蛋白質-蛋白質相互作用阻害剤の探索を指向した新規化合物評価システムの開発

• Project/Area Number: 17790091
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Drug development chemistry
• Research Institution: Kyoto University
• Principal Investigator: 大石 真也 Kyoto University, 薬学研究科, 助教 (80381739)
• Project Period (FY): 2005 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥3,400,000 (Direct Cost: ¥3,400,000); Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
• Keywords: 蛋白質-蛋白質相互作用 / スクリーニング / 膜融合 / 抗HIV剤 / ペプチド / HIV / gp41 / 化学合成ペプチド / HIV-1 / シグナル伝達 / 抗がん剤 / キナーゼ

Research Abstract

平成18年度に引き続き、HIV-1のウイルス膜と細胞膜の融合過程を阻害する抗HIV剤の効率的な探索法の確立を行った。HIV-1 gp41のHR1領域に親和性を有し、HR1-HR2のcoiled-coil構造の形成を阻害して抗HIV作用を示す化合物を選抜するために、まずHR1領域のペプチドであるN36のN末端タグを検証した。当初予定していたビオチンペプチドの利用では、ビーズ上のストレプトアビジンの担持量が少なく、期待されたHR2ペプチドのLC-MSでの分析が困難であった。このため、市販のNi-NTA樹脂およびこれに結合するHisタグ化HR1ペプチドを利用したところ、微量のペプチド混合物の中から活性体のペプチドが有意に選抜されることを確認した。
つづいて、すでに上市された膜融合阻害剤Fuzeonに対するHIV耐性株において認められるHR2領域138位の変異獲得メカニズムの解明と、昨年度の研究により見出したT-20EKの高活性誘導体の探索に本法を応用した。まず、HR2ペプチドSC35EKのSer138をCysを除く各種天然アミノ酸に置換したペプチドライブラリー(19種類の混合物)をスプリットーミックス法で構築した。この混合物にHisタグN36を添加して緩衝液中でインキュベーションした後、Ni-NTAを添加してHR1-HR2複合体をプルダウンにより回収し、酢酸水溶液処理後LC-MS分析に付したところ、138位置換HR2ペプチドは、置換アミノ酸がAla、Ser、Thr、Gly、Met、Leu、Valの順に多く回収された。この回収率は、別途パラレル法により合成して評価した抗HIV活性と一致し、HR1-HR2間の結合親和性が抗HIV活性と相関していることが示唆された。また、138位のAla変異によるT-20耐性獲得は、HR1-HR2間の結合親和性の向上によることが示唆された。

Research Products

• [Journal Article] Design of a novel HIV-1 fusion inhibitor that displays a minimal interface for binding affinity. (2008)
  • Author(s): Oishi S., et. al.
  • Journal Title: J. Med. Chem. 51 Pages: 388-391
  • Peer Reviewed
• [Journal Article] Synthesis and application of fluorescein-and biotin-labeled molecular probes for chemokine receptor CXCR4. (2008)
  • Author(s): Oishi S., et. al.
  • Journal Title: ChemBioChem. (in press)
  • Peer Reviewed
• [Journal Article] Structure-activity relationship study and NMR analysis of fluorobenzoyl pentapeptide GPR54 agonists. (2008)
  • Author(s): Tomita K., et. al.
  • Journal Title: Biopolymers (in press)
  • Peer Reviewed
• [Journal Article] Diastereoselective synthesis of highly functionalized fluoroalkene dipeptide isosteres and its application to fmoc-based solid phase synthesis of a cyclic pentapeptide mimetic. (2008)
  • Author(s): Narumi T., et. al.
  • Journal Title: Tetrahedron (in press)
  • Peer Reviewed
• [Journal Article] Efficient synthesis of trifluoromethyl and related trisubstituted alkene dipeptide isosteres by palladium-catalyzed carbonylation of amino acid-derived allylic carbonates. (2008)
  • Author(s): Inokuchi E., et. al.
  • Journal Title: J. Org. Chem. (in press)
  • Peer Reviewed
• [Journal Article] Versatile use of acid-catalyzed ring-opening of β-aziridinyl-α, βenoates to stereoselective synthesis of peptidomimetics. (2007)
  • Author(s): Tamamura H., et. al.
