Structural elucidation of Wnt signaling mechanism using unique chemical tools

• Project/Area Number: 20H00455
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• Research Institution: Osaka University
• Principal Investigator: Takagi Junichi 大阪大学, 蛋白質研究所, 教授 (90212000)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥45,500,000 (Direct Cost: ¥35,000,000、Indirect Cost: ¥10,500,000); Fiscal Year 2022: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000); Fiscal Year 2021: ¥14,170,000 (Direct Cost: ¥10,900,000、Indirect Cost: ¥3,270,000); Fiscal Year 2020: ¥17,290,000 (Direct Cost: ¥13,300,000、Indirect Cost: ¥3,990,000)
• Keywords: Wntシグナル / ケミカルバイオロジー / 立体構造解析 / 環状ペプチド / アンタゴニスト

Outline of Research at the Start

Wntは様々な組織幹細胞の分化状態を制御する鍵因子である。申請者はこれまでの研究で、ヒトWnt3の立体構造を世界で初めて解明するとともに、Wntが受容体と作る高次シグナリング複合体の構造モデルを提唱した。本研究では、モデルの実験的検証を、Wnt由来ペプチドや内因性アンタゴニストを利用した構造生物学的手法で達成する。さらに、Wntに結合して活性を修飾する環状ペプチドを中心としたケミカルバイオロジーツールを駆使し、それらの作用メカニズムの解明を通して、Wntシグナル伝達の分子論の理解を深めるとともに、環状ペプチドをプローブとしてこれまで不可能だった内因性Wntのイメージングや機能修飾を達成する。

Outline of Final Research Achievements

This study aimed to elucidate the action mechanism of the Wnt signaling pathway, which is a key factor controlling the differentiation state of various tissue stem cells, using chemical biology tools. In the process, we developed a drug candidate, a neutralizing antibody against the endogenous Wnt inhibitor, USAG-1, and identified its epitope. Although we have already discovered a macrocyclic peptide binders to Wnt3a (PD04) that inhibited Wnt signaling, we applied a block-mutagenesis scanning display to identify a mutant inhibitor, WAp-D04-W10P, with 5-fold greater potency. This work represents for the first instance of molecules capable of inhibiting Wnt signaling through direct interaction with a Wnt protein.

Academic Significance and Societal Importance of the Research Achievements

Wntシグナルは多細胞動物の発生・形態形成に必須なだけでなく、その異常はがんを含む様々な疾患に関わり、さらにはWntが組織幹細胞の生存と増殖に必須な増殖因子であることから再生医療を進めるためにも非常に重要な蛋白質である。本研究では、Wntシグナルを阻害することでがんに対する医薬のリードとなり得る環状ペプチド化合物の性質を詳細に調べるとともに、逆にWntシグナルを促進することで無歯症の治療に役立つ抗体の樹立とその作用原理の解明にも成功した。

