JP6078633B2 - 治療用のチオレドキシン相互作用タンパク質(txnip)のインヒビター - Google Patents
治療用のチオレドキシン相互作用タンパク質(txnip)のインヒビター Download PDFInfo
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- JP6078633B2 JP6078633B2 JP2015507410A JP2015507410A JP6078633B2 JP 6078633 B2 JP6078633 B2 JP 6078633B2 JP 2015507410 A JP2015507410 A JP 2015507410A JP 2015507410 A JP2015507410 A JP 2015507410A JP 6078633 B2 JP6078633 B2 JP 6078633B2
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Description
(a) プラスミド、ベクター又は天然/合成/変異ウイルス、種々の改変型 (たとえば、PTO, LNA, 2’F-ANA、タンパク質-ヌクレオチド複合体、shRNA(RNA干渉を通じて遺伝子発現をサイレンシングするために使用可能なタイトなヘアピンカーブを作り出すRNAの配列である「小ヘアピンRNA」または「ショートヘアビンRNA」)のオリゴヌクレオチド、RNAi, siRNA又はmikromiRNA、メチルメトキシ-、ホスホロアミダイト、PNA、モリフォリノ、ホスホラアミダイト、シクロヘキセン(CeNA)、gap-meres、リボザイム、アプタマ、CpG-オリゴ、DNA-ザイム、リボスイッチ、又は、脂質又は脂質含有分子、
(b) ペプチド、すべてのタイプのリンカーを含むペプチド複合体、
(c) 小分子、
(d) 抗体およびそれらの誘導体、特に、キメラ、Fabフラグメント、Fcフラグメント、
(e)担体、リポソーム、ナノ粒子、複合体、又は、上記構造物を含むその他すべてのデリバリシステム、又は、
(f)酸化剤又はスルフヒドリル(SH基)変性剤。
(a)候補化合物を、TXNIP又はその遺伝子を含むテストシステムとインキュベートする、そして、
(b)TXNIPの生物活性を分析する、
ここで、TXNIPの生物活性の抑制又は損失、特に、前記テスト化合物の不在のテストシステムとの比較における抑制又は損失は、前記所望特性を有する候補化合物の存在を示す。
(A) 統計学的分析
結果は、特に銘記されない限り、平均±SDとしてあらわされている。両側独立スチューデントt-検定を使用して統計学的比較を行った。2サンプルコルモゴロフ-スミルノフテストを使用して、生存曲線の有意性を決定した。統計的有意差異を、*p<0.05、**p<0.01、***p<0.001として定義した。
ジクロロヒドロフルオレシン二酢酸-(H2DCF-DA)を、Invitrogen (Carlsbad, CA, USA)から入手した。イオノマイシンは、Merk (Darmstadt, ドイツ)から購入した。その他すべての化学物質は、Sigma-Aldrich (ミュンヘン、ドイツ)によって供給された。蛍光色素共役抗体はBD Biosciences (ハイデルベルグ、ドイツ)から購入した。ヒトCD3に対するモノクローナル抗体(OKT3)は(13)に記載のように作成された。
全RNAを、RNeasy Mini Kit (Qiagen Hilden、ドイツ)又は、TRIzol試薬(Invitrogen)を製造業者の指示に従って使用して、一次T-細胞、Jurkat T細胞又は全ハエ溶解物から単離した。RNAを逆転写し、以前に報告されている(15)ようにしてqPCRを行った。正規化のためにコントロール遺伝子として、ハウスキーピング遺伝子GAPDHを使用した。プライマー配列は下記の通りである。GAPDH、 5’-GCA AAT TCC ATG GCA CCG T-3’および5’-TCG CCC CAC TTG ATT TTG G-3’、TXNIP、5’-TCA TGG TGA TGT TCA AGA AGA TC-3’および5’-ACT TCA CAC CTC CAC TAT C-3’、TXN、5’-GAC GCT GCA GGT GAT AAA C-3’および5’-CTG ACA GTC ATC CAC ATC TAC-3’、ARRB1、5’-AGT GGC CGT GGA ACT GCC CTT CA-3’および5’-GGA ACT TCC CGA TGC GGG GGT TC-3’、ARRB2、5’-GGG CAA GCG GGA CTT CGT AGA-3’および5’-TGC GGT CCT TCA GGT AGT CAG GG-3’。
