Development of a novel ligand screening system for G protein-coupled receptors and its application

• Project/Area Number: 24710247
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Living organism molecular science
• Research Institution: The University of Tokyo
• Principal Investigator: IIZUKA Ryo 東京大学, 薬学研究科(研究院), 助教 (90541954)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2014: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2013: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2012: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: Gタンパク質共役受容体 / 進化分子工学 / 出芽酵母 / マイクロ流体デバイス / エマルジョン / 分子進化 / リガンド探索

Outline of Final Research Achievements

G protein-coupled receptors (GPCRs) are particularly important drug targets. Thus, intense efforts have been devoted to screening novel ligands for GPCRs, which can lead to potential drug candidates. However, for many GPCRs, such efforts have failed to yield desired results. To overcome the problem, I envisioned a directed evolution system to generate agonistic peptides for GPCRs using water-in-oil emulsions. In this study, I established basic techniques for efficient implementation of the system.

Research Products

• [Journal Article] Nascent secM chain outside the ribosome reinforces translation arrest (2015)
  • Author(s): Zhuohao Yang, Ryo Iizuka, Takashi Funatsu
  • Journal Title: PLoS ONE Volume: 10 Issue: 3 Pages: e0122017-e0122017
  • DOI: 10.1371/journal.pone.0122017
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] PEPTIDE-BASED LIGAND SCREENING SYSTEM FOR G PROTEIN-COUPLED RECEPTORS USING WATER-IN-OIL MICRODROPLETS (2014)
  • Author(s): T. Sakurai, R. Iizuka, R. Sekine, Y. Nakamura, D. H. Yoon, T. Sekiguchi, J. Ishii, A. Kondo, S. Shoji, and T. Funatsu
  • Journal Title: Proceedings of the 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2014) Volume: なし
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] SENSITIVE FLUORESCENCE-ACTIVATED SORTING OF MICRODROPLETS CONTAINING SUBCELLUAR STRUCTURES BY THERMOREVERSIBLE GELATION POLYMER. (2014)
  • Author(s): H. Okada, A. Iguchi, R. Iizuka, D. H. Yoon, T. Sekiguchi, S. Shoji, and T. Funatsu
  • Journal Title: Proceedings of the 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2014) Volume: なし
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] SIMPLFIED MICROFLUIDIC-BASED SYSTEM TO GENERATE DOUBLE EMULSION MICRO DROPLETS FOR BIOMEDICAL APPLICATIONS. (2012)
  • Author(s): Rui Sekine, Haruka Okada, Takashi Sakurai, Yoon Donghyun, Ryo Iizuka, Tetsushi Sekiguchi, Takashi Funatsu and Shuichi Shoji
  • Journal Title: Proceedings of 1st International conference on MicroFluidic Handling Systems (MFHS2012) Pages: 127-130
  • Peer Reviewed
• [Journal Article] YEAST-BASED LIGAND ASSAY SYSTEM FOR DETECTING G PROTEIN-COUPLED RECEPTOR ACTIVATION IN WATER-IN-OIL DROPLETS. (2012)
  • Author(s): Takashi Sakurai, Ryo Iizuka, Yasuyuki Tanigaki, Rui Sekine, Dong Hyun Yoon, Tetsushi Sekiguchi, Jun Ishii, Akihiko Kondo, Naoto Nemoto, Shuichi Shoji and Takashi Funatsu
  • Journal Title: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (microTAS 2012) Pages: 1537-1539
  • Peer Reviewed
