JP6416828B2 - Tauタンパク質凝集阻害剤 - Google Patents
Tauタンパク質凝集阻害剤 Download PDFInfo
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- 230000002401 inhibitory effect Effects 0.000 claims description 13
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Description
<実施形態1>
<概要>
<構成>
<試験>
本試験に試料として用いたメトキシフラボノイドは、以下の通りである。図2は、各試料の構造式を示す図である。
(No.1)5,7-dimethoxy flavone(5,7-ジメトキシフラボン)
(No.2)5,7,4'-trimethoxy flavone(5,7,4'-トリメトキシフラボン)
(No.3)3,5,7-trimethoxy flavone(3,5,7-トリメトキシフラボン)
(No.4)3,5,7,4'-tetramethoxy flavone(3,5,7,4'-テトラメトキシフラボン)
(No.5)3,5,7,3',4'-pentamethoxy flavone(3,5,7,3',4'-ペンタメトキシフラボン)
Tauタンパク質は、His-tag融合タンパク質として発現するプラスミドベクター(3R MBD: 3repeat-microtubule binding domain)を用いて培養、精製したものを用いる。チオフラビンT(ThT)蛍光強度を測定することにより、Tauタンパク質凝集を評価する。
Tauタンパク質は凝集するとβ-sheet構造を形成するとされている。チオフラビンT(ThT)はタンパク質のβ-sheet構造を認識して蛍光を発するため、この特徴を利用してTauタンパク質の凝集が確認できる。さらに蛍光強度を比較することにより、凝集の程度を比較することができる。(参考文献1「Okuyama K, Nishiura C, Mizushima F, Minoura K, Sumida M, Taniguchi T, et al (2008).Linkage-dependent contribution of repeat peptides to self-aggregation of three- or four-repeat microtubule-binding domains in tau protein. FEBS J. 275(7): 1529-39」、参考文献2「Sugino E, Nishiura C, Minoura K, In Y, Sumida M, Taniguchi T, et al (2009).Three-/four-repeat-dependent aggregation profile of tau microtubule-binding domain clarified by dynamic light scattering analysis. Biochem Biophys Res Commun. 385(2): 236-40」)
≪4.−1リコンビナントTauタンパク質(3R-MBD)の準備≫
≪4.−1−1 Tau(3R MBD)の培養≫
プラスミドベクターをタンパク質大量発現用大腸菌BL21(DE3)に形質転換し、作成したグリセロールストックを用いる。(参考文献3「Yao TM, Tomoo K, Ishida T, Hasegawa H, Sasaki M, Taniguchi T (2003).Aggregation analysis of the microtubule binding domain in tau protein by spectroscopic methods. J Biochem. 134(1): 91-9」)
≪方法≫
培養・回収した菌体を、菌体量1Lあたり10 mLのBuffer(50 mM Tris-HCl、50 mM NaCl pH7.6)で懸濁し、超音波破砕を行う(超音波破砕:1秒+氷冷:2秒 を40回)。この懸濁液を遠心分離した後、上清を回収し、アフィニティークロマトグラフィー(Ni Chelating)を用いて単離精製を行う。
・結合Buffer:50 mM Tris-HCl/500 mM NaCl/10 mM Imidazole、pH7.6
・溶出Buffer:50 mM Tris-HCl/500 mM NaCl/100 mM Imidazole、pH7.6
凝集反応:
1.5 mlマイクロチューブ(BIO-BIK)に、以下の順に試料及びTau溶液を添加する。
(1)50 mM Tris-HCl (pH7.6) 150 μL
(2)試料溶液、陽性対照(Methylene Blue)、陰性対照(Water又は50%EtOH) 10 μL
(3)100 μM Tau (3R-MBD) 20 μL
(4)100 μM Heparin 20 μL
Total 200 μL