Jurkat J16-145細胞を、ヒトリンパ芽球細胞系Jurkat J16(13)から誘導した。Jurkat細胞を、10% FCSを含有するIMDM中で培養した。新しく単離された末梢ヒトT細胞を、10% FCSを含有するRPMI 1640(+1-グルタミン)中で2 x 106細胞/mlの濃度で培養した。培養物を、加湿された5%CO2インキュベータ中で37℃で保存した。一晩静置しておいた一次細胞に対して分析を行った。
T細胞を、20〜25歳(11名の若齢提供者)と55歳以上(16名の老齢提供者)の健常なヒト提供者の血液から単離した。研究への関与の前に、すべての被験者からインフォームドコンセントを得た。研究は、ドイツガン研究センター(German Cancer Research Center)の倫理的ガイドラインとヘルシンキ宣言に従って行われ、それは、Ruprecht-Karls大学、ハイデルベルグ、ドイツの倫理委員会によって認可された。
一次ヒトT細胞を、(13)に記載されているように精製した。調製されたT細胞の純度を、PE-共役抗-CD3抗体による染色と、その後の、FACS分析とによって確認した。
細胞死を、生細胞との比較による前方-側方拡散プロファイル(forward-to-side scatter profile)における減少として分析し、前述(13)のように、「特異的細胞死」に再計算した。
細胞を、H2DCF-DA(5μM)で10分間、染色した。次に、細胞を分け、プレート結合抗-CD3抗体(30μg/ml)で1時間刺激するか、又は未処理状態に放置した。細胞を氷冷PBSで二回洗浄し、ROS生成を、FACS分析によって測定した。ROS生成は、以前に報告されている(14)ようにして計算した平均蛍光強度(MFI)の増加として定量化された。
ウェスタンブロッティング分析のために、全細胞抽出物を、細胞又はハエから、氷冷RIPAリーシス緩衝液(50mM Tris-HCl、pH 8.0、120 mM NaCl, 1% NP-40、0.5% Na-デスオキシコール酸(Na-Desoxycholat)、0.1% SDS、2 mM EDTA、25 mM NaF、0.2 mM NaVO4、1 mM、DTT、Rocheのコンプリートプロテアーゼインヒビターカクテル)中での30分間の溶解によって作製した。タンパク質をSDS-PAGEによって分離し、タンパク質をニトロセルロース膜(Invitrogen Carlsbad, CA, USA)上にブロッティングし、その後、5%ミルク中でブロッキングした。下記の抗体を使用した。抗-TXNIP (1: 10000, T. Dickによって提供された)、抗-アクチン(1: 8000、Acris Herford、ドイツ)、抗-カタラーゼ(1: 10000、 Sigma-Aldrich, ミュンヘン、ドイツ)、および抗-TXN (1: 10000) (T.Dick博士、ドイツがん研究センター、ハイデルベルク、ドイツからの寄付)。
ヒトTXNIPcDNAを、プライマ対 5’-CCGGAATTCATGGTGATGTTCAAGAAGATCAAG-3’と5’-CGGGGTACCTCACTGCACATTGTTGTTGAGG-3’で、TXNIP発現ベクターIOH42128-pEFDEST51(Open Biosystemsハイデルベルグ、ドイツ)から増幅し、pRev-TRE-Tight (Clonetech, USA)にクローニングした。レトロウイルスを、pRev-TRE-TXNIPとのPhoenix細胞のトランスフェクションによって生成した。前記Dox-依存トランスアクチベータを保持するJurakat M2細胞を感染させ、100μg/mlハイグロマイシンを添加した培地中で、7日間、培養した。その結果得られた細胞を、二回サブクローニングし、ウェスタンブロッティングによってDox-誘導TXNIP発現に関してスクリーニングした。
