Development of new target discovery methods for circadian clock regulating compounds and elucidation of period and phase control mechanisms

• Project/Area Number: 19H03178
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 43030:Functional biochemistry-related
• Research Institution: Nagoya University
• Principal Investigator: Ohkawa Taeko 名古屋大学, 生命農学研究科, 准教授 (30432230)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,810,000 (Direct Cost: ¥13,700,000、Indirect Cost: ¥4,110,000); Fiscal Year 2021: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2020: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2019: ¥11,180,000 (Direct Cost: ¥8,600,000、Indirect Cost: ¥2,580,000)
• Keywords: 概日時計 / ケミカルバイオロジー / 標的同定 / ゲノムワイドノックアウトスクリーニング / 概日リズム / 標的探索

Outline of Research at the Start

生物は昼夜の環境変化への適応に概日時計を利用しており、様々な生理機能は概日時計に制御され約１日周期で活性が変動する。また概日時計は光により時計の時刻（位相）を調節できる。現在、周期が約1日になる仕組みや位相調節の機構についてはほとんど明らかになっていない。これまでに周期や位相を変化させる化合物が数多く得られており、これらが細胞内のどのタンパク質を標的として効果を示すのかが解明されれば、問題解決の手がかりになると期待される。本研究では、現行の手法の弱点をふまえた新たな手法を開発し化合物の標的同定に利用するとともに、得られた結果を数理生物学の専門家と共同で解析し、周期や位相の調節機構を理解する。

Outline of Final Research Achievements

For target identification by genetic methods, a genome-wide knockout library was introduced into a human diploid cell line, and the rhythms of approximately 3,000 clones were measured in 384-well plates. As a result, changes in the period and amplitude of the circadian rhythm were observed in about 0.5% of the clones. Using this system, target of the circadian clock-modulating compound can be identified by isolating clones whose response to the compound differs from that of wild-type cells. This screening system can also be used to identify novel circadian clock genes.
By derivatization of picrotoxinin, a compound that shortens the circadian rhythm cycle, we have obtained a compound that shows approximately 1,000-fold higher activity. We are going to perform affinity purification using the new derivative to identify the target.

Academic Significance and Societal Importance of the Research Achievements

様々な生理活性は内因性の振動体「概日時計」により制御され、約24時間周期の「概日リズム」を刻む。概日リズム異常は、心血管疾患や代謝異常等の生活習慣病、精神疾患、がんなどと密接な関連がある。また動物の繁殖の季節性は概日時計によって制御されている。概日時計調節化合物の標的の同定を通じて、概日時計発振機構の全貌が解明されれば、上記疾患の予防や治療法の開発、産業動物の繁殖効率の向上など様々な波及効果が期待される。

Research Products

• [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
• [Presentation] Levels of constitutively expressed Bmal1 affect the robustess of circadian oscillation (2021)
  • Author(s): Apirada PADLOM, RIo HAMASHIMA, Daisuke ONO, Yuko FURUKAWA, Takashi YOSHIMURA, Taeko OHKAWA
  • Organizer: 第28回日本時間生物学会学術大会