• [Presentation] Peptide-based ligand screening system for G protein-coupled receptors using water-in-oil droplets. (2015)
  • Author(s): Takashi Sakurai, Ryo Iizuka, Rui Sekine, Yasuyuki Nakamura, Dong Hyun Yoon, Tetsushi Sekiguchi, Jun Ishii, Akihiko Kondo, Shuichi Shoji, and Takashi Funatsu
  • Organizer: International Workshop on Extended-nano Fluidics
  • Place of Presentation: Koshiba Hall, The University of Tokyo (Tokyo)
  • Year and Date: 2015-03-26 – 2015-03-27
• [Presentation] 液滴を利用したGタンパク質共役型受容体作動性ペプチドリガンド創出技術の開発 (2015)
  • Author(s): 飯塚 怜，櫻井 貴志，中村 泰之，石井 純，関根 瑠威，井口 彩香，尹 棟鉉，関口 哲志，藤村 祐，赤木 仁，近藤 昭彦，庄子 習一，船津 高志
  • Organizer: 日本薬学会第135年会
  • Place of Presentation: 神戸学院大学（兵庫県神戸市）
  • Year and Date: 2015-03-25 – 2015-03-28
• [Presentation] 使い捨てマイクロ流路チップを使ったエマルジョン（Water-in-Oil）ソーティング技術の開発 (2015)
  • Author(s): 赤木 仁，飯塚 怜，藤村 祐
  • Organizer: 日本薬学会第135年会
  • Place of Presentation: 神戸サンボーホール（兵庫県神戸市）
  • Year and Date: 2015-03-25 – 2015-03-28
• [Presentation] PEPTIDE-BASED LIGAND SCREENING SYSTEM FOR G PROTEIN-COUPLED RECEPTORS USING WATER-IN-OIL MICRODROPLETS. (2014)
  • Author(s): T. Sakurai, R. Iizuka, R. Sekine, Y. Nakamura, D. H. Yoon, T. Sekiguchi, J. Ishii, A. Kondo, S. Shoji, and T. Funatsu
  • Organizer: The 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2014)
  • Place of Presentation: Henry B. Gonzalez Convention Center (San Antonio, TX, USA)
  • Year and Date: 2014-10-26 – 2014-10-30
• [Presentation] SENSITIVE FLUORESCENCE-ACTIVATED SORTING OF MICRODROPLETS CONTAINING SUBCELLUAR STRUCTURES BY THERMOREVERSIBLE GELATION POLYMER. (2014)
  • Author(s): H. Okada, A. Iguchi, R. Iizuka, D. H. Yoon, T. Sekiguchi, S. Shoji, and T. Funatsu
  • Organizer: The 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2014)
  • Place of Presentation: Henry B. Gonzalez Convention Center (San Antonio, TX, USA)
  • Year and Date: 2014-10-26 – 2014-10-30
• [Presentation] N-terminal region of SecM is essential for its stable translation arrest (2014)
  • Author(s): Zhuohao Yang, Ryo Iizuka, and Takashi Funatsu
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] Peptide-based ligand screening system for G protein-coupled receptors (GPCRs) using water-in-oil microdroplets (2014)
  • Author(s): Takashi Sakurai, Ryo Iizuka, Yasuyuki Nakamura, Jun Ishii, Rui Sekine, Dong Hyun Yoon, Tetsushi Sekiguchi, Akihiko Kondo, Shuichi Shoji, and Takashi Funatsu
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] Development of fluorescence-activated droplet sorting system using thermoreversible gelation polymer and its application (2014)
  • Author(s): Haruka Okada, Ryo Iizuka, Ayaka Iguchi, Dong Hyun Yoon, Tetsushi Sekiguchi, Shuichi Shoji, and Takashi Funatsu
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 札幌コンベンションセンター（北海道札幌市）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] 微小液滴を利用したGPCRリガンドアッセイシステムの開発 (2013)
  • Author(s): 飯塚 怜，櫻井 貴志，谷垣 保幸，関根 瑠威，尹 棟鉉，関口 哲志，石井 純, 中村 泰之，近藤 昭彦，根本 直人，庄子 習一，船津 高志
  • Organizer: 第10回GPCR研究会
  • Place of Presentation: 日本科学未来館みらいCANホール（東京都）
• [Presentation] Yeast-based fluorescence assay system for detecting G protein-coupled receptor activation in water-in-oil droplets (2013)
