JP6595470B2 - 脂肪酸代謝異常症を治療するための組成物および方法 - Google Patents
脂肪酸代謝異常症を治療するための組成物および方法 Download PDFInfo
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Description
この出願の発明に関連する先行技術文献情報としては、以下のものがある(国際出願日以降国際段階で引用された文献及び他国に国内移行した際に引用された文献を含む)。
(先行技術文献)
(特許文献)
(特許文献1) 米国特許出願公開第2010/0062059号明細書
(特許文献2) 米国特許出願公開第2003/0199529号明細書
(特許文献3) 米国特許出願公開第2011/0092594号明細書
(特許文献4) 米国特許出願公開第2011/0105458号明細書
(特許文献5) 国際公開第94/16740号
(非特許文献)
(非特許文献1) DOULIAS et al.Nitric Oxide Regulates Mitochondrial Fatty Acid Metabolism Through Reversible Protein S−Nitrosylation.Sci.Signal 66(256): 1−17,January 2013.[retrieved on 28.09.2014].Retrieved from the Internet.<URL: http://www.ncbi.nlm.nih.gov/pmc/articleslPMC4010156/>.
(非特許文献2) RUI−QIONG et al.Inhibition of nitric oxide synthase lowers fatty acid oxidation in preeclampsia−like mice at early gestational stage.Chinese Medical Journal 24(19): 3141−3147,2011.[retrieved on 01.10.2014].Retrieved from the Internet.<URL: http://www.cmj.orglch/reader/createJ)df.aspx?file_no=2011930343307 40&flag=1 &year _id=2011&quarteUd=19>.entire document
下記の定義は本発明の理解を簡単にするために提供される。
か、合成されたかによらず、前記プローブに相補的な配列を有する核酸とアニーリングす
るか、特異的にハイブリダイズすることができる、オリゴヌクレオチド、ポリヌクレオチ
ド、またはRNAまたはDNAいずれかの核酸を指す。プローブは一本鎖または二本鎖の
可能性がある。前記プローブの正確な長さは、温度、プローブ供給源、および前記方法を何に利用するかを含む多くの要素によって決まる。例えば、診断に応用する場合、標的配列の複雑さにより、前記オリゴヌクレオチドプローブは典型的に10〜30または15〜25、またはそれ以上のヌクレオチドを含むが、それよりも少ないヌクレオチドを含むこともある。本明細書のプローブは、特定の標的核酸配列の異なる鎖に相補的となるように選択される。これは、一連の事前に決定された条件下、前記プローブがそれぞれの標的鎖と「特異的にハイブリダイズする」またはアニーリングすることができるよう、十分相補的である必要があることを意味する。従って、前記プローブの配列は前記標的の厳密に相補的な配列を反映している必要はない。例えば、非相補的ヌクレオチドフラグメントが前記プローブの5’または3’末端に結合しており、前記プローブ配列の残りの部分が前記標的鎖と相補的であることも考えられる。または、前記プローブ配列は前記標的核酸の配列と十分相補的で、それと特異的にアニーリングするという前提条件下で、非相補的塩基またはそれよりも長い配列が前記プローブ内に点在していることも可能である。
化学薬品および試薬
パルミトイルCoAリチウム塩および六フッ化リン酸フェリシニウムはSigma−Aldrichより得られた。マウスモノクローナル抗FLAG(登録商標)、グリセルアルデヒド3リン酸脱水素酵素、およびシトクロムcオキシダーゼサブユニットI抗体はそれぞれStratageneおよびAbcamより得られた。抗VLCADのウサギおよびヤギ(クローンG−16)ポリクロ―ナル抗体は、GeneTexおよびSanta Cruz Biotechnologyよりそれぞれ得られた。すべての薬品および試薬は分析グレードのものである。
