• 文部科学省
  • JSPS
  • 科研費
  • ユビキチンネオバイオロジー
  • ユビキチンネオバイオロジー
研究チーム

研究チーム

Team

研究チーム

研究項目A02 計画研究:革新的ユビキチンケモテクノロジーの創出

ケミカルプロテインノックダウン技術の開発と細胞制御

内藤 幹彦

研究代表者 内藤 幹彦

東京大学大学院薬学系研究科 特任教授

出水 庸介

研究分担者 出水 庸介

国立医薬品食品衛生研究所 有機化学部 部長

石川 稔

研究分担者 石川 稔

東北大学大学院 生命科学研究科 活性分子動態分野 教授

伊藤 拓水

研究分担者 伊藤 拓水

東京医科大学・ナノ粒子先端医学応用講座 准教授

本計画研究の概要

私たちは、SNIPER(Specific and Nongenetic IAP-dependent Protein Eraser)やセレブロン(CRBN)モジュレーター(別名:IMiDs免疫調節剤)等の化合物を利用して細胞内の標的タンパク質をユビキチン化し、プロテアソームで分解するケミカルプロテインノックダウン技術を確立してきました。SNIPERはIAPに結合するリガンド(IAPアンタゴニスト)と標的タンパク質に結合するリガンド(分子弾頭)を繋いだキメラ化合物であり、ユビキチンリガーゼ(E3)活性を持つIAPを標的タンパク質にリクルートする事によりユビキチン化を引き起こします。分子弾頭を置換することによって様々な標的タンパク質を狙って分解する事ができる汎用性の高い技術です。一方CRBNモジュレーターは複合体型E3の基質認識分子であるCRBNに結合して基質特異性を変化させる化合物です。これらの化合物は、分解だけでなく細胞膜受容体の内在化などユビキチンが関与するさまざまな細胞応答を誘導できることが最近わかってきました。
本研究では、これらのケミカルプロテインノックダウン技術を利用して様々な標的タンパク質をユビキチン化し、領域内の研究班と協力して細胞応答等を解析する事により、ユビキチンコードの作動機構の解明に挑戦します。

1. SNIPERテクノロジーの開発と拡充

様々な標的タンパク質をIAPでユビキチン化するSNIPER化合物を各種合成します。また様々なE3リガーゼで標的タンパク質をユビキチン化する各種の化合物と実験系を開発します。これらの化合物で引き起こされる細胞応答とそのメカニズムを解明します。

2. SNIPER等の化合物を利用したユビキチンコード作動機構の解析

上記で開発したSNIPER等を利用して、形成されるユビキチン鎖、デコーダー分子、細胞応答等を解析し、ユビキチンコード作動機構の一端を解明します。

3. CRBNモジュレーターの開発と催奇形性・治療効果のメカニズム

サリドマイド等の存在下におけるCRBN結合因子の網羅的探索を質量分析計で行い、催奇形性や治療効果に関与する基質とメカニズムを解明します。

4. SNIPER等によって形成されるE3リガーゼとネオ基質複合体の構造解析

SNIPERやCRBNモジュレーターによって形成されるE3リガーゼとネオ基質複合体の結晶構造を解析し、ネオ基質がE3リガーゼに認識される機構を分子レベルで解明します。