  • Author(s): Takashi Sakurai, Ryo Iizuka, Yasuyuki Tanigaki, Rui Sekine, Dong Hyun Yoon, Tetsushi Sekiguchi, Jun Ishii, Akihiko Kondo, Naoto Nemoto, Shuichi Shoji, Takashi Funatsu
  • Organizer: 8th Asian Biophysics Association Symposium
  • Place of Presentation: Ramada Plaza Jeju, Korea
• [Presentation] Yeast-based fluorescence assay system for detecting human G protein-coupled receptor activation in water-in-oil droplets (2013)
  • Author(s): Takashi Sakurai, Ryo Iizuka, Rui Sekine, Dong Hyun Yoon, Tetsushi Sekiguchi, Jun Ishii, Akihiko Kondo, Shuichi Shoji, Takashi Funatsu
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館（京都府）
• [Presentation] Simple and Efficient Approach for Proteomic Analysis of Subcellular Structures using Droplet-Based Microfluidics (2013)
  • Author(s): Haruka Okada, Ryo Iizuka, Rui Sekine, Dong Hyun Yoon, Tetsushi Sekiguchi, Shuichi Shoji, Takashi Funatsu
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館（京都府）
• [Presentation] Yeast-based ligand assay system for detecting G protein-coupled receptor activation in water-in-oil droplets. (2012)
  • Author(s): Takashi Sakurai, Ryo Iizuka, Yasuyuki Tanigaki, Rui Sekine, Dong H. Yoon, Tetsushi Sekiguchi, Jun Ishii, Akihiko Kondo, Naoto Nemoto, Shuichi Shoji and Takashi Funatsu
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学東山キャンパス（愛知県）
• [Presentation] SIMPLFIED MICROFLUIDIC-BASED SYSTEM TO GENERATE DOUBLE EMULSION MICRO DROPLETS FOR BIOMEDICAL APPLICATIONS. (2012)
  • Author(s): Rui Sekine, Haruka Okada, Takashi Sakurai, Yoon Donghyun, Ryo Iizuka, Tetsushi Sekiguchi, Takashi Funatsu and Shuichi Shoji
  • Organizer: 1st International conference on MicroFluidic Handling Systems (MFHS2012)
  • Place of Presentation: Holland Casino Enschede（オランダ）
• [Presentation] YEAST-BASED LIGAND ASSAY SYSTEM FOR DETECTING G PROTEIN-COUPLED RECEPTOR ACTIVATION IN WATER-IN-OIL DROPLETS. (2012)
  • Author(s): Takashi Sakurai, Ryo Iizuka, Yasuyuki Tanigaki, Rui Sekine, Dong Hyun Yoon, Tetsushi Sekiguchi, Jun Ishii, Akihiko Kondo, Naoto Nemoto, Shuichi Shoji and Takashi Funatsu
  • Organizer: The 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (microTAS 2012)
  • Place of Presentation: 沖縄コンベンションセンター（沖縄県）
• [Presentation] Yeast-based ligand assay system for detecting G protein-coupled receptor activation in water-in-oil droplets
  • Author(s): 櫻井 貴志，飯塚 怜，谷垣 保幸，関根 瑠威，Dong Hyun Yoon，関口 哲志，石井 純，近藤 昭彦，根本 直人，庄子 習一，船津 高志
  • Organizer: 第12回東京大学生命科学シンポジウム
  • Place of Presentation: 東京大学本郷キャンパス（東京都）
• [Presentation] Yeast-based ligand assay system to detect G protein-coupled receptor activation in water-in-oil droplets.
  • Author(s): Ryo Iizuka
  • Organizer: 海洋・極限環境生物圏領域海洋生物多様性研究プログラムセミナー
  • Place of Presentation: 海洋開発研究機構（神奈川県）
  • Invited
• [Presentation] Yeast-based ligand assay system to detect G protein-coupled receptor activation in water-in-oil droplets.
  • Author(s): Ryo Iizuka, Takashi Sakurai, Yasuyuki Tanigaki, Rui Sekine, Dong Hyun Yoon, Tetsushi Sekiguchi, Jun Ishii, Akihiko Kondo, Naoto Nemoto, Shuichi Shoji and Takashi Funatsu
  • Organizer: 細胞アッセイ技術の現状と将来（Symposium on New Technology for Cell-based Drug Assay）
  • Place of Presentation: 東京大学弥生講堂一条ホール（東京都）