Research Products

• [Int'l Joint Research] Seoul National University(韓国)
• [Journal Article] Celastrol suppresses humoral immune responses and autoimmunity by targeting the COMMD3/8 complex (2023)
  • Author(s): Shirai T, Nakai A, Ando E, Fujimoto J, Leach S, Arimori T, Higo D, van Eedem FJ, Tulyeu J, Liu YC, Okuzaki D, Murayama MA, Miyata H, Nunomura K, Lin B, Tani A, Kumanogoh A, Ikawa M, Wing JB, Standley DM, Takagi J, Suzuki K.
  • Journal Title: Science Immunology Volume: 8 Issue: 81
  • DOI: 10.1126/sciimmunol.adc9324
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Wnt 2022 EMBO | the Company of Biologists workshop and Yamada conference (2023)
  • Author(s): Kikuchi Akira、Takagi Junichi、Takada Shinji、Ishitani Tohru、Minami Yasuhiro
  • Journal Title: Genes to Cells Volume: 28 Issue: 4 Pages: 249-257
  • DOI: 10.1111/gtc.13014
  • Peer Reviewed / Open Access
• [Journal Article] Advances in tooth agenesis and tooth regeneration (2023)
  • Author(s): Ravi V.、Murashima-Suginami A.、Kiso H.、Tokita Y.、Huang C.L.、Bessho K.、Takagi J.、Sugai M.、Tabata Y.、Takahashi K.
  • Journal Title: Regenerative Therapy Volume: 22 Pages: 160-168
  • DOI: 10.1016/j.reth.2023.01.004
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 spike protein (2023)
  • Author(s): Kuhn Christopher Cyrus、Basnet Nirakar、Bodakuntla Satish、Alvarez-Brecht Pelayo、Nichols Scott、Martinez-Sanchez Antonio、Agostini Lorenzo、Soh Young-Min、Takagi Junichi、Biert?mpfel Christian、Mizuno Naoko
  • Journal Title: Nature Communications Volume: 14 Issue: 1 Pages: 620-620
  • DOI: 10.1038/s41467-023-36279-5
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Designing receptor agonists with enhanced pharmacokinetics by grafting macrocyclic peptides into fragment crystallizable regions (2022)
  • Author(s): Sakai Katsuya、Sugano-Nakamura Nozomi、Mihara Emiko、Rojas-Chaverra Nichole Marcela、Watanabe Sayako、Sato Hiroki、Imamura Ryu、Voon Dominic Chih-Cheng、Sakai Itsuki、Yamasaki Chihiro、Tateno Chise、Shibata Mikihiro、Suga Hiroaki、Takagi Junichi、Matsumoto Kunio
  • Journal Title: Nature Biomedical Engineering Volume: 7 Issue: 2 Pages: 164-176
  • DOI: 10.1038/s41551-022-00955-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] ラッソ・グラフト法を用いた抗体の高付加価値化技術 (2022)
  • Author(s): 高木淳一、菅 裕明
  • Journal Title: 実験医学 Volume: 40 Pages: 820-824
• [Journal Article] Engineered fast-dissociating antibody fragments for multiplexed super-resolution microscopy (2022)
  • Author(s): Qianli Zhang, Akitoshi Miyamoto, Shin Watanabe, Takao Arimori, Masanori Sakai, Madoka Tomisaki, Tai Kiuchi, Junichi Takagi, Naoki Watanabe
  • Journal Title: Cell Reports Methods Volume: 2 Issue: 10 Pages: 100301-100301
  • DOI: 10.1016/j.crmeth.2022.100301
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structure-Based Discovery of a Novel Class of Small-Molecule Pure Antagonists of Integrin αVβ3 (2022)
  • Author(s): Sen Soumyo、Spasic Aleksandar、Sinha Anjana、Wang Jialing、Bush Martin、Li Jihong、Ne?i? Dragana、Zhou Yuchen、Angiulli Gabriella、Morgan Paul、Salas-Estrada Leslie、Takagi Junichi、Walz Thomas、Coller Barry S.、Filizola Marta
  • Journal Title: Journal of Chemical Information and Modeling Volume: 62 Issue: 22 Pages: 5607-5621
  • DOI: 10.1021/acs.jcim.2c00999
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Hybrid in vitro/in silico analysis of low-affinity protein-protein interactions that regulate signal transduction by Sema6D (2022)
  • Author(s): Tanaka Tsubasa、Ekimoto Toru、Nagatomo Meri、Neyazaki Makiko、Shimoji Erena、Yamane Tsutomu、Kanagawa Sakura、Oi Rika、Mihara Emiko、Takagi Junichi、Akashi Satoko、Ikeguchi Mitsunori、Nogi Terukazu
  • Journal Title: Protein Science Volume: 31 Issue: 11
  • DOI: 10.1002/pro.4452
  • Peer Reviewed
• [Journal Article] De novo Fc-based receptor dimerizers differentially modulate PlexinB1 function (2022)