上記を添加後、ボルテックスミキサーで撹拌し、アルミホイルで覆って遮光後37℃、16時間インキュベートする。
インキュベート終了後、各反応液を96 well plateへ135 μLずつ添加し(n=3)、Perkin Elmer ARVO Wallac1420にて自家蛍光を測定する。(励起波長:440nm、測定波長:486 nm)さらにこの反応液に100 μM ThT溶液を15 μLずつ添加し、プレートミキサーにかけた後、同波長で測定する。
陰性対照の測定値を100%として各被験物質の測定値を%換算し、さらに100%からこの%換算値を引いて凝集阻害率とする。
図3は、No.1からNo.5までの各試料について、3μM,10μM,30μMの各濃度における凝集阻害率を示す表である。併せて陽性対照としてメチレンブルーの凝集阻害率を示したものである。なお、メチレンブルーについては、0.3μM,1μM,3μMの各濃度における凝集阻害率を示した。
<効果>
<実施形態2>
<概要>
<試験>
チオフラビンT(ThT)蛍光強度を測定することにより、Aβタンパク質凝集を評価する。
Aβタンパク質は凝集するとβ-sheet構造を形成するとされている。チオフラビンT(ThT)はタンパク質のβ-sheet構造を認識して蛍光を発するため、この特徴を利用してAβタンパク質の凝集が確認できる。さらに蛍光強度を比較することにより、凝集の程度を比較することができる。(参考文献4「Hudson SA, Ecroyd H, Kee TW, Carver JA. (2009) The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds FEBS J.; 276 (20): 5960-72.」、参考文献5「HARRY LEVINE. (1992) Thioflavine T interaction with synthetic Alzheimer's disease β-amyloid peptides:Detection of amyloid aggregation in solution. Department of Neuroscience Pharmacology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48106-1047」)
≪3.1 Aβタンパク質の準備≫
Aβタンパク質1-42:
インキュベート直前にAβタンパク質1-42(ペプチド研 大阪 4349-v Lot:611204)に0.1% NH3水を240 μL加え溶解後(500 μM)、PBSを960μL加える。この溶液をPBSにて20 μMに希釈する。
凝集反応:
反応系終濃度の100倍の被験物質溶液を蒸留水または50%EtOHで希釈し作成し、これをPBSにて50倍希釈する。384well plateに調製した各被験物質溶液を25μL添加し、さらに希釈したAβタンパク質1-42を25uLずつ添加する(n=3)。撹拌後アルミホイルで遮光し、37 ℃で24 時間インキュベートする。
インキュベート終了後、Perkin Elmer ARVO Wallac1420にて自家蛍光を測定する(励起389 nm、測定488 nm)。さらに反応液に6 μM ThT溶液を50 μLずつ添加し、プレートミキサーに30秒かけた後、同波長で測定する。
陰性対照の測定値を100%として各被験物質の測定値を%換算し、さらに100%からこの%換算値を引いて凝集阻害率とする。
図5は、No.1からNo.5までの各試料について、3μM,10μM,30μMの各濃度におけるAβタンパク質の凝集阻害率を示している。併せて陽性対照としてメチレンブルーの凝集阻害率を示したものである。なお、メチレンブルーについては、0.3μM,1μM,3μMの各濃度における凝集阻害率を示した。
(HPLC条件)
カラム:InertsilODS-4, 3μm
カラムサイズ(長さx内径):100×4.6(mm)
溶離液:0.1%ギ酸/ 0.1%ギ酸-ACN= 70/ 30
流速:1.0mL/min
検出波長:UV 260nm
カラム温度:25℃
注入量:10μL(抽出液をエタノールで2倍希釈)
<効果>
Claims (5)
- 5,7−ジメトキシフラボン、5,7,4’−トリメトキシフラボン、3,5,7−トリメトキシフラボン、及び3,5,7,4’−テトラメトキシフラボンからなる群より選択される1種以上のメトキシフラボノイドを有効成分として含有するTauタンパク質凝集阻害剤。
- Aβタンパク質の凝集抑制作用を有する請求項1に記載のTauタンパク質凝集阻害剤。
- 神経原線維化阻害剤として用いられる、請求項1又は2に記載のTauタンパク質凝集阻害剤。
- 医薬品又は医薬部外品である請求項1〜3のいずれか1項に記載のTauタンパク質凝集阻害剤。
- 食品又は食品添加物である請求項1〜3のいずれか1項に記載のTauタンパク質凝集阻害剤。
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