ケージド時計調節化合物を用いた概日リズムの光制御と細胞間相互作用の解析

• Project/Area Number: 20K21269
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 38:Agricultural chemistry and related fields
• Research Institution: Nagoya University
• Principal Investigator: 廣田 毅 名古屋大学, トランスフォーマティブ生命分子研究所, 特任准教授 (50372412)
• Project Period (FY): 2020-07-30 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2021: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2020: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: 概日時計 / ケミカルバイオロジー / ケージド化合物 / 細胞間相互作用

Outline of Research at the Start

申請者は概日リズムの周期を変化させる新規化合物を発見して鍵となる制御機構を明らかにしてきた。多細胞から成る概日時計システムの理解に向けて解明すべき重要課題が、細胞間の相互作用である。本研究では時計調節化合物のケージド誘導体を用い、狙ったタイミングに狙った細胞で概日時計を定量的に操作する新技術を生み出す。これを概日リズムの1細胞イメージングに応用し、細胞間相互作用の時空間的な解析に挑戦する。

Outline of Annual Research Achievements

概日時計は分子から個体の各階層を時間・空間的に統合して多様な生理現象の日内リズムを生み出す。申請者はケミカルバイオロジーの手法を応用し、概日リズムの周期を変化させる新規化合物を発見して鍵となる制御機構を明らかにしてきた。多細胞から成る概日時計システムの理解に向けた次のステップとして解明すべき重要課題が、細胞間の相互作用である。これまでに時計調節化合物longdaysinをもとにケージド誘導体を開発し、時間的な精密制御を実現した。本研究ではこのようなケージド時計調節化合物を用い、狙ったタイミングに狙った細胞で概日時計を定量的に操作する新技術を生み出す。これを概日リズムの1細胞イメージングに応用し、細胞間相互作用の時空間的な解析に挑戦する。本年度は1細胞レベルの概日リズムを測定する顕微鏡システムにおいて、ケージド化合物の活性化に必要な紫色光の照射による細胞毒性の問題を、マウス組織の培養系を用いて検討した。しかし、培養細胞を用いた実験と同様に、細胞毒性の問題を避けることができなかった。一方、時計タンパク質CRY1に作用する光スイッチ化合物を用いた条件検討では、緑色光を用いることで細胞毒性の問題を回避できたが、化合物による周期変化の度合いがケージドlongdaysinと比べて大幅に小さく、局所的な変化の検出が困難であることが判明した。そこで誘導体の探索を行い、KL101よりも高い活性を持つ化合物を見出した。この化合物をもとに光スイッチ化合物を改変することで、効果の上昇が期待される。