  • Author(s): Sugano-Nakamura Nozomi、Matoba Kyoko、Hirose Mika、Bashiruddin Nasir K.、Matsunaga Yukiko、Yamashita Keitaro、Hirata Kunio、Yamamoto Masaki、Arimori Takao、Suga Hiroaki、Takagi Junichi
  • Journal Title: Structure Volume: 30 Issue: 10 Pages: 1411-1423.e4
  • DOI: 10.1016/j.str.2022.07.008
  • Peer Reviewed
• [Journal Article] Engineering ACE2 decoy receptors to combat viral escapability (2022)
  • Author(s): Arimori Takao、Ikemura Nariko、Okamoto Toru、Takagi Junichi、Standley Daron M.、Hoshino Atsushi
  • Journal Title: Trends in Pharmacological Sciences Volume: 43 Issue: 10 Pages: 838-851
  • DOI: 10.1016/j.tips.2022.06.011
  • Peer Reviewed
• [Journal Article] Functional role of the Frizzled linker domain in the Wnt signaling pathway (2022)
  • Author(s): Ko Seung-Bum、Mihara Emiko、Park Yedarm、Roh Kyeonghwan、Kang Chanhee、Takagi Junichi、Bang Injin、Choi Hee-Jung
  • Journal Title: Communications Biology Volume: 5 Issue: 1 Pages: 421-421
  • DOI: 10.1038/s42003-022-03370-4
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo (2022)
  • Author(s): Ikemura Nariko、Taminishi Shunta、Inaba Tohru、Arimori Takao、Motooka Daisuke、Katoh Kazutaka、Kirita Yuhei、Higuchi Yusuke、Li Songling、Suzuki Tatsuya、Itoh Yumi、Ozaki Yuki、Nakamura Shota、Matoba Satoaki、Standley Daron M.、Okamoto Toru、Takagi Junichi、Hoshino Atsushi
  • Journal Title: Science Translational Medicine Volume: 14 Issue: 650
  • DOI: 10.1126/scitranslmed.abn7737
  • Peer Reviewed / Open Access
• [Journal Article] Selective targeting of multiple myeloma cells with a monoclonal antibody recognizing the ubiquitous protein CD98 heavy chain (2022)
  • Author(s): Hasegawa Kana et al.
  • Journal Title: Science Translational Medicine Volume: 14 Issue: 632 Pages: 7706-7706
  • DOI: 10.1126/scitranslmed.aax7706
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Serum autoantibodies against the extracellular region of α6β4 integrin in a patient with dipeptidyl peptidase-4 inhibitor?induced bullous pemphigoid (2022)
  • Author(s): Yamashita Chigusa、Arase Noriko、Higuchi Shuhei、Arase Hisashi、Takagi Junichi、Nojima Satoshi、Tanemura Atsushi、Fujimoto Manabu
  • Journal Title: JAAD Case Reports Volume: 20 Pages: 65-68
  • DOI: 10.1016/j.jdcr.2021.12.021
  • Peer Reviewed / Open Access
• [Journal Article] De novo peptide grafting to a self-assembling nanocapsule yields a hepatocyte growth factor receptor agonist (2021)
  • Author(s): Komatsu Yamato、Terasaka Naohiro、Sakai Katsuya、Mihara Emiko、Wakabayashi Risa、Matsumoto Kunio、Hilvert Donald、Takagi Junichi、Suga Hiroaki
  • Journal Title: iScience Volume: 24 Issue: 11 Pages: 103302-103302
  • DOI: 10.1016/j.isci.2021.103302
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 環状ペプチドとタンパク質の融合による新しいバイオ医薬品モダリティの創成 (2021)
  • Author(s): 菅野(中村) 希、高木 淳一
  • Journal Title: Farumashia Volume: 57 Issue: 9 Pages: 820-824
  • DOI: 10.14894/faruawpsj.57.9_820
  • NAID: 130008082215
  • ISSN: 0014-8601, 2189-7026
• [Journal Article] Structural mechanism of laminin recognition by integrin (2021)
  • Author(s): Arimori Takao、Miyazaki Naoyuki、Mihara Emiko、Takizawa Mamoru、Taniguchi Yukimasa、Caba?as Carlos、Sekiguchi Kiyotoshi、Takagi Junichi
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 4012-4012
  • DOI: 10.1038/s41467-021-24184-8
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2 (2021)
  • Author(s): Higuchi Yusuke、Suzuki Tatsuya、Arimori Takao、Ikemura Nariko、Mihara Emiko、Kirita Yuhei、Ohgitani Eriko、Mazda Osam、Motooka Daisuke、Nakamura Shota、Sakai Yusuke、Itoh Yumi、Sugihara Fuminori、Matsuura Yoshiharu、Matoba Satoaki、Okamoto Toru、Takagi Junichi、Hoshino Atsushi
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 3802-3802
  • DOI: 10.1038/s41467-021-24013-y
  • Peer Reviewed / Open Access
• [Journal Article] Structural insights into integrin α5β1 opening by fibronectin ligand (2021)
  • Author(s): Schumacher Stephanie、Dedden Dirk、Nunez Roberto Vazquez、Matoba Kyoko、Takagi Junichi、Biert?mpfel Christian、Mizuno Naoko