  • Journal Title: Tetrahedron 63 Pages: 9243-9254
  • Peer Reviewed
• [Journal Article] SAR and QSAR studies on the N-terminally acylated pentapeptide agonists for GPR54. (2007)
  • Author(s): Tomita K., et. al.
  • Journal Title: J. Med. Chem. 50 Pages: 3222-3228
  • Peer Reviewed
• [Journal Article] Facile synthesis of fluoroalkenes by palladium-catalyzed reductive defluorination of allylic gem-difluorides. (2007)
  • Author(s): Narumi T., et. al.
  • Journal Title: Org. Lett. 9 Pages: 3465-3468
  • Peer Reviewed
• [Journal Article] ペプチドをリードとした創薬 (2007)
  • Author(s): 大石真也, et. al.
  • Journal Title: 蛋白質 核酸 酵素 増刊 ケミカルバイオロジー 長野哲雄・長田裕之・菊地和也・上杉志成編集 52 Pages: 1696-1701
  • Peer Reviewed
• [Journal Article] ケミカルバイオロジーを基盤とするペプチド創薬 (2007)
  • Author(s): 大石真也, et. al.
  • Journal Title: 遺伝子医学MOOK8ペプチドと創薬 寒川賢治・南野直人編集 Pages: 87-91
  • Peer Reviewed
• [Journal Article] Cyclic peptide analysis of the biologically active loop region in the laminin R3 chain LG4 module demonstrates the importance of peptide conformation on biological activity. (2007)
  • Author(s): Kato Takagaki, K., Oishi, S., Nomizu, M.et al.
  • Journal Title: Biochemistry 46・7 Pages: 1952-1960
• [Journal Article] Examination of acylated 4-aminopiperidine-4-carboxylic acid residues in the pTyr+1 position of Grb2 SH2 domain-binding tripeptides. (2007)
  • Author(s): Kang, S.-U., Oishi, S., Burke, T.R., Jr.et al.
  • Journal Title: J.Med.Chem. 50・8 Pages: 1978-1982
• [Journal Article] Fmoc-based solid-phase synthesis of GPR54-agonistic pentapeptide derivatives containing alkene-and fluoroalkene-dipeptide isosteres. (2007)
  • Author(s): Tomita, K., Oishi, S., Fujii, N.et al.
  • Journal Title: Biopolymers 88・2 Pages: 272-278
• [Journal Article] Direct Synthesis of 2-(Aminomethyl)indoles through Copper(I)-Catalyzed Domino Three-Component Coupling and Cyclization Reactions. (2007)
  • Author(s): Ohno, H., Oishi, S., Fujii, N., et al.
  • Journal Title: Angew.Chem.Int.Ed.Engl. 46・13 Pages: 2295-2298
• [Journal Article] Structure-activity relationships of cyclic Peptide-based chemokine receptor CXCR4 antagonists : disclosing the importance of side-chain and backbone functionalities. (2007)
  • Author(s): Ueda, S., Oishi, H., Fujii, N.et al.
  • Journal Title: J.Med.Chem. 50・2 Pages: 192-198
• [Journal Article] A novel oxazolidine linker for the synthesis of peptide aldehydes (2007)
  • Author(s): Tanaka, M., Oishi, H., Fujii, N.et al.
  • Journal Title: Int.J.Pep.Res.Ther. (in press)
• [Journal Article] Structure-activity relationship study on small peptidic GPR54 agonists. (2006)
  • Author(s): Tomita, K., Oishi, S., Fujii, N.et al.
  • Journal Title: Bioorg.Med.Chem. 45・22 Pages: 7595-7603
• [Journal Article] Synthesis of (Z)-alkene and (E)-fluoroalkene-containing diketopiperazine mimetics utili-zing organocopper-mediated reduction-alkylation and diastereoselectivity examination using DFT calculations. (2006)
  • Author(s): Niida, A., Oishi, S., Fujii, N.et al.