全てのマウス実験はInstitutional Animal Care and Use Committee of the Children‘s Hospital of Philadelphia Research Instituteによる審査および承認を経ている。野生型C57BL/6J、Nos3tm1Unc(eNOS−/−)C57BL/6J、およびLepob(ob/ob)C57BL/6Jの成体マウスはジャクソン研究所より、得られた。一日置きにPBSまたは5mMのGSNOが注射されたob/obマウスにおいて4週間にわたり食餌摂取量および体重の記録をとった。PBSおよびGSNO注射されたob/obマウスの平均食餌摂取量はそれぞれ42±9と41±10gであった(各遺伝子型につきn=4匹ずつ)。同じ期間における、平均体重変化はPBS注射されたob/obマウスとGSNO注射されたob/obマウスにおいて、それぞれ28.2±1.0と28.1±4.0であった(各遺伝子型にn=4匹ずつ)。マウスはCO2によって麻酔にかけ、左心室に浸み込む前に血液を回収した。完全な状態で臓器を回収し、すぐに液体窒素によって凍結して、使用されるまで−80℃で保存された。組織は3mlのリシスバッファー[1mMジエチレントリアミン五酢酸、0.1mネオクプロイン、1%TritonX−100、およびプロテアーゼ阻害剤を含む250mMヘぺス−水酸化ナトリウム(pH7.7)]中でテフロン乳棒およびJumbo Stirrer(Fisher Scientific)によってホモジナイズした。このホモジネートはその後、4℃において13,000gで30分遠心分離した。この可溶性タンパク質層を回収して、このタンパク質濃度をブラッドフォード分析によって同定した。試料およびネガティブコントロール試料の作成は、(Doulias et al. (2010) Proc. Natl. Acad. Sci., 107:16958−16963)に説明されるよう行った。
カラムの調製および活性化、有機水銀レジンとの反応のためのホモジネート調製、に関する詳細な実験操作は(Doulias et al. (2010) Proc. Natl. Acad. Sci., 107:16958−16963)で説明される。各臓器から3つの生物学的複製が解析された。各試料には対応する同条件下で解析されたUV前処理済みのネガティブコントロールも用意した。偽発見率は脳において<6%、他の全ての臓器において<3%とした。洗浄については、六臓器間での脂質含有量に差があるため厳しい条件を選んだ。カラムは最初50ベッド体積の300mM NaClおよび0.5%SDSを含む50mM トリス塩酸(pH7.4)で、その後50ベッド体積の0.05%SDSを含む同バッファーを用いて洗浄した。カラムは300mMのNaCl、1%TritonX−100、そして1M尿素を含む50ベッド体積の50mMのトリス塩酸(pH 7.4)で、その後0.1%TritonX−100および0.1M尿素を含む50ベッド体積の同バッファーで洗浄をした。最後に、カラムは200ベッド体積の水で洗浄されて、タンパク質を10mlの50mM β−メルカプトエタノール水により溶出した。試料を濃縮しゲル−LC(liquid chromatography)−MS/MS分析によって分析をした。最後の水による洗浄後のカラム中消化には、前記カラムを10ベッド体積の0.1M炭酸水素アンモニウムによって洗浄した。吸着したタンパク質はTrypsin Gold(1μg/ml)(Promega)を含む1ベッド体積の0.1M炭酸水素アンモニウムを添加することによって暗中において16時間室温で消化した。前記レジンは次に、40ベッド体積の300mM NaClを含む0.1M炭酸水素アンモニウム(pH7.4)で、その後40体積のNaClを除いた同バッファーによって洗浄をした。カラムはその後40体積の0.1M炭酸水素アンモニウムで、そして200体積の脱イオン水で洗浄した。吸着ペプチドの溶出のため、前記レジンは、1ベッド体積の過ギ酸水(過ギ酸は1%のギ酸と0.5%の過酸化水素を少なくとも60分間、遮光されたガラスバイアル中で振盪させることによって反応させて合成される)中に30分間室温にて置いた(Doulias et al. (2010) Proc. Natl. Acad. Sci., 107:16958−16963)。溶出されたペプチドは、前記レジンを1ベッド体積の脱イオン水で洗浄することにより回収した。溶出物は一晩−80℃で保存し、凍結乾燥後して300μlの0.1%ギ酸中で再懸濁した。ペプチド懸濁液は低吸着性試験管(Axygen)に移して、高速バキューム機によって体積を30μlまで減らした。20マイクロリットルのペプチド懸濁液は高速液体クロマトグラフィーの用のバイアルに移されてLC−MS/MS分析に出した。