5. マイトファジー誘導技術の開発

マイトファジー誘導に必要なユビキチンコードを明らかにし、マイトファジーを誘導する技術及び化合物の開発を進めます。

研究概要を示す模式図

研究概要を示す模式図

本領域での研究成果

  1. Nishiyama Y, Fujii S, Makishima M, Hashimoto Y, Ishikawa M.
    Efficient lead finding, activity enhancement and preliminary selectivity control of nuclear receptor ligands bearing a phenanthridinone skeleton.
    Int. J. Mol. Sci. 19, 2090 (2018)
    PMID: 30021999
  2. Shibata N, Shimokawa K, Nagai K, Ohoka N, Hattori T, Miyamoto N, Ujikawa O, Sameshima T, Nara H, Cho N, Naito M.
    Pharmacological difference between degrader and inhibitor against oncogenic BCR-ABL kinase.
    Sci. Rep. 8, 13549 (2018)
    PMID: 30202081
  3. Shioi R, Toyota Y, Noguchi-Yachide T, Ishikawa M,Yamaguchi T, Makishima M, Hashimoto Y, Ohgane K.
    Unexpected emergence of luciferase-inhibitory activity during structural development study of phenyloxadiazole-based PPAR ligands.
    Heterocycles 97, 854-864 (2018)
  4. Misawa T, Goto C, Shibata N, Hirano M, Kikuchi Y, Naito M, Demizu Y.
    Rational design of novel amphipathic antimicrobial peptides focused on distribution of cationic amino acid residues.
    MedChemComm 10, 896-900 (2019)
    PMID: 31303986
  5. Ohoka N, Ujikawa O, Shimokawa K, Sameshima T, Shibata N, Hattori T, Nara H, Cho N, Naito M.
    Different Degradation Mechanisms of Inhibitor of Apoptosis Proteins (IAPs) by the Specific and Nongenetic IAP-Dependent Protein Eraser (SNIPER).
    Chem. Pharm. Bull. 67, 203-209 (2019)
    PMID: 30369550
  6. Ando H, Sato T, Ito T, Yamamoto J, Sakamoto S, Nitta N, Asatsuma-Okumura T, Shimizu N, Mizushima R, Aoki I, Imai T, Yamaguchi Y, Berk AJ, Handa H.
    Cereblon Control of Zebrafish Brain Size by Regulation of Neural Stem Cell Proliferation.
    iScience 15, 95-108 (2019)
    PMID: 31055217
    プレスリリース
  7. Misawa T, Ohoka N, Oba M, Yamashita H, Tanaka M, Naito M, Demizu Y.
    Development of 2-aminoisobutyric acid (Aib)-rich cell-penetrating foldamers for efficient siRNA delivery.
    Chem. Commun. 55, 7792-7795 (2019)
    PMID: 31210205
  8. Asatsuma-Okumura T, Ando H, De Simone M, Yamamoto J, Sato T, Shimizu N, Asakawa K, Yamaguchi Y, Ito T, Guerrini L, Handa H.
    p63 is a cereblon substrate involved in thalidomide teratogenicity.
    Nat. Chem. Biol. 15, 1077-1084 (2019)
    PMID: 31591562
    プレスリリース
  9. Ohoka N, Tsuji G, Shoda T, Fujisato T, Kurihara M, Demizu Y, Naito M.
    Development of Small Molecule Chimeras That Recruit AhR E3 Ligase to Target Proteins.
    ACS Chem. Biol. 14, 2822-2832 (2019)
    PMID: 31580635
  10. Goto C, Hirano M, Hayashi K, Kikuchi Y, Hara-Kudo Y, Misawa T, Demizu Y.
    Development of amphipathic antimicrobial peptide foldamers based on Magainin 2 sequence.
    ChemMedChem 14, 1911-1916 (2019)
    PMID: 31667994
  11. Yamashita H, Tomoshige S, Nomura S, Ohgane K, Hashimoto Y, Ishikawa M.
    Application of Protein Knockdown Strategy Targeting β-Sheet Structure to Multiple Disease-associated Polyglutamine Proteins.
    Bioorg. Med. Chem. 28, 115175 (2020)
    PMID: 31767406
  12. Tateno S, Iida M, Fujii S, Suwa T, Katayama M, Tokuyama H, Yamamoto J, Ito T, Sakamoto S, Handa H, Yamaguchi Y.
    Genome-wide screening reveals a role for subcellular localization of CRBN in the anti-myeloma activity of pomalidomide.
    Sci. Rep. 10, 4012 (2020)
    PMID: 32132601
  13. *Shoda T, Ohoka N, Tsuji G, Fujisato T, Inoue H, Demizu Y, Naito M, Kurihara M.
    Targeted protein degradation by chimeric compounds using hydrophobic E3 ligands and adamantane moiety.
    Pharmaceuticals 13, 34 (2020)
    PMID: 32106507
  14. Shibata N, Ohoka N, Tsuji G, Demizu Y, Akiyama T, *Naito M.
    Deubiquitylase USP25 prevents degradation of BCR-ABL protein and ensures proliferation of Ph-positive leukemia cells.
    Oncogene 39, 3867-3878 (2020)
    PMID: 32203161
  15. Yokoo H, Ohoka N, Naito M, *Demizu Y.
    Design and synthesis of peptide-based chimeric molecules to induce degradation of the estrogen and androgen receptors.
    Bioorg. Med. Chem. 28, 115595 (2020)
    PMID: 32631565
