非天然アミノ酸の導入を可能にする新型リボソーム翻訳系の構築

• Project/Area Number: 18H02080
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 37010:Bio-related chemistry
• Research Institution: The University of Tokyo
• Principal Investigator: 加藤 敬行 東京大学, 大学院理学系研究科(理学部), 准教授 (90567760)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,160,000 (Direct Cost: ¥13,200,000、Indirect Cost: ¥3,960,000); Fiscal Year 2021: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000); Fiscal Year 2020: ¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000); Fiscal Year 2019: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2018: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
• Keywords: ペプチド医薬 / 翻訳 / tRNA / 非天然アミノ酸 / リボソーム / 翻訳合成 / ペプチド創薬

Outline of Annual Research Achievements

本研究では、リボソーム翻訳系を人工的に改変することによりD-アミノ酸、β-アミノ酸、γ-アミノ酸、N-メチルアミノ酸等の特殊骨格を持つ非天然アミノ酸のペプチド鎖中への導入を目指した。今年度は、アミノアシル-tRNAのT-ステム領域の配列を最適化することにより、N-メチルアミノ酸の導入効率の向上に成功した。また、翻訳因子の１つであるEF-GがD-アミノ酸の連続導入の際にペプチドのトランケーションを生じさせることを明らかにし、D-アミノ酸導入において翻訳系中のEF-Gの濃度を最適化することが必要であることを示した。
さらに、環状β-アミノ酸の一種である2-aminobenzoic acidと3-aminothiophene-2-carboxylic acidおよび２種類のD-アミノ酸を導入した大環状ペプチドライブラリの構築に成功し、RaPID display法を用いて２種類の標的タンパク質（FXIIaおよびIFNGR1）に対する阻害剤ペプチドのセレクションを行った。その結果、FXIIaおよびIFNGR1に対する強力な阻害剤を取得することに成功した。いずれの阻害剤ペプチドにおいても2-aminobenzoic acidおよび3-aminothiophene-2-carboxylic acidが阻害活性に寄与しており、なおかつペプチドの血中安定性の向上にも寄与していることが確認された。これらのβ-アミノ酸は剛直な環状構造を持つことからペプチドのターン構造を強く誘起することが知られており、今後フォルダマーペプチドの新しいビルディングブロックとして活用できることが期待される。

