Creation and crystallographic studies of artificial medium-sized aptamers that modulate membrane protein functions

• Project/Area Number: 18K05334
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 37020:Chemistry and chemical methodology of biomolecules-related
• Research Institution: Kyoto University
• Principal Investigator: Nomura Norimichi 京都大学, 医学研究科, 准教授 (10314246)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 膜タンパク質 / 中分子創薬 / X線結晶構造解析 / クライオ電子顕微鏡単粒子解析 / ファージディスプレイ / 中分子 / 抗体 / 創薬 / 創薬ターゲット / 中分子ペプチド / アロステリックモジュレーター / 阻害剤

Outline of Final Research Achievements

Many therapeutic drugs for various diseases such as cancer, hypertension and chronic inflammation target membrane proteins. In this study, I have established a new methodology useful for the creation of medium-sized peptides that regulate the function of medically relevant membrane proteins. In order to validate the technology, we have obtained medium-sized cystein-rich peptides that specifically recognize and bind to several drug-target membrane proteins, such as PD-1, an inhibitory receptor for immune checkpoints, human membrane protein A, which functions as an entry receptor for pathogenic viral infections, and human membrane protein B, which is involved in the acquisition of metastatic potential and progression of malignant transformation in cancer. To elucidate the mode of action of the artificial peptides, I am conducting the structural studies of the membrane protein/medium-sized peptide complexes by cryo-electron microscopy single particle analysis.

Academic Significance and Societal Importance of the Research Achievements

本研究は、最先端の膜タンパク質構造研究と合成生物学の知見を融合させて中分子創薬に有用なワンストップ技術プラットフォームを創造した点が重要である。研究代表者が既に保有すしていた抗体技術を用いた膜タンパク質構造解析技術の能力を最大限に活用して、取得した中分子アプタマーの結合様式の構造学的検証までを行なった学術的意義と独自性がある。本研究により創出された中分子ペプチドは各種の疾病の治療のための医薬品開発リードとなりうるため、研究成果の社会実装が期待される点からも意義深い。