レンチウイルス粒子の作成のために、予め25μMのクロロキンで1時間、前処理しておいたHEK293T細胞を、TXNIPに対するshRNAを含有するベクター(Open Biosystems, ハイデルベルグ)とgag、pol、envおよびVSV-G(シュードタイピング(psuedotyping)用)のためのプラスミド混合物とトランスフェクションした。トランスフェクションの8時間後、培地を、パッケージング細胞から取り換えた。二日後、上清を、0.45μMフィルタに通した。これは、ポリブレン(Polybrene)(8μg/ml)が添加されている。1x 105の標的細胞を、1mlのウイルス上清とのspin occulationによって感染させた。安定的に形質導入されたJurkat細胞を、ピューロマイシン耐性(1μg/mlピューロマイシン)によって選択し、Dox依存発現をウェスタンブロッティングによってチェックした。
TXN活性の分析を、一次T細胞と全ハエのタンパク質溶解物中で分析した。
ハエにおけるRNAi-媒介TXNIPノックダウン、w1118に関して、P(GD4976)v15203メスハエをウイーンショウジョウバエRNAiセンター(Vienna Drosophila RNAi Center: VDRC、ウイーン、オーストリア)から入手し、B.A. Edgarによって提供されたtubGa 14オスと交配させた。ハエは、標準培地では25℃で生育した。
産卵行動の分析のために、10匹の新たに孵化したメス(孵化の3日後)と5匹のオス(孵化の3日後)とをバイアルに共に入れ、グレープジュースを含む寒天プレート上に24時間置いた。各メスによって寒天プレート上に産卵された卵を計数した。
新たに孵化したハエを、標準的なエサを含むバイアル中(各バイアルにつき10匹のハエ、オスとメスとを分離)で25℃で維持した。2〜3日毎に、ハエを新しいバイアルに移し、死んだハエを記録した。
以前、酸化信号がT細胞媒介免疫応答の調節において重要な役割を果たすことが示された(13-15)。ROS生成の年齢関連変化を分析するために、老齢(>55歳)および若齢(20-25歳)の個人からの一次T細胞を、単離し、これら二つのグループ間で、活性-誘導ROS生成を比較した。驚くべきことに、T細胞受容体(TCR)トリガー時に、老齢提供者のT細胞中にROS生成の増加が観察された(図1a)。老齢者におけるROSを調節する分子を求めて、mRNAとタンパク質レベルとの両方における老齢者のT細胞でのネガティブ酸化還元調節因子TXNIPの発現の大幅な増大が同定された(図1b,c)。若齢提供者のT細胞におけるTCRトリガー時に観察された完全な活性-誘導ダウンレギュレーションは、老齢者のT細胞においては、効率的ではなかった。その結果、段レギュレーションは、グルコース取り込みと、関連する細胞成長とのために必要であることが示されている(16)ことから、残りのTXNIP発現は、適切な細胞の活性化を妨げる可能性がある。連続調査によって、老齢者の、いくつかの一次細胞タイプ、即ち、単球、肝細胞と、更に、間葉又は造血幹細胞とにおける発現を増加が示された(図5a、b、c)。これは、老齢者の皮膚におけるTXNIP発現の増大を示す従来の報告と一致している(17)。更に、免疫系が加速老齢化表現型に関連するリウマチ性関節炎患者(図5d)のCD4+T細胞においてもTXNIPレベルの増大が見られた(18)。興味深いことに、主要なTXNIP相互作用パートナー、チオレドキシン(TXN)の分析は、老齢者のT細胞におけるTXN発現のダウンレギュレーションを示した(図1c、d)。従って、若齢提供者と比較して老齢者から単離されたT細胞におけるTXN活性の大幅な低下が観察された(図1e)。これらのデータは、高いTXNIP発現が、老齢者のT細胞のTXP抗酸化能力の減少に関連していることを示している。
T細胞におけるTXNIPの役割を調べるために、ヒトモデルT細胞系Jurkatにおける誘発TXNIP過剰発現システムを使用した。TXNIP発現を、1μg/mlのドキシサイクリン(Dox)の24時間、約2倍、の使用によって誘導した(図2a、b)。老齢者のT細胞において得られた結果と一致して、誘導されたTXNIP過剰発現によって、1時間のCDトリガー時に活性-誘導ROS遊離の大幅な増大が起こった(図2c)。正常T細胞ホメオスタシスの維持のためには、免疫応答後の抗原特異性T細胞の除去がきわめて重要である。これは、TCRの再刺激時にアポトーシス経路を介して活性-誘導細胞死(AICD)をトリガーすることによって達成される(19, 20)。ここで、TCRトリガーされた酸化信号がAICDの誘導を調節において重要な役割を果たす(13)。従って、AICDに対するTXNIP発現の増大を調べた。TXNIP誘導Jurkat細胞を、プレート結合α-CD3抗体で24時間刺激してAICDを誘導した。図2dに図示されているように、TXNIP過剰発現によってAICDの大幅な増加が生じた。これらを総合すると、これらの結果は、TXNIP発現の増大がJurkat T細胞における活性-誘導ROS遊離と細胞死とに影響する、ことを示している。
