screening kinase inhibitors using cultured cells.

Academic Significance and Societal Importance of the Research Achievements

マイクロモル濃度以下のリン酸化生体分子を生理条件下で選択的に捕捉する低分子亜鉛錯体化合物を用いて、生体機能制御を担うリン酸化シグナル分子の蛍光分析法やリン酸基やチオール基と親和性のあるマイクロビーズ法を開発した。その研究手法は，現在汎用されているリン酸化シグナルの分析法(放射性同位体法やリン酸化分子の抗体を用いる方法)と比較して，迅速な定量定性解析を可能にし，簡便な操作性や安全性などの利点をもち，癌やアルツハイマーなどの治療薬や次世代の抗菌薬やiPS細胞などの研究に適用可能な遺伝子制御試薬の開発に役立つオリジナルな分析技術である。

Research Products

• [Journal Article] Characterization of Zinc(II) Complex of 1,4,7,10-Tetrazacyclododecane and Deprotonated 5-Fluorouracil (FU) in Crystalline/Solution States and Evaluation of Anticancer Activity: Approach for Improving the Anticancer Activity of FU (2023)
  • Author(s): Yoshimi Ichimaru, Koichi Kato, Rina Nakatani, Kirara Sugiura, Hideki Mizutani, Emiko Kinoshita-Kikuta, Tohru Koike, Wanchun Jin, Masanori Imai, Hiromasa Kurosaki
  • Journal Title: Inorganic Chemistry Communications Volume: 147 Pages: 110221-110221
  • DOI: 10.1016/j.inoche.2022.110221
  • Peer Reviewed / Open Access
• [Journal Article] Crystal Structure of [{<i>m,m</i>-bis(Zn^II-cyclen)}₃(μ₂-CO₃)₂(H₂O)₂](ClO₄)₈·<i>n</i>H₂O (2022)
  • Author(s): Y. Ichimaru, K. Kato, K. Sugiura, C. Hirata, W. Jin, H. Fujioka, H. Kurosaki, and T. Koike
  • Journal Title: X-ray Structure Analysis Online Volume: 38 Issue: 0 Pages: 53-55
  • DOI: 10.2116/xraystruct.38.53
  • ISSN: 1883-3578
  • Year and Date: 2022-03-10
  • Peer Reviewed / Open Access
• [Journal Article] Bis(nitrato-κO)(1,4,8,11-tetraazacyclotetradecane-κ4N)zinc(II) methanol monosolvate (2022)
  • Author(s): Ichimaru Yoshimi、Kato Koichi、Kurihara Masaaki、Jin Wanchun、Koike Tohru、Kurosaki Hiromasa
  • Journal Title: IUCrData Volume: 7 Issue: 8 Pages: 220854-220863
  • DOI: 10.1107/s2414314622008549
  • Peer Reviewed / Open Access
• [Journal Article] Use of <i>Escherichia coli</i> expression system for analyzing kinase motifs (2022)
  • Author(s): Kinoshita-Kikuta, E., Ino, Y., Kimura, Y., Akiyama, T., Kinoshita, E., Koike, T.
  • Journal Title: Journal of Electrophoresis Volume: 66 Issue: 1 Pages: 13-20
  • DOI: 10.2198/jelectroph.66.13
  • ISSN: 1349-9394, 1349-9408
  • Peer Reviewed / Open Access
• [Journal Article] Phos-tag-based phosphate affinity chromatographic techniques (2022)
  • Author(s): E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Journal Title: Journal of Chromatography Open Volume: 2 Pages: 10051-10061
  • DOI: 10.1016/j.jcoa.2022.100051
  • Peer Reviewed / Open Access
• [Journal Article] Crystal Structure of Bis{1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-​2-olato-dizinc(II)}orthophosphate Tris(perchlorate) Octahydrate, [(Phos-tag)₂-PO₄³⁻][ClO₄⁻]₃·8H₂O (2021)
  • Author(s): Ichimaru Yoshimi、Kato Koichi、Jin Wanchun、Sugiura Kirara、Kinoshita-Kikuta Emiko、KINOSHITA Eiji、Kurosaki Hiromasa、Koike Tohru
  • Journal Title: X-ray Structure Analysis Online Volume: 37 Issue: 0 Pages: 87-88
  • DOI: 10.2116/xraystruct.37.87
  • NAID: 130008127785
  • ISSN: 1883-3578
  • Year and Date: 2021-12-10
  • Peer Reviewed / Open Access
• [Journal Article] Crystal Structure of [1¹,1⁴,1⁷,1¹⁰,5¹,5⁴,5⁷,5¹⁰-Octaaza-1,5(1,4)-dicyclododecana-3,7(1,3)-dibenzenacyclooctaphane]zinc(II) tetrakis(nitrate), [<i>m,m</i>-bis(Zn^II-cyclen)](NO₃)₄ (2021)
  • Author(s): Y.Ichimaru, J. Wanchun, Y. Yamaguchi, K. Sugiura, M.Imai H. Kurosaki, H. Fujioka, Tohru Koike, Y. Hieda, and K. Kato
  • Journal Title: X-ray Structure Analysis Online Volume: 37 Issue: 0 Pages: 15-16
  • DOI: 10.2116/xraystruct.37.15
  • NAID: 130007996645
  • ISSN: 1883-3578
  • Year and Date: 2021-03-10
  • Peer Reviewed / Open Access
• [Journal Article] An assay of human tyrosine protein kinase ABL activity using an Escherichia coli protein expression system. (2021)