Research Progress Status

令和3年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和3年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] IECB(フランス)
• [Int'l Joint Research] IECB(フランス)
• [Journal Article] Drop-off-reinitiation triggered by EF-G-driven mistranslocation and its alleviation by EF-P (2022)
  • Author(s): Tajima Kenya、Katoh Takayuki、Suga Hiroaki
  • Journal Title: Nucleic Acids Research Volume: 50 Pages: 2736-2753
  • DOI: 10.1093/nar/gkac068
  • Peer Reviewed / Open Access
• [Journal Article] In Vitro Selection of Foldamer-Like Macrocyclic Peptides Containing 2-Aminobenzoic Acid and 3-Aminothiophene-2-Carboxylic Acid (2022)
  • Author(s): Katoh Takayuki、Suga Hiroaki
  • Journal Title: Journal of the American Chemical Society Volume: 144 Pages: 2069-2072
  • DOI: 10.1021/jacs.1c12133
  • Peer Reviewed
• [Journal Article] Consecutive Ribosomal Incorporation of α-Aminoxy/α-Hydrazino Acids with D/L-Configurations into Nascent Peptide Chains (2021)
  • Author(s): Katoh Takayuki、Suga Hiroaki
  • Journal Title: Journal of the American Chemical Society Volume: 143 Pages: 18844-18848
  • DOI: 10.1021/jacs.1c09270
  • Peer Reviewed / Open Access
• [Journal Article] In Vitro Selection of Thioether-Closed Macrocyclic Peptide by Means of the RaPID System (2021)
  • Author(s): Katoh Takayuki、Goto Yuki、Suga Hiroaki
  • Journal Title: Methods in Molecular Biology Volume: 2371 Pages: 247-259
  • DOI: 10.1007/978-1-0716-1689-5_13
  • Peer Reviewed
• [Journal Article] Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation (2021)
  • Author(s): Iwane Yoshihiko、Kimura Hiroyuki、Katoh Takayuki、Suga Hiroaki
  • Journal Title: Nucleic Acids Research Volume: 49 Pages: 10807-10817
  • DOI: 10.1093/nar/gkab288
  • Peer Reviewed / Open Access
• [Journal Article] Macrocyclic peptides exhibit antiviral effects against influenza virus HA and prevent pneumonia in animal models (2021)
  • Author(s): Saito, M Itoh Y, Yasui F, Munakata T, Yamane D, Ozawa M, Ito R, Katoh T, Ishigaki H, Nakayama M, Shichinohe S, Yamaji K, Yamamoto N, Ikejiri A, Honda T, Sanada T, Sakoda Y, Kida H, Le TH, Kawaoka Y, Ogasawara Tsukiyama-Kohara T, Suga H, & Kohara M
  • Journal Title: Nat Commun. Volume: - Pages: 2654-2654
  • DOI: 10.1038/s41467-021-22964-w
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] In Vitro Selection of Macrocyclic <scp>d</scp>/<scp>l</scp>-Hybrid Peptides against Human EGFR (2021)
  • Author(s): Imanishi Sayaka、Katoh Takayuki、Yin Yizhen、Yamada Mituhiro、Kawai Marina、Suga Hiroaki
  • Journal Title: Journal of the American Chemical Society Volume: 143 Pages: 5680-5684
  • DOI: 10.1021/jacs.1c02593
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Ribosomal synthesis and de novo discovery of bioactive foldamer peptides containing cyclic β-amino acids (2020)
  • Author(s): Katoh Takayuki、Sengoku Toru、Hirata Kunio、Ogata Kazuhiro、Suga Hiroaki
  • Journal Title: Nature Chemistry Volume: 12 Pages: 1081-1088
  • DOI: 10.1038/s41557-020-0525-1
  • Peer Reviewed
• [Journal Article] Ribosomal Elongation of Aminobenzoic Acid Derivatives (2020)
  • Author(s): Katoh Takayuki、Suga Hiroaki
  • Journal Title: Journal of the American Chemical Society Volume: 142 Pages: 16518-16522
  • DOI: 10.1021/jacs.0c05765
  • Peer Reviewed
• [Journal Article] Ribosomal Elongation of Cyclic γ-Amino Acids using a Reprogrammed Genetic Code (2020)
  • Author(s): Katoh Takayuki、Suga Hiroaki
  • Journal Title: Journal of the American Chemical Society Volume: 142 Pages: 4965-4969
  • DOI: 10.1021/jacs.9b12280
  • Peer Reviewed
• [Journal Article] Flexizyme-catalyzed synthesis of 3'-aminoacyl-NH-tRNAs. (2019)
  • Author(s): Katoh T, Suga H.
  • Journal Title: Nucleic Acids Research Volume: pii
  • DOI: 10.1093/nar/gkz143
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] In vitro expression of genetically encoded non-standard peptides consisting of exotic amino acid building blocks. (2019)
  • Author(s): Hirose H, Tsiamantas C, Katoh T, Suga H.
  • Journal Title: Current Opinions in Biotechnology Volume: 58 Pages: 26-28
  • DOI: 10.1016/j.copbio.2018.10.012
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Engineering Translation Components Improve Incorporation of Exotic Amino Acids (2019)
  • Author(s): Katoh Takayuki、Suga Hiroaki
  • Journal Title: International Journal of Molecular Sciences Volume: 20 Pages: 522-522
  • DOI: 10.3390/ijms20030522
  • Peer Reviewed / Open Access
• [Journal Article] Ribosomal Incorporation of Consecutive beta-Amino Acids. (2018)
  • Author(s): Katoh T, Suga H.
  • Journal Title: Journal of the American Chemical Society Volume: 140 Pages: 12159-12167
  • DOI: 10.1021/jacs.8b07247
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] 細胞膜透過型Helixペプチドライブラリーの構築とその薬剤候補探索への適用 (2022)
  • Author(s): 大城 裕亮・加藤 敬行・菅裕明
  • Organizer: 日本化学会
• [Presentation] In vitro selection of antibiotic peptides that inhibit the bacterial ribosome (2022)
  • Author(s): Rei Takahashi, Takayuki Katoh, Axel Innis, Hiroaki Suga
  • Organizer: 日本化学会
• [Presentation] 環状γ-アミノ酸含有特殊ペプチドライブラリの構築と薬剤候補探索への適用 (2020)
  • Author(s): 三浦敬、加藤敬行、菅裕明
  • Organizer: 日本化学会
• [Presentation] 環状β-アミノ酸を含むヘリックスペプチドライブラリを用いた薬剤候補スクリーニング (2020)
  • Author(s): 川合茉利奈、加藤敬行、菅裕明
  • Organizer: 日本化学会
• [Presentation] Ribosomal incorporation of consecutive D- and β-amino acids (2019)
  • Author(s): Katoh Takayuki、Suga Hiroaki
  • Organizer: RNA 2019
  • Int'l Joint Research
• [Presentation] Ribosomal synthesis and de novo discovery of bioactive foldamer peptides containing β- and D-amino acids (2019)
  • Author(s): Takayuki Katoh
  • Organizer: 12th International Symposium on Aminoacyl-tRNA Synthetases
  • Int'l Joint Research / Invited
• [Presentation] Ribosomal incorporation of consecutive D- and β-amino acids (2019)
  • Author(s): Takayuki Katoh, Hiroaki Suga
  • Organizer: Ribosome 2019
  • Int'l Joint Research
• [Presentation] Logical engineering of tRNA for incorporation of D- and β-amino acids into macrocyclic peptides (2018)
  • Author(s): Takayuki Katoh
  • Organizer: 4th International Conference on Circular Proteins and Peptides (ICCPP2018)
  • Int'l Joint Research / Invited
• [Presentation] Logical engineering of D-arm and T-stem of tRNA for incorporation of non-proteinogenic amino acids (2018)
  • Author(s): Takayuki Katoh, Hiroaki Suga
  • Organizer: 27th tRNA conference
  • Int'l Joint Research