  • Journal Title: J.Org.Chem. 71・11 Pages: 4118-4129
• [Journal Article] A novel one-pot reaction involving organocopper-mediated reduction/transmetalation/asymmetric alkylation, leading to the diastereoselective synthesis of functionalized (Z)-fluoroalkene dipeptide isosteres. (2006)
  • Author(s): Narumi, T., Oishi, S., Fujii, N.et al.
  • Journal Title: Chem.Commun. 45 Pages: 4720-2722
• [Journal Article] Folliculin encoded by the BHD gene interacts with a novel binding protein, FNIPl, and AMPK, and is involved in AMPK and mTOR signaling. (2006)
  • Author(s): Baba, M., Oishi, S., Zbar, B.et al.
  • Journal Title: Proc.Natl.Acad.Sci. 103・42 Pages: 15552-15557
• [Journal Article] Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity (2006)
  • Author(s): Niida, A. et al.
  • Journal Title: Bioorg.Med.Chem.Lett. 16・1 Pages: 134-137
• [Journal Article] Application of tri- and tetrasubstituted alkene dipeptide mimetics to conformational studies of cyclic RGD peptides (2006)
  • Author(s): Oishi, S. et al.
  • Journal Title: Tetrahedron 62・7 Pages: 1416-1424
• [Journal Article] Unequivocal synthesis of (Z)-alkene and (E)-fluoroalkene dipeptide isosteres to probe structural requirements of the peptide transporter PEPT1 (2006)
  • Author(s): Niida, A. et al.
  • Journal Title: Org.Lett. 8・4 Pages: 613-616
• [Journal Article] Ring-closing metathesis of C-terminal allylglycine residues with an N-terminal β-vinyl-substituted phosphotyrosyl mimetic as an approach to novel Grb2 SH2 domain-binding macrocycles (2005)
  • Author(s): Oishi, S. et al.
  • Journal Title: ChemBioChem 6・4 Pages: 668-674
• [Journal Article] Utilization of a nitrobenzoxadiazole (NBD) fluorophore in the design of a Grb2 SH2 domain-binding peptide mimetic (2005)
  • Author(s): Shi, Z-D. et al.
  • Journal Title: Bioorg.Med.Chem.Lett. 15・5 Pages: 1385-1388
• [Journal Article] Design and synthesis of conformationally constrained Grb2 SH2 domain binding peptides employing y-methylphenylalanyl based phosphotyrosyl mimetics (2005)
  • Author(s): Oishi, S. et al.
  • Journal Title: J.Med.Chem. 48・3 Pages: 764-772
• [Journal Article] Evaluation of macrocyclic Grb2 SH2 domain-binding peptide mimetics prepared by ring-closing metathesis of C-terminal allylglycines with an N-terminal β-vinyl-substituted phosphotyrosyl mimetic (2005)
  • Author(s): Oishi, S. et al.
  • Journal Title: Bioorg.Med.Chem. 13・7 Pages: 2431-2438
• [Journal Article] Identification of neurite outgrowth active sites on the laminin α4 chain G domain (2005)
  • Author(s): Ichikawa, N. et al.
  • Journal Title: Biochemistry 44・15 Pages: 5755-5762
• [Journal Article] Identification of novel low molecular weight CXCR4 antagonists by structural tuning of cyclic tetrapeptide scaffolds. (2005)
  • Author(s): Tamamura, H. et al.
  • Journal Title: J.Med.Chem. 48・9 Pages: 3280-3289
• [Journal Article] Facile access to (Z)-alkene-containing diketopiperazine mimetics utilizing organocopper-mediated anti-SN2' reactions (2005)
  • Author(s): Niida, A. et al.