Research Products

• [Int'l Joint Research] University of Southern California(米国)
• [Int'l Joint Research] University of Southern California(米国)
• [Int'l Joint Research] University of Padova(イタリア)
• [Int'l Joint Research] University of Groningen(オランダ)
• [Int'l Joint Research] University of Southern California(米国)
• [Int'l Joint Research] Indian Institute of Technology(インド)
• [Int'l Joint Research] University of Groningen(オランダ)
• [Int'l Joint Research] University of Padova(イタリア)
• [Journal Article] Introduction (2024)
  • Author(s): Cornelissen Germaine、Otsuka Kuniaki、Hirota Tsuyoshi
  • Journal Title: Chronobiology and Chronomedicine Volume: 1 Pages: 1-11
  • DOI: 10.1039/bk9781839167553-00001
  • ISBN: 9781839162008, 9781839167560, 9781839167553
  • Int'l Joint Research
• [Journal Article] Quo Vadis (2024)
  • Author(s): Cornelissen Germaine、Hirota Tsuyoshi
  • Journal Title: Chronobiology and Chronomedicine Volume: 1 Pages: 648-664
  • DOI: 10.1039/bk9781839167553-00648
  • ISBN: 9781839162008, 9781839167560, 9781839167553
  • Int'l Joint Research
• [Journal Article] Isoform-selective compounds against mammalian circadian clock proteins CRY1 and CRY2 (2023)
  • Author(s): 廣田 毅、羽鳥 恵
  • Journal Title: 生化学 Volume: 95 Issue: 5 Pages: 604-608
  • DOI: 10.14952/SEIKAGAKU.2023.950604
  • ISSN: 0037-1017
  • Year and Date: 2023-10-25
  • Peer Reviewed / Open Access
• [Journal Article] Development of a novel PET ligand, [11C]GO289 targeting CK2 expressed in the brain (2023)
  • Author(s): Ogata Aya、Yamada Takashi、Hattori Saori、Ikenuma Hiroshi、Abe Junichiro、Tada Mari、Ichise Masanori、Suzuki Masaaki、Ito Kengo、Kato Takashi、Amaike Kazuma、Hirota Tsuyoshi、Kakita Akiyoshi、Itami Kenichiro、Kimura Yasuyuki
  • Journal Title: Bioorganic & Medicinal Chemistry Letters Volume: 90 Pages: 129327-129327
  • DOI: 10.1016/j.bmcl.2023.129327
  • Peer Reviewed
• [Journal Article] Regulation of mammalian Cryptochrome with small molecule compounds from in vitro to in vivo (2023)
  • Author(s): Hirota Tsuyoshi
  • Journal Title: Circadian Clocks Volume: 1 Pages: 265-281
• [Journal Article] Analysis of the phosphoproteome of CK2α(-/-)/Δα' C2C12 myoblasts compared to the wild-type cells (2023)
  • Author(s): Borgo Christian、Cesaro Luca、Hirota Tsuyoshi、Kuwata Keiko、D'Amore Claudio、Ruppert Thomas、Blatnik Renata、Salvi Mauro、Pinna Lorenzo A.
  • Journal Title: Open Biology Volume: 13 Issue: 2 Pages: 220220-220220
  • DOI: 10.1098/rsob.220220
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Phosphorylation of RNA Polymerase II by CDKC;2 Maintains the Arabidopsis Circadian Clock Period (2022)
  • Author(s): Uehara Takahiro N、Nonoyama Takashi、Taki Kyomi、Kuwata Keiko、Sato Ayato、Fujimoto Kazuhiro J、Hirota Tsuyoshi、Matsuo Hiromi、Maeda Akari E、Ono Azusa、Takahara Tomoaki T、Tsutsui Hiroki、Suzuki Takamasa、Yanai Takeshi、Kay Steve A、Itami Kenichiro、Kinoshita Toshinori、Yamaguchi Junichiro、Nakamichi Norihito
  • Journal Title: Plant and Cell Physiology Volume: 63 Issue: 4 Pages: 450-462
  • DOI: 10.1093/pcp/pcac011
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Cell-Based Phenotypic Screens to Discover Circadian Clock-Modulating Compounds (2022)
  • Author(s): Hatori Megumi、Hirota Tsuyoshi
  • Journal Title: Methods Mol Biol Volume: 2482 Pages: 95-104