  16. Yamamoto J, Suwa T, Murase Y, Tateno S, Mizutome H, Asatsuma-Okumura T, Shimizu N, Kishi T, Momose S, Kizaki M, Ito T, *Yamaguchi Y, *Handa H.
    ARID2 is a pomalidomide-dependent CRL4CRBN substrate in multiple myeloma cells.
    Nat. Chem. Biol. 16, 1208-1217 (2020)
    PMID: 32958952
  17. *Yamano K, Kikuchi R, Kojima W, Hayashida R, Koyano F, Kawakami J, Shoda T, Demizu Y, Naito M, Tanaka K, Matsuda N.
    Critical role of mitochondrial ubiquitination and the OPTN-ATG9A axis in mitophagy.
    J. Cell Biol. 219, e201912144 (2020)
    PMID: 32556086
  18. Hirano M, Saito C, Goto C, Yokoo H, Kawano R, *Misawa T, *Demizu Y.
    Rational design of helix-stabilized antimicrobial peptide foldamers containing a,a-disubstituted amino acids or side-chain stapling.
    ChemPlusChem 85,2731-2736 (2020)
    PMID: 33369262
  19. Yokoo H,# Shibata N,# Naganuma M, Murakami Y, Ito T, *Aritake K, *Naito M, *Demizu Y.
    Development of hematopoietic prostaglandin D synthase-degradation inducer.
    ACS Med. Chem. Lett. 12,236-241 (2021)
    PMID: 33603969
    #These authors equally contributed to this work.
    <Selected as Supplementary Cover>
  20. Hirano M, Saito C, Yokoo H, Goto C, Kawano R, *Misawa T, *Demizu Y.
    Structure-activity relationship analysis of Mag2-based peptide foldamers.
    Molecules 26,444 (2021)
    PMID: 33466998
  21. Soma-Kaiho A, Akizuki Y, Igarashi K, Endo A, Kawase Y, Shoda T, Demizu Y, Naito M, Saeki Y, Tanaka K, *Ohtake F.
    TRIP12 enhances small molecule-induced degradation of BRD4 through K29/K48 branched ubiquitin chains.
    Mol. Cell, 81, 1411-1424.e7 (2021)
    PMID: 33567268
    プレスリリース
  22. Ichikawa Y, Hiramatsu M, Mita Y, Makishima M, Matsumoto Y, Masumoto Y, Muranaka A, Uchiyama M, Hashimoto Y, *Ishikawa M.
    meta-Non-flat substituents: a novel molecular design to improve aqueous solubility in small molecule drug discovery.
    Org. Biomol. Chem. 19, 446-456 (2021)
    PMID: 33331380
  23. Tsukumo Y,# Tsuji G,# Yokoo H, Shibata N, Ohoka N, Demizu Y, *Naito M.
    Protocols for synthesis of SNIPERs and the methods to evaluate the anticancer effects.
    Methods Mol. Biol. 2365, 331-347 (2021)
    PMID: 34432253
    #These authors equally contributed to this work.
  24. Xu H,# Ohoka N,# Yokoo H, Nemoto K, Ohtsuki T, Matsufuji H, Naito M, Inoue T, *Tsuji G, *Demizu Y.
    Development of agonist-based PROTACs targeting liver X receptor.
    Front. Chem. 9, 674967 (2021)
    PMID: 34124002
    #These authors equally contributed to this work.
  25. Nakane K, *Sato S, Niwa T, Tsushima M, Tomoshige S, Taguchi H, Ishikawa M, Nakamura H.
    Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen.
    J. Am. Chem. Soc. 143, 7726-7731 (2021)
    PMID: 33904715
  26. Morimoto J, Miyamoto K, Ichikawa Y, Uchiyama M, Makishima M, Hashimoto Y, *Ishikawa M.
    Improvement in aqueous solubility of achiral symmetric cyclofenil by modification to a chiral asymmetric analog.
    Sci. Rep. 11, 12697 (2021)
    PMID: 34135380
  27. *Yokoo H, Hirano M, Ohoka N, Misawa T, *Demizu Y.
    Structure-activity relationship study of amphipathic antimicrobial peptides using helix-destabilizing sarcosine.
    J. Pept. Sci. e3360, 1-6 (2021)
    PMID: 34164880
  28. Obara S, Nakane K, Fujimura C, Tomoshige S, Ishikawa M, *Sato S.
    Functionalization of Human Serum Albumin by Tyrosine Click.
    Int. J. Mol. Sci. 22, 8676-8676 (2021)
    PMID: 34445381
  29. Yokoo H,# Ohoka N,# Takyo M, Ito T, Tsuchiya K, Kurohara T, Fukuhara K, Inoue T, Naito M, *Demizu Y.
    Peptide stapling improves the sustainability of a peptide-based chimeric molecule that induces targeted protein degradation.
    Int. J. Mol. Sci. 22, 8772 (2021)
    PMID: 34445478
  30. Yokoo H, *Shibata N, Endo A, Ito T, Yanase Y, Murakami Y, Fujii K, Hamamura K, Saeki Y, *Naito M, *Aritake K, *Demizu Y.
    Discovery of a highly potent and selective PROTAC targeting hematopoietic prostaglandin D synthase via in silico design.
    J. Med. Chem. AOP (2021)
    <Selected as Supplementary Cover>