  • Journal Title: Tetrahedron Lett. 46・24 Pages: 4183-4186
• [Presentation] ヘリックス誘起モチーフを導入した抗HIVペプチドのデザインと合成 (2008)
  • Author(s): 渡辺健太郎
  • Organizer: 日本薬学会第128年会(2008)
  • Place of Presentation: 横浜
  • Year and Date: 2008-03-27
• [Presentation] CXCR4受容体アンタゴニストの蛍光標識体の合成と応用 (2008)
  • Author(s): 増田亮
  • Organizer: 日本薬学会第128年会(2008)
  • Place of Presentation: 横浜
  • Year and Date: 2008-03-27
• [Presentation] T-20(Fuzeon)耐性HIV-1変異株における6-ヘリカルバンドル構造のX線結晶構造解析 (2008)
  • Author(s): 渡部毅
  • Organizer: 日本化学会第88春季年会(2008)
  • Place of Presentation: 池袋
  • Year and Date: 2008-03-27
• [Presentation] ペプチド・蛋白質化学を基盤とする新興・再興感染症治療薬の創製 (2008)
  • Author(s): 大石真也
  • Organizer: 第5回バイオメディカル研究会
  • Place of Presentation: 大阪
  • Year and Date: 2008-02-13
• [Presentation] CXCR4受容体アンタゴニストの蛍光標識体の合成と応用 (2007)
  • Author(s): 増田亮
  • Organizer: 第44回ペプチド討論会
  • Place of Presentation: 富山
  • Year and Date: 2007-11-08
• [Presentation] ヘリックス誘起モチーフを導入した抗HIVペプチドのデザインと合成 (2007)
  • Author(s): 渡辺健太郎
  • Organizer: 第44回ペプチド討論会
  • Place of Presentation: 富山
  • Year and Date: 2007-11-07
• [Presentation] 有機金属による炭素-フッ素結合切断を伴うフルオロアルケン骨格の合成:多価官能基を有するフルオロアルケン型ジペプチドイソスターの高立体選択的合成への展開 (2007)
  • Author(s): 鳴海哲夫
  • Organizer: 第57回日本薬学会近畿支部大会
  • Place of Presentation: 大阪
  • Year and Date: 2007-10-27
• [Presentation] Development of novel FIV membrane fusion inhibitors. (2007)
  • Author(s): Hiroki Nishikawa
  • Organizer: Virus Molecular Interactions: Therapeutic Targets
  • Place of Presentation: Oxford
  • Year and Date: 2007-09-17
• [Presentation] Design and synthesis of novel HIV fusion inhibitors. (2007)
  • Author(s): 〓inya Oishi
  • Organizer: Virus Molecular Interactions: Therapeutic Targets
  • Place of Presentation: Oxford
  • Year and Date: 2007-09-17
• [Presentation] Development of a Novel Fusion Inhibitor against T-20-resistant HIV-1. (2007)
  • Author(s): Oishi S.
  • Organizer: 20th American Peptide Symposium
  • Place of Presentation: Montreal
  • Year and Date: 2007-06-29
• [Presentation] ネコ免疫不全ウイルス(FIV)膜融合阻害剤の創製研究 (2007)
  • Author(s): 西川裕樹
  • Organizer: 日本ケミカルバイオロジー研究会第2回年会
  • Place of Presentation: 京都
  • Year and Date: 2007-05-10
• [Presentation] EK-コンセプトを利用した高活性HIV膜融合阻害剤の創製研究 (2007)
  • Author(s): 田中理紀
  • Organizer: 日本ケミカルバイオロジー研究会第2回年会
  • Place of Presentation: 京都
  • Year and Date: 2007-05-09
• [Remarks]
  • URL: http://www.pharm.kyoto-u.ac.jp/seizo/publication.htm
• [Patent(Industrial Property Rights)] GPR54アゴニスト活性を有する化合物 (2006)
  • Inventor(s): 藤井信孝 他
  • Industrial Property Rights Holder: 京都大学
  • Industrial Property Number: 2006-122305
  • Filing Date: 2006-04-26
• [Patent(Industrial Property Rights)] 抗FIV剤 (2006)
  • Inventor(s): 藤井信孝 他
  • Industrial Property Rights Holder: 京都大学, 東京大学
  • Industrial Property Number: 2006-242428
  • Filing Date: 2006-09-07
• [Patent(Industrial Property Rights)] 抗HIV剤 (2006)
  • Inventor(s): 藤井信孝 他
  • Industrial Property Rights Holder: 京都大学
  • Industrial Property Number: 2006-290241
  • Filing Date: 2006-10-25