  • Journal Title: Science Advances Volume: 7 Issue: 19
  • DOI: 10.1126/sciadv.abe9716
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Molecular recognition of a single-chain Fv antibody specific for GA-pyridine, an advanced glycation end-product (AGE), elucidated using biophysical techniques and synthetic antigen analogues. (2021)
  • Author(s): Kobashigawa, Y., Ohara, T., Morita, K., Toyota, Y., Nakamura, T., Kotani. S., Arimori, T., Yamauchi, S., Liu, C., Kitazaki, M., Wakeyama-Miyazaki, Y., Suwa, Y., Uchida-Kamekura, M., Fukuda, N., Sato, T., Nakajima, M., Takagi, J., Yamagata, Y., and Morioka, H
  • Journal Title: J. Biochem Volume: 170 Issue: 3 Pages: 379-387
  • DOI: 10.1093/jb/mvab056
  • NAID: 40022721879
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural insights into the integrin α5β1 opening by fibronectin ligand. (2021)
  • Author(s): Schumacher S, Dedden D, Nunez RV, Matoba K, Takagi J, Biertumpfel C, Mizuno N.
  • Journal Title: Science Advances Volume: -
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Anti-USAG-1 therapy for tooth regeneration through enhanced BMP signaling (2021)
  • Author(s): Murashima-Suginami A.、Kiso H.、Tokita Y.、Mihara E.、Nambu Y.、Uozumi R.、Tabata Y.、Bessho K.、Takagi J.、Sugai M.、Takahashi K.
  • Journal Title: Science Advances Volume: 7 Issue: 7
  • DOI: 10.1126/sciadv.abf1798
  • NAID: 120006958083
  • Peer Reviewed / Open Access
• [Journal Article] Lasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins (2021)
  • Author(s): Mihara E, Watanabe S, Bashiruddin N, Nakamura N, Matoba K, Maini R, Yin Y, Sakai K, Arimori T, Matsumoto K, Suga H, and Takagi J.
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 1543-1543
  • DOI: 10.1038/s41467-021-21875-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A feasibility study of inverse contrast-matching small-angle neutron scattering method combined with size exclusion chromatography using antibody interactions as model systems (2021)
  • Author(s): Sato Nobuhiro、Yogo Rina、Yanaka Saeko、Martel Anne、Porcar Lionel、Morishima Ken、Inoue Rintaro、Tominaga Taiki、Arimori Takao、Takagi Junichi、Sugiyama Masaaki、Kato Koichi
  • Journal Title: The Journal of Biochemistry Volume: 0 Issue: 6 Pages: 1-8
  • DOI: 10.1093/jb/mvab012
  • NAID: 40022687772
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural studies of reelin N-terminal region provides insights into a unique structural arrangement and functional multimerization (2020)
  • Author(s): Nagae Masamichi、Suzuki Kei、Yasui Norihisa、Nogi Terukazu、Kohno Takao、Hattori Mitsuharu、Takagi Junichi
  • Journal Title: The Journal of Biochemistry Volume: mvaa144 Issue: 5 Pages: 1-41
  • DOI: 10.1093/jb/mvaa144
  • NAID: 40022623693
  • Peer Reviewed
• [Journal Article] High affinity modified ACE2 receptors protect from SARS-CoV-2 infection. (2020)
  • Author(s): Higuchi Y, Suzuki T, Arimori T, Ikemura N, Kirita Y, Ohgitani E, Mazda O, Motooka D, Nakamura S, Matsuura Y, Matoba S, Okamoto T, Takagi J, and Hoshino A.
  • Journal Title: bioRxiv Volume: -
  • DOI: 10.1101/2020.09.16.299891
  • Open Access
• [Journal Article] Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation (2020)
  • Author(s): Bashiruddin Nasir K.、Hayashi Mikihito、Nagano Masanobu、Wu Yan、Matsunaga Yukiko、Takagi Junichi、Nakashima Tomoki、Suga Hiroaki
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 117 Issue: 49 Pages: 31070-31077
  • DOI: 10.1073/pnas.2012266117
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Dissection of α 4 β 7 integrin regulation by Rap1 using novel conformation-specific monoclonal anti-β 7 antibodies. (2020)
  • Author(s): Sato T, Ishihara S, Marui R, Takagi J, Katagiri K
  • Journal Title: Sci Rep. Volume: 10 Issue: 1 Pages: 13221-13221
  • DOI: 10.1038/s41598-020-70111-0