関連する代表的な論文

  1. Ito T, Ando H, Suzuki T, Ogura T, Hotta K, Imamura Y, Yamaguchi Y, *Handa H.
    Identification of a primary target of thalidomide teratogenicity.
    Science 327, 1345-1350 (2010)
    PMID: 20223979
  2. Itoh Y, Ishikawa M, Naito M, *Hashimoto Y.
    Protein knockdown using methyl bestatin-ligand hybrid molecules: design and synthesis of inducers of ubiquitination-mediated degradation of cellular retinoic acid-binding proteins.
    J. Am. Chem. Soc. 132, 5820-5826 (2010)
    PMID: 20369832
  3. Matyskiela ME, *Lu G, Ito T (co-1st author), Pagarigan B, Lu C-C, Miller K, Fang W, Wang NY, Nguyen D, Houston J, Carmel G, Tran T, Riley M, Nosaka L, Lander GC, Gaidarova S, Xu S, Ruchelman AL, Handa H, Carmichael J, Daniel TO, Cathers BE, Lopez-Girona A, *Chamberlain PP.
    A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase.
    Nature 535, 252-257 (2016)
    PMID: 27338790
  4. Yamashita H, Kato T, Oba M, Misawa T, Hattori T, Ohoka N, Tanaka M, Naito M, *Kurihara M, *Demizu Y.
    Development of a cell-penetrating peptide that exhibits responsive changes in its secondary structure in the cellular environment.
    Sci. Rep. 6, 33003 (2016)
    PMID: 27609319
  5. Tomoshige S, Nomura S, Ohgane K, Hashimoto Y, *Ishikawa M.
    Discovery of small molecules that induce the degradation of huntingtin.
    Angew. Chem. Int. Ed. 56, 11530-11533 (2017)
    PMID: 28703441
  6. Okuhira K, Shoda T, Omura R, Ohoka N, Hattori T, Shibata N, Demizu Y, Sugihara R, Ichino A, Kawahara H, Itoh Y, Ishikawa M, Hashimoto Y, Kurihara M, Itoh S, Saito H, *Naito M.
    Targeted degradation of proteins localized in subcellular compartments by hybrid small molecules.
    Mol. Pharmacol. 91, 159-166 (2017)
    PMID: 27965304
  7. Ohoka N, Okuhira K, Ito M, Nagai K, Shibata N, Hattori T, Ujikawa O, Shimokawa K, Sano O, Koyama R, Fujita H, Teratani M, Matsumoto H, Imaeda Y, Nara H, Cho N, *Naito M.
    In vivo knockdown of pathogenic proteins via specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs).
    J. Biol. Chem. 292, 4556-4570 (2017)
    PMID: 28154167
  8. Mori T, Ito T (co-1st author), Liu S, Ando H, Sakamoto S, Yamaguchi Y, Tokunaga E, Shibata N, *Handa H, *Hakoshima T.
    Structural basis of thalidomide enantiomer binding to cereblon.
    Sci. Rep. 8, 1294 (2018)
    PMID: 29358579
  9. Okitsu K, Hattori T, Misawa T, Shoda T, Kurihara M, *Naito M, *Demizu Y.
    Development of a small hybrid molecule that mediates degradation of His-tag fused proteins.
    J. Med. Chem. 61, 576-582 (2018)
    PMID: 28460164
  10. Shibata N, Nagai K, Morita Y, Ujikawa O, Ohoka N, Hattori T, Koyama R, Sano O, Imaeda Y, Nara H, Cho N, *Naito M.
    Development of protein degradation inducers of androgen receptor by conjugation of androgen receptor ligands and inhibitor of apoptosis protein ligands.
    J. Med. Chem. 61, 543-575 (2018)
    PMID: 28594553
  11. Nishiyama Y, Mori S, Makishima M, Fujii S, Kagechika H, Hashimoto Y, *Ishikawa M.
    Novel nonsteroidal progesterone receptor (PR) antagonists with a phenanthridinone skeleton.
    ACS Med. Chem. Lett. 9, 641-645 (2018)
    PMID: 30034593