VLCADの酵素活性は(Lehman et al. (1990) Anal. Biochem., 186:280−284)に説明されるように解析した。簡単に述べると、フェリシニウムイオンを、VLCADを介するパルミトイルCoA脱水素化の人工的な電子アクセプターとして使った。0.2%TritonX−100および0.1mM EDTAを含む100mMリン酸カリウムバッファー(pH 7.2)中の肝臓のホモジネート(最終濃度0.03μg/μl)または細胞溶解液(最終濃度0.09μg/μl)を、150mMのフェリシニウムイオンと混合し、そしてパルミトイルCoA(最終的な体積は130μl)を添加することによって反応を開始させた。300nmにおけるフェリシニウム吸光度の減少を時間の関数として記録して、時間0(パルミトイルCoAが添加された時点)から吸光度の総変化の5%に対応する時間までの曲線の傾きを、前記酵素の初速度(Vo)とした。各動物のVLCADの見かけ上のVmaxおよびKMの実験による算出に、0.015〜2mMの範囲で、少なくとも9つの濃度のパルミトイルCoAを用いた。これらの実験データはGraphPad Prismソフトによって、ミカエリス・メンテン式に非線形回帰により当てはめられた。酵素活性の単位は室温で1分あたり1mmolのフェリシニウムの減少に繋がる酵素量で定義される(300nmにおいてε=4.3mM−1cm−1)(Izai et al. (1992) J. Biol. Chem., 267:1027−1033)。この分析法の特異性を確かめる実験として、10mgの抗VLCAD抗体を、事前に肝臓のホモジネートと20分反応させた。UV光分解をS−ニトロソシステインを除去するために使用するときは、前記肝臓のホモジネートを氷上で10分間、従来型のUVトランスイルミネーターによって照射した。肝臓ホモジネート(図2H)および細胞溶解液(図4)におけるVLCADの特異活性測定は、それぞれ0.25および0.125mMのパルミトイルCoA存在下において実施した。
ホルマリン固定された肝臓の切片をトリクローム染色キット(Trichrome Stain Kit (Sigma))を用い製造業者の説明書に従い染色した。トリグリセリドはFolch法(Folch et al. (1957) J. Biol. Chem., 226:497−509)によって抽出した。血清および肝臓のトリグリセリド濃度をtriglyceride quantification kit(Abcam)を使用し製造業者の説明書に従って測定した。
脂肪酸を含まないウシ血清アルブミン(2.5 mg/ml)、2.5mMパルミチン酸、10mMカルチニン、および4mCiの9,10−3H−パルミトイルCoA(Biomedicals)を含むKrebs−Ringer重炭酸イオンバッファー中に、1ミリグラムのタンパク質懸濁液を加えた。この混合物は、暗中の37℃において二時間振盪して、その後Folch法を用いて、9,10−3H−パルミトイルCoAと3H2Oを分離した(Folch et al. (1957) J. Biol. Chem., 226:497−509)。この水相を回収し、10%TCAの添加後に10分間室温で8000gにおいて遠心分離することにより、タンパク質を回収した。残りの放射性パルミトイルCoAはAG 1−X8ギ酸塩レジン(Life Science)を用いて 強アニオン交換クロマトグラフィーによって除去した。3H2Oを含む流出液は回収してシンチレーション検出に用いた。各実験は、バックグラウンド値の測定と共に行うため、タンパク質を含まない試料も用いて行われた。このバックグラウンド測定値は対応する実験用の試料の各測定値から引いた。