  • DOI: 10.1007/978-1-0716-2249-0_6
  • ISBN: 9781071622483, 9781071622490
  • Peer Reviewed
• [Journal Article] CRY2 isoform selectivity of a circadian clock modulator with antiglioblastoma efficacy (2022)
  • Author(s): Miller Simon、Kesherwani Manish、Chan Priscilla、Nagai Yoshiko、Yagi Moeri、Cope Jamie、Tama Florence、Kay Steve A.、Hirota Tsuyoshi
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 119 Issue: 40
  • DOI: 10.1073/pnas.2203936119
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A methylbenzimidazole derivative regulates mammalian circadian rhythms by targeting Cryptochrome proteins (2022)
  • Author(s): Yagi Moeri、Miller Simon、Nagai Yoshiko、Inuki Shinsuke、Sato Ayato、Hirota Tsuyoshi
  • Journal Title: F1000Research Volume: 11 Pages: 1016-1016
  • DOI: 10.12688/f1000research.124658.2
  • Peer Reviewed / Open Access
• [Journal Article] 概日時計の分子機構をケミカルバイオロジーで紐解く (2022)
  • Author(s): 松田 智宏、廣田 毅
  • Journal Title: 化学と生物 Volume: 60 Pages: 212-214
  • Peer Reviewed
• [Journal Article] Structural and Chemical Biology Approaches Reveal Isoform-Selective Mechanisms of Ligand Interactions in Mammalian Cryptochromes (2022)
  • Author(s): Miller Simon、Hirota Tsuyoshi
  • Journal Title: Frontiers in Physiology Volume: 13 Pages: 837280-837280
  • DOI: 10.3389/fphys.2022.837280
  • Peer Reviewed / Open Access
• [Journal Article] Chemical biology of the circadian clock and its application to drug discovery (2021)
  • Author(s): 廣田 毅、松田 智宏
  • Journal Title: MEDCHEM NEWS Volume: 31 Issue: 2 Pages: 62-67
  • DOI: 10.14894/medchem.31.2_62
  • NAID: 130008033651
  • ISSN: 2432-8618, 2432-8626
  • Year and Date: 2021-05-01
• [Journal Article] Effects of Cryptochrome-modulating compounds on circadian behavioral rhythms in zebrafish (2021)
  • Author(s): Iida M, Nakane Y, Yoshimura T, Hirota T
  • Journal Title: The Journal of Biochemistry Volume: Sep Issue: 5 Pages: 501-507
  • DOI: 10.1093/jb/mvab096
  • Peer Reviewed / Open Access
• [Journal Article] Reversible modulation of circadian time with chronophotopharmacology (2021)
  • Author(s): Kolarski Dusan、Mir-Vinyals Carla、Sugiyama Akiko、Srivastava Ashutosh、Ono Daisuke、Nagai Yoshiko、Iida Mui、Itami Kenichiro、Tama Florence、Szymanski Wiktor、Hirota Tsuyoshi、Feringa Ben L.
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 3164-3164
  • DOI: 10.1038/s41467-021-23301-x
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation (2021)
  • Author(s): Miller Simon、Srivastava Ashutosh、Nagai Yoshiko、Aikawa Yoshiki、Tama Florence、Hirota Tsuyoshi
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 118 Issue: 26
  • DOI: 10.1073/pnas.2026191118
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Modulation of circadian clock by crude drug extracts used in Japanese Kampo medicine (2021)
  • Author(s): Zhang M, Kobayashi K, Atsumi H, Katada Y, Nakane Y, Chen J, Nagano R, Kadofusa N, Nishiwaki-Ohkawa T, Kon N, Hirota T, Sato A, Makino T, Yoshimura T
  • Journal Title: Scientific Reports Volume: 11 Issue: 1 Pages: 21038-21038
  • DOI: 10.1038/s41598-021-00499-w
  • Peer Reviewed / Open Access
• [Journal Article] 概日時計を標的とした化合物と創薬の最前線 (2021)
  • Author(s): 飯田 夢惟、廣田 毅
  • Journal Title: Medical Science Digest Volume: 47 Pages: 23-25
• [Journal Article] Structural insights into isoform-selective regulators of mammalian Cryptochromes (2021)
