Development of specific inhibitors targeting a deubiquitinating enzyme CYLD and application of these inhibitors for chemical probe.

• Project/Area Number: 15K18468
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: System genome science
• Research Institution: Ehime University
• Principal Investigator: Takahashi Hirotaka 愛媛大学, プロテオサイエンスセンター, 講師 (70432804)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2016: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2015: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: CYLD / 脱ユビキチン化酵素 / NF-kBシグナル / 低分子阻害剤探索 / Subquinocin / NF-κB経路 / 低分子化合物スクリーニング / NF-κB活性化経路 / ケミカルプローブ / ユビキチン

Outline of Final Research Achievements

Many deubiquitinating enzymes (DUBs) are involved in NF-kB signal transduction. To clarify the individual role of CYLD, which is one of a main negative regulators of the NF-kB signal, we aimed to develop small chemical compounds specifically inhibited CYLD activity.
Using a cleavage assay system based on wheat cell-free protein expression system and AlphaScreen technology, one chemical compound with high inhibitory activity to CYLD and low cytotoxicity was identified from 9,600 chemical compound library. This compound significantly inhibited NF-kB signal transduction in cultured cell, but not in CYLD-knockout cells. These results indicated that we succeeded in development of small chemical compound inhibitor targeting the activity of CYLD.