  • Peer Reviewed / Open Access
• [Journal Article] Negatively charged amino acids in the stalk region of membrane proteins reduce ectodomain shedding. (2020)
  • Author(s): Iwagishi R, Tanaka R, Seto M, Takagi T, Norioka N, Ueyama T, Kawamura T, Takagi J, Ogawa Y, Shirakabe K.
  • Journal Title: Journal of Biological Chemistry Volume: 295 Issue: 35 Pages: 12343-12352
  • DOI: 10.1074/jbc.ra120.013758
  • Peer Reviewed / Open Access
• [Journal Article] Site-specific epitope insertion into recombinant proteins using the MAP tag system. (2020)
  • Author(s): Wakasa A, Kaneko MK, Kato Y, Takagi J, and Arimori T.
  • Journal Title: The Journal of Biochemistry Volume: 168 Issue: 4 Pages: 375-384
  • DOI: 10.1093/jb/mvaa054
  • NAID: 40022393375
  • Peer Reviewed / Open Access
• [Journal Article] Macrocyclic peptides that inhibit Wnt signallingviainteraction with Wnt3a (2020)
  • Author(s): Otero-Ramirez Manuel E.、Matoba Kyoko、Mihara Emiko、Passioura Toby、Takagi Junichi、Suga Hiroaki
  • Journal Title: RSC Chemical Biology Volume: 1 Issue: 1 Pages: 26-34
  • DOI: 10.1039/d0cb00016g
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] 受容体構造生物学から創薬へ -LassoGraft Technologyと受容体作動薬の開発- (2022)
  • Author(s): 高木淳一
  • Organizer: 量子構造生物学委員会第8回研究会
  • Invited
• [Presentation] Fcを基盤としたバイオ医薬モダリティ“Mirabody”による受容体機能の修飾 (2022)
  • Author(s): 高木淳一
  • Organizer: 第一回日本抗体学会学術集会
  • Invited
• [Presentation] The "Lasso-grafted" Fc Design Platform Offers Creation of Novel Receptor Agonists and Antagonists (2022)
  • Author(s): Junichi Takagi
  • Organizer: The 68th NIBB Conference
  • Int'l Joint Research / Invited
• [Presentation] LassoGraft Technologyによる新規バイオ医薬品モダリティの創成 (2022)
  • Author(s): 高木淳一
  • Organizer: 次世代モダリティ研究会
  • Invited
• [Presentation] 高親和性受容体デコイによる“エスケープフリー”のCOVID-19治療薬創製 (2022)
  • Author(s): 高木淳一
  • Organizer: 創薬薬理フォーラム
  • Invited
• [Presentation] LassoGraft Technologyによる新規バイオ医薬品モダリティの創成 (2022)
  • Author(s): 高木淳一
  • Organizer: 大阪大学工学研究科テクノアリーナ・フォーラム
  • Invited
• [Presentation] ラッソ・グラフト法を用いた抗体の高付加価値化技術 (2022)
  • Author(s): 高木淳一
  • Organizer: 第８回蛋白質創薬学分野講演会
  • Invited
• [Presentation] 受容体の構造生物学研究から新しいバイオ医薬の創製へ (2022)
  • Author(s): 高木淳一
  • Organizer: 12th INA Chemical Biology Seminar
  • Invited
• [Presentation] 受容体の構造生物学研究から新しいバイオ医薬の創製へ (2022)
  • Author(s): 高木 淳一
  • Organizer: 2021年度 中性子構造生物学研究会 「タンパク質科学・構造生物学と創薬への展開」
  • Invited
• [Presentation] 受容体の構造生物学研究から新しいバイオ医薬の創製へ (2022)
  • Author(s): 高木 淳一
  • Organizer: 新適塾「未来創薬への誘い」
  • Invited
• [Presentation] BINDS支援と高度化で進んだ「日本発のバイオ医薬」創出 (2021)
  • Author(s): 高木 淳一
  • Organizer: 2021年度AMED/BINDSセミナー（構造解析ユニット）―成果と今後の展望―
  • Invited
• [Presentation] Structural Mechanism of Laminin Recognition by Integrins (2021)
  • Author(s): 高木 淳一
  • Organizer: American Society for Matrix Biology Biennial Meeting
  • Int'l Joint Research / Invited
• [Presentation] LassoGraft Technology allows rapid and simple generation of engineered AAV vectors with defined receptor dependency (2021)
  • Author(s): 高木 淳一
  • Organizer: 第27回日本遺伝子細胞治療学会学術集会 プレナリーセッション
  • Int'l Joint Research / Invited
• [Presentation] "Neobiologics" - New modality of multi-functional protein biologics with simple design concept - (2021)
  • Author(s): 高木 淳一
  • Organizer: 蛋白研セミナー「Antibody engineering with AI towards next generation drug discovery」
  • Invited
• [Presentation] LassoGraft Technology による新規バイオ医薬品モダリティの創成 (2021)
  • Author(s): 高木 淳一
  • Organizer: 第21回日本蛋白質科学会年会 シンポジウムS「蛋白質科学が社会へ与えるインパクト ーAMED-BINDSから次のステージへー」
  • Invited
• [Presentation] LassoGraft Technology による新規バイオ医薬品モダリティの創成 (2021)
  • Author(s): 高木 淳一
  • Organizer: 第114回未来医療セミナー（招待講演）
  • Invited
• [Presentation] Instant formulation of multispecific binders using LassoGraft Technology, (2021)
  • Author(s): Junichi Takagi
  • Organizer: PepTalk2021( Characterization of Biotherapeutics)
  • Int'l Joint Research / Invited