Research Products

• [Int'l Joint Research] Fudan University(中国)
• [Int'l Joint Research] University of Sydney/Centenary Institute(オーストリア)
• [Int'l Joint Research] Martin-Luther Universitat(ドイツ)
• [Journal Article] Cryo-EM Structure of the Prostaglandin E Receptor EP4 Coupled to G Protein. (2021)
  • Author(s): Nojima S, Fujita Y, Kimura KT, Nomura N, Suno R, Morimoto K, Yamamoto M, Noda T, Iwata S, Shigematsu H, Kobayashi T.
  • Journal Title: Structure Volume: 29 Issue: 3 Pages: 252-260
  • DOI: 10.1016/j.str.2020.11.007
  • Peer Reviewed / Open Access
• [Journal Article] The structure of MgtE in the absence of magnesium provides new insights into channel gating. (2021)
  • Author(s): Jin F, Sun M, Fujii T, Yamada Y, Wang J, Maturana AD, Wada M, Su S, Ma J, Takeda H, Kusakizako T, Tomita A, Nakada-Nakura Y, Liu K, Uemura T, Nomura Y, Nomura N, Ito K, Nureki O, Namba K, Iwata S, Yu Y, Hattori M.
  • Journal Title: PLoS Biology Volume: 19 Issue: 4 Pages: e3001231-e3001231
  • DOI: 10.1371/journal.pbio.3001231
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural insights into tetraspanin CD9 function (2020)
  • Author(s): Umeda Rie、Satouh Yuhkoh、Takemoto Mizuki、Nakada-Nakura Yoshiko、Liu Kehong、Yokoyama Takeshi、Shirouzu Mikako、Iwata So、Nomura Norimichi、Sato Ken、Ikawa Masahito、Nishizawa Tomohiro、Nureki Osamu
  • Journal Title: Nature Communications Volume: 11 Issue: 1 Pages: 643-651
  • DOI: 10.1038/s41467-020-15459-7
  • NAID: 120006957458
  • Peer Reviewed / Open Access
• [Journal Article] Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion (2020)
  • Author(s): Matoba Kazuaki、Kotani Tetsuya、Tsutsumi Akihisa、Tsuji Takuma、Mori Takaharu、Noshiro Daisuke、Sugita Yuji、Nomura Norimichi、Iwata So、Ohsumi Yoshinori、Fujimoto Toyoshi、Nakatogawa Hitoshi、Kikkawa Masahide、Noda Nobuo N.
  • Journal Title: Nature Structural & Molecular Biology Volume: 27 Issue: 12 Pages: 1185-1193
  • DOI: 10.1038/s41594-020-00518-w
  • Peer Reviewed / Open Access
• [Journal Article] Isolation and thermal stabilization of mouse ferroportin (2020)
  • Author(s): Deshpande Chandrika N.、Azucenas Corbin R.、Qiao Bo、Nomura Norimichi、Xin Vicky、Font Josep、Iwata So、Ganz Tomas、Nemeth Elizabeta、Mackenzie Bryan、Jormakka Mika
  • Journal Title: FEBS Open Bio Volume: 11 Issue: 1 Pages: 26-34
  • DOI: 10.1002/2211-5463.13039
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone (2020)
  • Author(s): Im Dohyun、Inoue Asuka、Fujiwara Takaaki、Nakane Takanori、Yamanaka Yasuaki、Uemura Tomoko、Mori Chihiro、Shiimura Yuki、Kimura Kanako Terakado、Asada Hidetsugu、Nomura Norimichi、Tanaka Tomoyuki、Yamashita Ayumi、Nango Eriko、Tono Kensuke、Kadji Francois Marie Ngako、Aoki Junken、Iwata So、Shimamura Tatsuro
  • Journal Title: Nature Communications Volume: 11 Issue: 1 Pages: 1-11
  • DOI: 10.1038/s41467-020-20221-0
  • NAID: 120006943309
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone (2020)
  • Author(s): Asada Hidetsugu、Inoue Asuka、Ngako Kadji Francois Marie、Hirata Kunio、Shiimura Yuki、Im Dohyun、Shimamura Tatsuro、Nomura Norimichi、Iwanari Hiroko、Hamakubo Takao、Kusano-Arai Osamu、Hisano Hiromi、Uemura Tomoko、Suno Chiyo、Aoki Junken、Iwata So
  • Journal Title: Structure Volume: 28 Issue: 4 Pages: 418-425.e4
  • DOI: 10.1016/j.str.2019.12.003
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics (2019)
  • Author(s): Kinjo Tomoaki、Terai Kenta、Horita Shoichiro、Nomura Norimichi、Sumiyama Kenta、Togashi Kaori、Iwata So、Matsuda Michiyuki
  • Journal Title: Nature Methods Volume: 16 Issue: 10 Pages: 1029-1036
  • DOI: 10.1038/s41592-019-0541-5
  • Peer Reviewed / Open Access
• [Journal Article] Structures of the 5-HT2A receptor in complex with the antipsychotics risperidone and zotepine (2019)
  • Author(s): Kimura Kanako Terakado、Asada Hidetsugu、Inoue Asuka、Kadji Francois Marie Ngako、Im Dohyun、Mori Chihiro、Arakawa Takatoshi、Hirata Kunio、Nomura Yayoi、Nomura Norimichi、Aoki Junken、Iwata So、Shimamura Tatsuro
  • Journal Title: Nature Structural & Molecular Biology Volume: 26 Issue: 2 Pages: 121-128
  • DOI: 10.1038/s41594-018-0180-z
  • Peer Reviewed
• [Journal Article] Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface (2019)
  • Author(s): Toyoda Y, Morimoto K, Suno R, et al.
  • Journal Title: Nature Chemical Biology Volume: 15 Issue: 1 Pages: 18-26
  • DOI: 10.1038/s41589-018-0131-3
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural basis for tumor necrosis factor blockade with the therapeutic antibody golimumab (2018)
  • Author(s): Ono Masatsugu、Horita Shoichiro、Sato Yumi、Nomura Yayoi、Iwata So、Nomura Norimichi
  • Journal Title: Protein Science Volume: 印刷中 Issue: 6 Pages: 1038-1046
  • DOI: 10.1002/pro.3407
  • NAID: 120006471155
  • Peer Reviewed
• [Journal Article] Crystal structure of the human angiotensin II type 2 receptor bound to an angiotensin II analog (2018)
  • Author(s): Asada H, Horita S, Hirata K, Shiroishi M, Shiimura Y, Iwanari H, Hamakubo T, Shimamura T, Nomura N, Kusano-Arai O, Uemura T, Suno C, Kobayashi T, Iwata S.
  • Journal Title: Nature Structural and Molecular Biology Volume: 25 Issue: 7 Pages: 570-576
  • DOI: 10.1038/s41594-018-0079-8
  • Peer Reviewed
• [Journal Article] Outward open conformation of a Major Facilitator Superfamily multidrug/H+ antiporter provides insights into switching mechanism (2018)
  • Author(s): Nagarathinam K, Nakada-Nakura Y, Parthier C, Terada T, Juge N, Jaenecke F, Liu K, Hotta Y, Miyaji T, Omote H, Iwata S, Nomura N, Stubbs M, Tanabe M
  • Journal Title: Nature Communications Volume: 9 Issue: 1 Pages: 4005-4005
  • DOI: 10.1038/s41467-018-06306-x
  • NAID: 120006528362
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Book] “The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments” in Methods Mol Biol. (2021)
  • Author(s): *Nomura N, Nomura Y, Sato Y and *Iwata S.
  • Total Pages: 340
  • Publisher: Humana Press
• [Book] Methods in Enzymology, Vol. 629, "Tumor Immunology and Immunotherapy; Molecular Methods" (Edited by Lorenzo Galluzzi, Nils-Petter Rudqvist) (2019)
  • Author(s): Norimichi Nomura, Yayoi Nomura, Yumi Sato, So Iwata
  • Total Pages: 511
  • Publisher: Academic Press
• [Book] Methods Molecular Biology, Vol. 1700, Yamaguchi, A. and Nishino, K. (Eds.) “Bacterial Multidrug Exporters” (2018)
  • Author(s): Jaenecke F., Nakada-Nakura, Y., Nagarathinam, K., Ogasawara, S., Liu, K., Hotta, Y., Iwata, S., Nomura, N., Tanabe, M
  • Total Pages: 355
  • Publisher: Springer
  • ISBN: 9781493974528
• [Remarks] 薬剤耐性の原因「薬剤汲み出しタンパク質」の排出メカニズムを解明
  • URL: http://www.kyoto-u.ac.jp/ja/research/research_results/2018/181001_2.html
• [Remarks] 分子の立体構造から血圧調節の仕組みを原子レベルで解明 －高血圧症の新しい治療法の開発へ－
  • URL: http://www.kyoto-u.ac.jp/ja/research/research_results/2018/180702_2.html
• [Remarks] プロスタグランジン受容体の立体構造を世界初解明
  • URL: http://www.kyoto-u.ac.jp/ja/research/research_results/2018/181204_2.html
• [Remarks] 統合失調症やパーキンソン病の治療薬の標的の構造解明 －副作用を抑えた薬の合理的な探索・設計が可能に－
  • URL: http://www.kyoto-u.ac.jp/ja/research/research_results/2018/190205_1.html