Research Products

• [Journal Article] HTLV-1 Tax induces formation of the active macromolecular IKK complex by generating Lys63- and Met1-linked hybrid polyubiquitin chains. (2017)
  • Author(s): Shibata Y, Tokunaga F, Goto E, Komatsu G, Gohda J, Saeki Y, Tanaka K, Takahashi H, Sawasaki T, Inoue S, Oshiumi H, Seya T, Nakano H, Tanaka Y, Iwai K, and Inoue J.
  • Journal Title: PLoS Pathog. Volume: 13 Issue: 12 Pages: e1006162-e1006162
  • DOI: 10.1371/journal.ppat.1006162
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Dysregulation of a potassium channel, THIK-1, targeted by caspase-8 accelerates cell shrinkage (2016)
  • Author(s): Sakamaki K, Ishii TM, Sakata T, Takemoto K, Takagi C, Takeuchi A, Morishita R, Takahashi H, Nozawa A, Shinoda H, Chiba K, Sugimoto H, Saito A, Tamate S, Satou Y, Jung SK, Matsuoka S, Koyamada K, Sawasaki T, Nagai T, Ueno N
  • Journal Title: Biochim Biophys Acta Volume: 1863 Issue: 11 Pages: 2766-2783
  • DOI: 10.1016/j.bbamcr.2016.08.010
  • Peer Reviewed
• [Journal Article] Linear ubiquitination is involved in the pathogenesis of optineurin-associated amyotrophic lateral sclerosis. (2016)
  • Author(s): Nakazawa S, Oikawa D, Ishii R, Ayaki T, Takahashi H, Takeda H, Ishitani R, Kamei K, Takeyoshi I, Kawakami H, Iwai K, Hatada I, Sawasaki T, Ito H, Nureki O, and Tokunaga F.
  • Journal Title: Nat. Commun. Volume: 7 Issue: 1 Pages: 12547-12547
  • DOI: 10.1038/ncomms12547
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] MyoD reprogramming requires Six1 and Six4 homeoproteins: genome-wide cis-regulatory module analysis. (2016)
  • Author(s): Santolini M, Sakakibara I, Gauthier M, Ribas-Aulinas F, Takahashi H, Sawasaki T, Mouly V, Concordet JP, Defossez PA, Hakim V, Maire P.
  • Journal Title: Nucleic Acids Research Volume: 44 Issue: 18 Pages: 8621-8640
  • DOI: 10.1093/nar/gkw512
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins. (2016)
  • Author(s): Takahashi H, Uematsu A, Yamanaka S, Imamura M, Nakajima T, Doi K, Yasuoka S, Takahashi C, Takeda H, Sawasaki T.
  • Journal Title: PLoS One Volume: 11 Issue: 6 Pages: e0156718-e0156718
  • DOI: 10.1371/journal.pone.0156718
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis. (2016)
  • Author(s): Yano T, Takeda H, Uematsu A, Yamanaka S, Nomura S, Nemoto K, Iwasaki T, Takahashi H, Sawasaki T.
  • Journal Title: PLoS One Volume: 11 Issue: 6 Pages: e0156716-e0156716
  • DOI: 10.1371/journal.pone.0156716
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Characterization of RyDEN (C19orf66) as an interferon-stimulated cellular inhibitor against dengue virus replication. (2016)
  • Author(s): Suzuki Y, Chin WX, Han Q, Ichiyama K, Lee CH, Eyo ZW, Ebina H, Takahashi H, Takahashi C, Tan BH, Hishiki T, Ohba K, Matsuyama T, Koyanagi Y, Tan YJ, Sawasaki T, Chu1 JJ, Vasudevan SG, Sano K, Yamamoto N.
  • Journal Title: PLoS Pathogen Volume: 12 Issue: 1 Pages: 275-275
  • DOI: 10.1186/s12870-015-0660-9
  • NAID: 120005763386
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] コムギ無細胞タンパク質アレイ解析によって見出された、NF-kB活性を抑制する新規DUBの機能解析 (2016)
  • Author(s): 高橋 宏隆, 桒田 翔平, 後藤 栄治, 徳永 文稔, 澤崎 達也
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-12-02
• [Presentation] Regulation of the stability of Dengue virus non-structural protein 4B by ubiquitin-proteasome pathway. (2016)
  • Author(s): Takahashi H, Sameshima Y, Vasudevan SG, Suzuki Y, Yamamoto N. Sawasaki T.
  • Organizer: 23th International Symposium on Hepatitis C Virus and Related Viruses.
  • Place of Presentation: 京都国際会議場（京都府京都市）
  • Year and Date: 2016-10-12
  • Int'l Joint Research
• [Presentation] コムギ無細胞系を基盤としたプロテインアレイ技術の開発と応用 (2016)
  • Author(s): 高橋 宏隆
  • Organizer: AMED-創薬等支援技術基盤プラットフォーム解析拠点-領域間技術交流会
  • Place of Presentation: 名古屋大学・創薬科学研究館（愛知県名古屋市）
  • Year and Date: 2016-09-12
  • Invited
• [Presentation] コムギ無細胞タンパク質合成系を用いたフラビウイルス タンパク質の宿主相互作用タンパク質の探索 (2016)
  • Author(s): 高橋宏隆
  • Organizer: 関西ウイルスクラブ
  • Place of Presentation: 大阪大学微生物病研究所（大阪府吹田市）
  • Year and Date: 2016-07-30
  • Invited
• [Presentation] Establishment of deubiquitinating enzyme (DUB) protein array based on wheat cell-free system for a novel biochemical tool. (2016)
  • Author(s): Takahashi H, Doi K, Yamanaka S, Kuwada S, Goto E, Tokunaga F, Sawasaki T.
  • Organizer: Ubiquitin signaling joint with the meeting on NF-kB and MAP kinase signaling in inflammation, Keystone Symposia on Molecular and Cellular Biology
  • Place of Presentation: Whistler, British Columbia, Canada
  • Year and Date: 2016-03-13
  • Int'l Joint Research
• [Presentation] コムギ無細胞系を用いた287種類のヒトとマウスからなるE3ユビキチンリガーゼプロテインアレイの作製と生化学的解析への応用 (2015)
  • Author(s): 高橋 宏隆, 中島 達朗, 今村 芽依, 高橋 千佳子, 澤崎 達也
  • Organizer: 第38回日本分子生物学会年会, 第88回日本生化学会大会 合同大会
  • Place of Presentation: 神戸ポートピアホテルおよび神戸国際会議場他, 兵庫県神戸市中央区港島中町
  • Year and Date: 2015-12-01
• [Presentation] Regulation of the stability of dengue virus non-structural protein 4B by ubiquitin-proteasome pathway. (2015)
  • Author(s): Takahashi H, Suzuki Y, Vasudevan SG, Sawasaki T, Yamamoto N.
  • Organizer: 第63回ウイルス学会学術集会
  • Place of Presentation: 福岡国際会議場, 福岡県福岡市博多区石城町
  • Year and Date: 2015-11-22
• [Presentation] Identification of novel E3 ligase that degrades HCV nonstructural protein 4B and inhibits viral replication in Huh7.5.1 cells using wheat cell-free protein production system. (2015)
  • Author(s): Takahashi H, Imamura M, Takeda H, Ito M, Suzuki T, Wakita T, Sawasaki T
  • Organizer: 22nd International Symposium on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Strasbourg, France
  • Year and Date: 2015-10-09
  • Int'l Joint Research
• [Book] Cellular control pf dengue virus replication: Role of interferon-inducible genes. in Dengue (2017)
  • Author(s): Takahashi H, Suzuki Y
  • Total Pages: 23
  • Publisher: InTech
• [Book] Technology of wheat cell-free based protein array for biochemical analyses of protein kinases and ubiquitin E3 ligases. in Protein Modifications in Pathogenic Dysregulation of Signaling. (2015)
  • Author(s): Takahashi H, Nemoto K, Ramadan A, Sawasaki T
  • Total Pages: 18
  • Publisher: Springer Japan