  • Author(s): Miller Simon、Hirota Tsuyoshi
  • Journal Title: SPring-8 /SACLA Research Frontiers 2020 Volume: - Pages: 22-23
  • Open Access
• [Journal Article] Reductive stability evaluation of 6-azopurine photoswitches for the regulation of CKIα activity and circadian rhythms (2021)
  • Author(s): Kolarski Dusan、Sugiyama Akiko、Rodat Theo、Schulte Albert、Peifer Christian、Itami Kenichiro、Hirota Tsuyoshi、Feringa Ben L.、Szymanski Wiktor
  • Journal Title: Organic & Biomolecular Chemistry Volume: 19 Issue: 10 Pages: 2312-2321
  • DOI: 10.1039/d1ob00014d
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Comparing the efficacy and selectivity of Ck2 inhibitors. A phosphoproteomics approach (2021)
  • Author(s): Borgo Christian、Cesaro Luca、Hirota Tsuyoshi、Kuwata Keiko、D’Amore Claudio、Ruppert Thomas、Blatnik Renata、Salvi Mauro、Pinna Lorenzo A.
  • Journal Title: European Journal of Medicinal Chemistry Volume: 214 Pages: 113217-113217
  • DOI: 10.1016/j.ejmech.2021.113217
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock (2021)
  • Author(s): Kolarski Dusan、Miller Simon、Oshima Tsuyoshi、Nagai Yoshiko、Aoki Yugo、Kobauri Piermichele、Srivastava Ashutosh、Sugiyama Akiko、Amaike Kazuma、Sato Ayato、Tama Florence、Szymanski Wiktor、Feringa Ben L.、Itami Kenichiro、Hirota Tsuyoshi
  • Journal Title: Journal of the American Chemical Society Volume: 143 Issue: 4 Pages: 2078-2087
  • DOI: 10.1021/jacs.0c12280
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 化合物を使って体内時計を操る (2020)
  • Author(s): 松田 智宏、廣田 毅
  • Journal Title: 化学 Volume: 75 Pages: 66-67
• [Presentation] 時計タンパク質CRYの機能制御化合物と構造ダイナミクス (2023)
  • Author(s): 廣田 毅
  • Organizer: 1.日本睡眠学会第45回定期学術集会・第30回日本時間生物学会学術大会合同大会
  • Invited
• [Presentation] Regulation of mammalian Cryptochrome with small molecule compounds from in vitro to in vivo (2022)
  • Author(s): Hirota Tsuyoshi
  • Organizer: Sapporo Symposium on Biological Rhythm 2022
  • Int'l Joint Research / Invited
• [Presentation] Isoform-selective compounds for mammalian CRY proteins (2021)
  • Author(s): Tsuyoshi Hirota
  • Organizer: 5th Asian Forum on Chronobiology
  • Int'l Joint Research / Invited
• [Presentation] 低分子化合物を用いた概日時計タンパク質の機能制御の最先端 (2021)
  • Author(s): 廣田 毅
  • Organizer: 日本睡眠学会第46回定期学術集会
  • Invited
• [Presentation] 哺乳類の階層時計システムの理解に向けた時計タンパク質の調節化合物の開発 (2021)
  • Author(s): 廣田 毅
  • Organizer: 第28回日本時間生物学会学術大会
  • Invited
• [Presentation] Chemical biology of the circadian clock and its potential to improve health (2021)
  • Author(s): Tsuyoshi Hirota
  • Organizer: The 94th Japanese Biochemical Society Meeting
  • Invited
• [Presentation] 時計タンパク質に作用する低分子化合物の開発 (2020)
  • Author(s): 廣田 毅
  • Organizer: 第41回日本臨床薬理学会学術総会
  • Invited
• [Book] Chronobiology and Chronomedicine (2024)
  • Author(s): Cornelissen, G. and Hirota, T.
  • Total Pages: 716
  • Publisher: Royal Society of Chemistry
  • ISBN: 9781839162008
• [Book] Circadian Clocks (2022)
  • Author(s): Hirota, T., Hatori, M., and Panda, S
  • Total Pages: 429
  • Publisher: Springer Nature
  • ISBN: 9781071625767
• [Remarks] 名古屋大学トランスフォーマティブ生命分子研究所Kay-廣田グループ
  • URL: https://www.itbm.nagoya-u.ac.jp/ja/kay-hirota_group/
• [Remarks] 名古屋大学トランスフォーマティブ生命分子研究所Kay-廣田グループ
  • URL: http://www.itbm.nagoya-u.ac.jp/ja/kay-hirota_group/