TXNIPダウンレギュレーションの作用を調べるために、誘導shRNAシステムを使用した。Doxとの24時間のインキュベーションによって、TXNIP shRNAの発現が誘導され、その結果、TXNIP mRNAとタンパク質レベルのダウンレギュレーションがもたらされた(図3a、b)。Jurkat T細胞におけるTXNIP発現の喪失によって、コントロール細胞又は非誘導細胞との比較において、CD3に対するアゴニスト抗体でのトリガー時に、活性-誘導ROS生成の速度が低下した(図3c)。若齢提供者における低いTXNIPレベルは低い活性-誘導ROS生成に相関したので、これらの結果は、一次T細胞に関して観察されたデータに対応している。次に、AICD誘導におけるTXNIPダウンレギュレーションの作用を調べた。Dox処理によるTXNIPに対する特異的shRNAの誘導後、Jurkat細胞を、プレート結合α-CD3抗体で24時間刺激し、細胞死を分析した。コントロール形質転換又は非形質転換細胞と比較して、特異的TXNIPダウンレギュレーションによって、AICDにおける大幅な減少がもたらされ(図3d)、TXNIP発現が免疫細胞の細胞死調節に関連していることが更に示された。これらの知見を更に裏付けるために、特異的TXNIPダウンレギュレーション後の種々の刺激による細胞死誘導を、フローサイトメトリーを使用して分析した。若齢 対 老齢提供者の一次T細胞で得られた結果に一致して(図1f)、TXNIPの特異的ダウンレギュレーションによって、H2O2処置による酸化ストレス誘導時の生存が改善された(図3e)。しかしながら、スタウロスポリン等の他の細胞ストレッサや、アゴニスト抗体によるCD95トリガーによる細胞死誘導は、TXNIPがダウンレギュレーションされようがされまいが細胞死レベルの変化を示さず(データ図示なし)、CD95L転写の上流の近位側ROS依存シグナリング経路はTXNIPによって影響されることを示している。総合すると、これらのデータは、Jurkat T細胞のTXNIPノックダウンは酸化還元均衡に影響を与えただけでなく、酸化ストレス時の細胞死誘導に対する耐性を増大させたことを示唆している。
TXNIPが、その発現が老齢化と相関するマーカーであるのか、又は、TXNIPは寿命の決定的な調節因子であるのかを分析するために、TXNIPの役割をイン・ヴィトロで調べた。ショウジョウバエゲノムは、約47%の全体的類似性を有するヒトTXNIPのホモログを含む。ショウジョウバエにおいて、それは神経系の発達において役割を有することが報告されている(21、22)。TXNIP欠損ハエを、UAS-TXNIP RNAiハエをtubulin-GAL4ドライバーと交配して、TXNIPの遍在的ダウンレギュレーションを作り出すことによって調製した。TXNIPに対するRNAiはqPCRとウェスタンブロッティングによる測定で、TXNIP発現の顕著な低減をもたらした (図6a、b)。前記ハエの顕微鏡検査は、TXNIP欠損ハエはより大きいことが示された(図6c)。これは、コントロールハエに比べてTXNIP欠損のより大きな体重により、もたらされた(図6a)。われわれは、種々のヒト細胞タイプにおけるTXNIP発現のアップレギュレーションを観察していたので、老齢のコントロールハエが若齢のハエと比較して高いTXNIPレベルを示すかどうかも問うた。事実、コントロールのハエからの全ハエタンパク質抽出物の免疫ブロットは、生後一日の若齢ハエと比較して、47日齢のハエにおいてはTXNIPタンパク質の顕著な増加を示した。TXNIP欠損ハエは、タンパク質発現が終生欠如し、高効率のTXNIPノックダウンを確認した(図6d)。次に、TXNIPは、TXNIP欠損Jurkat T細胞において観察されたものに類似して、種々のストレス誘導物に対する耐性において重要な役割を果たすものであるか否かを調べた。従って、TXNIP欠損ハエの、飢餓状態、パラコート、酸化ストレスの誘導体での処理における生存についてテストした。ハエを、摂食妨害分析用の寒天のみのバイアル、又は、酸化ストレス耐性のテストのための1mMパラコートを添加した寒天に移した。TXNIP欠損は、飢餓とパラコートに対する曝露との両方においてハエの生存を顕著に増大させた(図4b、c)。