Screening for small molecule competitors of the phosphorylated substrates of beta-TrCP and their application for the induction of targeted protein degradation

• Project Area: New frontier for ubiquitin biology driven by chemo-technologies
• Project/Area Number: 19H05302
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Complex systems
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: 渡邉 信元 国立研究開発法人理化学研究所, 環境資源科学研究センター, ユニットリーダー (90221689)
• Project Period (FY): 2019-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥8,060,000 (Direct Cost: ¥6,200,000、Indirect Cost: ¥1,860,000); Fiscal Year 2020: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2019: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
• Keywords: ユビキチン化 / 小分子探索 / タンパク質分解 / タンパク質間相互作用 / タンパク質リン酸化 / PROTAC / Fボックスタンパク質 / 蛍光タンパク質 / ユビキチン

Outline of Research at the Start

β-TrCP(β-Transducin repeat Containing Protein)は、SCF型E3ユビキチン化酵素の基質認識サブユニットF-BOXタンパク質のひとつであり、基質のリン酸化を認識して結合する。本研究計画では、β-TrCPの基質へのリン酸化依存結合に拮抗する小分子リガンドを見出し、β-TrCPリガンドと細胞内の分解させたい標的タンパク質の特異的リガンドとの結合キメラ化合物を合成し、標的タンパク質を結合キメラ化合物によってプロテアソーム依存分解させるシステムの構築研究を行う。

Outline of Annual Research Achievements

昨年度までの研究で、これまでに代表者が確立してきた、他のタンパク質にリン酸化に依存して結合するタンパク質のリン酸化ペプチドへの結合をハイスループットに測定する探索系の技術を応用して、β-TrCPのリン酸化基質への結合に拮抗する小分子探索系を構築した。具体的には、蛍光タンパク質(monomeric Azami Green; mAG)と融合したβ-TrCP1あるいはβ-TrCP2をバキュロウイルスに導入して昆虫細胞に感染することでこれらの融合タンパク質を大量発現する系を構築した。さらにβ-TrCPの標的リン酸化ペプチドを96穴プレートに共有結合し、上述の感染細胞の細胞抽出液を入れたのちに洗浄し、結合を蛍光プレートリーダーで測定することで、融合タンパク質の結合をハイスループットに測定できることを示した。これらの結果からβ-TrCPの標的への結合阻害物質を直接ハイスループットに探索できる系を計画通り構築できたと結論した。β-TrCPの標的への結合を直接検出する阻害物質探索系はこれが初めての例であり、阻害物質の探索に極めて有効であると考えている。
本年度は、次にこの系の有効性の検証をさらに進めた。β-TrCPの阻害物質は文献上知られているものはほとんど無いが、その一つであるGS143について阻害能の確認を行い、それほど強くはないが(IC50=数十μM)、濃度依存の阻害が確認された。このシステムをもちいて、理研天然物ライブラリー(RIKEN NPDepo Library)の化合物について阻害物質の探索を行った。約５００の物質を探索し、結合を有意に阻害出来る物質を12化合物得ることができた。これらについてその類縁体の解析、Gleevecの融合化合物がBcr-Ablタンパク質のProtacとして機能できる可能性へと研究を進める事への基盤作りに成功した。

Research Progress Status

令和2年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和2年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] Max Planck Institute(ドイツ)
• [Int'l Joint Research] North East Normal University/Guangzhou Medical University(中国)
• [Int'l Joint Research] Universiti Sains Malaysia(マレーシア)
• [Int'l Joint Research] Max Planck Institute(ドイツ)
• [Int'l Joint Research] North East Normal University(中国)
• [Int'l Joint Research] Universiti Sains Malaysia(マレーシア)
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• [Journal Article] Discovery of small-molecule modulator of heterotrimeric Gi-protein by integrated phenotypic profiling and chemical proteomics (2020)
  • Author(s): 16)Kawamura, T., Futamura, Y., Shang, E., Muroi, M., Janning, P., Ueno, M., Wilke, J., Takeda, S., Kondoh, Y., Ziegler, S., Watanabe, N., Waldmann, H., Osada, H.
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• [Journal Article] Measurement of ATPase Activity of Valosin-containing Protein/p97 (2020)
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• [Journal Article] Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry (2020)
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  • Journal Title: BIO-PROTOCOL Volume: 10 Issue: 3
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• [Journal Article] A novel inhibitor of tumorspheres reveals the activation of the serine biosynthetic pathway upon mitochondrial inhibition. (2019)
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• [Journal Article] 2-Sulfonylpyrimidines target the kinesin HSET via cysteine alkylation. (2019)
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• [Journal Article] Comparison of microplate- and bottle-based methods to age yeast for chronological life span assays. (2019)
  • Author(s): Kwong MMY, Lee JW, Samian MR, Watanabe N, Osada H, Ong EBB.
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• [Journal Article] SUMO3 modification by PIAS1 modulates androgen receptor cellular distribution and stability (2019)
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• [Journal Article] Curcumol: From Plant Roots to Cancer Roots (2019)
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  • Journal Title: International Journal of Biological Sciences Volume: 15 Issue: 8 Pages: 1600-1609
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• [Presentation] Target identification of a small-molecule inhibitor of cancer cell-accelerated fibroblast migration as valosin-containing protein/p97 (2019)
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  • Organizer: URICAS Symposium 2019
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• [Presentation] Small molecule inhibitors of cancer cell-accelerated fibroblast migration and their mechanism of action. (2019)
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  • Int'l Joint Research / Invited