  • Author(s): Kinoshita-Kikuta E, Yoshimoto M, Yano M, Eiji Kinoshita, Koike T.
  • Journal Title: Biotechniques Volume: 70(4) Issue: 4 Pages: 209-217
  • DOI: 10.2144/btn-2020-0154
  • Peer Reviewed / Open Access
• [Journal Article] Aqua(1,4,7,10-tetraazacyclodecane =cyclen)zinc(II) diperchlorate (2021)
  • Author(s): Y.Ichimaru, K. Kato, H. Kurosaki, H. Fujioka, M. Sakai, Y. Yamaguchi, J. Wanchun, K. Sugiura, M.Imai, and Tohru Koike
  • Journal Title: IUCrData Volume: 6 Issue: 4 Pages: 397-415
  • DOI: 10.1107/s2414314621003977
  • Peer Reviewed / Open Access
• [Journal Article] Characterization of the binding of adenosine-5’-monophosphate to a mu-type alkoxide-linked dinuclear zinc(II) complex in crystal and solution state. (2021)
  • Author(s): Kinoshita-Kikuta, E., Ichimaru, Y., Yamano, Y., Kato, K., Kurosaki, H. Eiji Kinoshita, Koike, T.
  • Journal Title: Bull. Chem. Soc. Jpn. Volume: 94(11) Issue: 11 Pages: 2670-2677
  • DOI: 10.1246/bcsj.20210280
  • NAID: 130008137574
  • Peer Reviewed / Open Access
• [Journal Article] Characterization of phosphorylation status and kinase activity of Src family kinases expressed in cell-based and cell-free protein expression system. (2021)
  • Author(s): Kinoshita-Kikuta E, Eiji Kinoshita, Suga M, Higashida M, Yamane Y, Nakamura T, Koike T.
  • Journal Title: Biomolecules Volume: 11(10) Issue: 10 Pages: 1448-1448
  • DOI: 10.3390/biom11101448
  • Peer Reviewed / Open Access
• [Journal Article] Evaluation of four phosphopeptide enrichment strategies for mass spectrometry‐based proteomic analysis (2021)
  • Author(s): Ino Yoko、Kinoshita Eiji、Kinoshita‐Kikuta Emiko、Akiyama Tomoko、Nakai Yusuke、Nishino Kohei、Osada Makoto、Ryo Akihide、Hirano Hisashi、Koike Tohru、Kimura Yayoi
  • Journal Title: PROTEOMICS Volume: 22 Issue: 7 Pages: 2100216-2100216
  • DOI: 10.1002/pmic.202100216
  • Peer Reviewed / Open Access
• [Journal Article] An immuno-dot blot assay for screening histidine kinase inhibitors (2020)
  • Author(s): Kinoshita-Kikuta Emiko、Maruta Shino、Eguchi Yoko、Igarashi Masayuki、Okajima Toshihide、Utsumi Ryutaro、Kinoshita Eiji、Koike Tohru
  • Journal Title: Analytical Biochemistry Volume: 600 Pages: 113765-113765
  • DOI: 10.1016/j.ab.2020.113765
  • Peer Reviewed / Open Access
• [Journal Article] Phos-tag-based micropipette-tip method for analysis of phosphomonoester-type impurities in synthetic oligonucleotides (2020)
  • Author(s): M. Tsunehiro, K. Sasaki, E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Journal Title: Journal of Chromatography B Volume: 1151 Pages: 122198-122206
  • Peer Reviewed / Open Access
• [Journal Article] Crystal Structure of [3-(1,4,7,10-Tetraazacyclododecan-1-yl)propan-1-amine]zinc(II) Bis(perchlorate) (2020)
  • Author(s): Y. Ichimaru, M. Imai, H. Fujioka, T. Koike, H. Kurosaki, and K. Kato
  • Journal Title: X-ray Structure Analysis Online Volume: 36 Issue: 0 Pages: 43-44
  • DOI: 10.2116/xraystruct.36.43
  • NAID: 130007925228
  • Peer Reviewed / Open Access
• [Journal Article] Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic (2020)
  • Author(s): Kinoshita-Kikuta Emiko、Utsumi Toshihiko、Miyazaki Aya、Tokumoto Chiharu、Doi Kyosuke、Harada Haruna、Kinoshita Eiji、Koike Tohru
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 16273-16273
  • DOI: 10.1038/s41598-020-73248-0
  • NAID: 120007169771
  • Peer Reviewed / Open Access
• [Journal Article] A dot-blot-staining method for detecting phosphoproteins with a Phos-tag Aqua fluorescent dye (2020)
  • Author(s): Kinoshita-Kikuta Emiko、Akayama Keisuke、Kinoshita Eiji、Koike Tohru
  • Journal Title: Journal of Electrophoresis Volume: 64 Issue: 1 Pages: 7-11
  • DOI: 10.2198/jelectroph.64.7
  • NAID: 130007961315
  • ISSN: 1349-9394, 1349-9408
  • Peer Reviewed / Open Access