更に、イン・ヴィトロデータに対応して、TXNIP欠損は、全ハエタンパク質溶解物におけるTXN活性の増大をもたらし(図4d)、従って、ショウジョウバエの抗酸化能力にも影響した。TXNIP欠損が老齢化の調節に直接関与しているか否かを調べるために、TXNIP欠損ハエとコントロールのハエの寿命を測定した。驚くべきことに、TXNIPの遺伝障害によってオスとメスのショウジョウバエの健康寿命が大幅に延伸され、TXNIPの寿命の決定的調節因子としての役割が示された。平均寿命は、それぞれコントロールのハエと比較して、メスのTXNIP RNAiハエにおいて18%増加し、これに対して、最大寿命はオスにおいて5%(p<0.01)、メスRNAiハエでは13%(p<0.001)増加した(図4e)。これらのデータに対応して、過去の研究も、メスとオスの生物間の寿命延伸の差を報告している(23, 24)。従って、このことは、TXNIPを、イン・ヴィヴォで、寿命の直接的調節因子として同定するものである。更に、TXNIP発現の欠損時における産卵頻度による測定でのメスの繁殖性の増大が観察された。TXNIP欠損ハエは、24時間の観察時間内において、産卵数の最大30%の増加を示した(図4f)。これらのデータは、TXNIP欠損は、過去の報告書(23)におけるショウジョウバエのラパマイシン処理時にも見られるような、寿命と繁殖性との脱共役をもたらすということを示している。総合すると、上述した表現型は、TXNIPのハエの老齢化と長命においてのみならず、ストレス耐性と繁殖性の調節における役割を示している。
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Claims (3)
- 女性(メス)の生殖能の改善のための医薬組成物であって、
(a)チオレドキシン相互作用タンパク質(TXNIP)の生物活性、又は、
(b)TXNIPをコードする遺伝子の発現を低減又は抑制することが可能な化合物を含み、
前記化合物は、
(i)TXNIPをコードする遺伝子の発現を低減又は抑制するアンチセンスオリゴヌクレオチド又はshRNA、若しくは、
(ii)TXNIP、又はそのフラグメントに対する抗体、であることを特徴とする、医薬組成物。 - TXNIPの生物活性、又はTXNIPをコードする遺伝子の発現を低減又は抑制することによって 女性(メス)の生殖能の改善する化合物を同定する方法であって、以下の工程を有する、
(a)候補化合物と、TXNIP又はTXNIPをコードする遺伝子を含むテストシステムとをインキュベートする、そして、
(b)TXNIPの生物活性を分析する、
ここで、TXNIPの生物活性の抑制又は損失は、 女性(メス)の生殖能を改善する特性を有する候補化合物の存在を示す、方法。 - 前記TXNIPの生物活性の抑制又は損失は、テスト化合物の不在によって特徴付けられるテストシステムとの比較によって判定される請求項2に記載の方法。
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EP12002914.5 | 2012-04-25 | ||
PCT/EP2013/001143 WO2013159879A1 (en) | 2012-04-25 | 2013-04-17 | Inhibitors of thioredoxin-interacting protein (txnip) for therapy |
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US6355426B2 (en) | 1996-10-31 | 2002-03-12 | Smithkline Beecham Corporation | Methods for the characterization and selection of RNA target motifs that bind compounds of pharmaceutical use |
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US8999944B2 (en) * | 2005-01-20 | 2015-04-07 | University Of Rochester | Thioredoxin interacting protein (TXNIP) as regulator of vascular function |
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