QuikChange(登録商標) Lightning Site−Directed Mutagenesis Kit(Agilent Technologies)を使用してVLCADをコードするcDNAに1アミノ酸変異の導入を行った。アミノ酸位置238番目の、システインからアラニンへの変異は、マウスVLCADをコードするプラスミドと3個のFLAG(登録商標)タグをこのタンパク質のC末端部位で結合させた(GeneCopoeia)、Ex−Mm01013−M14(以下、pVLCAD−3xFLAG(登録商標))をテンプレートとして行った。フォワードプライマー:5′−TCAGCCATACCCAGCCCCGCTGGAAAATATTACACTCTC−3′(配列ID番号:1)およびリバースプライマー:5′−GAGAGTGTAATATTTTCCAGCGGGGCTGGGTATGGCTGA−3′(配列ID番号:2)をpVLCAD−3xFLAG(登録商標)においてシステインのコドンをアラニンのコドンに置換する工程に用いて、これによりpVLCAD−C238A−3xFLAG(登録商標)を作成した。pVLCAD−3xFLAG(登録商標)およびpVLCAD−C238A−3xFLAG(登録商標)の配列は、次の実験に移る前に確認した。
Hepa1〜6細胞を、10%ウシ胎児血清、2mMグルタミン、ペニシリン(100U/ml)、およびストレプトマイシン(100ng/ml)を含むダルベッコ改変イーグル培地(Dulbecco’s modified Eagle’s medium:DMEM)を用い、37℃で5%CO2を含む空気中で培養した。細胞は5×104細胞/cm2の濃度において通常の条件下で24時間培養した。細胞にLipofectamine(登録商標) 2000剤(Life Technologies)を用いてFLAG(登録商標)標識された野生型VLCADまたはFLAG(登録商標)標識されたC238AVLCADを製造者の説明書に従いトランスフェクトした。トランスフェクションの48時間後、増殖培地は血清なしDMEMと交換されて、新たに調製された250mMのGSNOが30分間添加され、その後この細胞はPBSにより複数回洗浄をした。細胞溶解液はGSNO除去後すぐ、60、120、および240分後回収された。これらの試料はUV光から保護されていた。溶解液はタンパク質濃度が分析され、試料あたり等量のタンパク質を有機水銀支援捕捉に用いた。
ヒトVLCADの結晶構造はPDB(ID 2UXW)よりダウンロードした。異常または欠損残基に関してはPymol(www.pymol.org)のMutagenesisツールによって補填した。S−ニトロソシステインはMolecular Modeling ToolkitにおいてTimerghazin研究室によるPythonスクリプト「S−nitrosator」を使用して生成した。S−nitrosatorはS−ニトロソシステイン残基をへリックスに関してPCM−ONIOM(PBE0/def2−TZVPPD:AmberFF)計算によって算出されたチオレドキシンおよびχ3値を座標として使用している。ElNemo(Suhre et al. (2004) Nucleic Acids Res., 32:W610−W614)を最も低い100周波数モードを観察するために用い、VLCADの初めの重要な5モードについて摂動したモデルの生成を行った。残基の平均二乗変位(r2)をタンパク質の移動を同定するために用いた。
データはGraphPad Prism 5.0dソフトによって解析した。全ての正規分布データは、平均±標準偏差で示される。群のものは一元配置分散分析によって解析した。
マウスS−ニトロソシステインのプロテオーム
野生型マウスの脳、心臓、腎臓、肝臓、肺、および胸腺で、647個のタンパク質中に1011個のS−ニトロソシステインを含むペプチドを同定した。広範な文献検索によれば、この拡張されたS−ニトロソシステインプロテオームは、既に生理学的条件下で修飾されていると報告されている46のタンパク質を同定し、971のこれまで知られていなかった内在性のS−ニトロシル化部位を明らかにしていた。全6臓器で、S−ニトロシル化部位の数はタンパク質の数を超えており、in vivoで複数のS−ニトロシル化がタンパク質制御に関わっている可能性を示唆した(Simon et al. (1996) Proc. Natl. Acad. Sci., 93:4736−4741)。前記6臓器で同定されたタンパク質を各臓器少なくとも3つの生物学的複製を用いて比較したところ、平均で、72%のタンパク質が2臓器以上で同定されており、S−ニトロシル化がin vivoにおいて全体的な機能を持っていることを示唆している。さらに、これらのデータはこれらのプロテオームを取得するのに用いた方法が正確かつ再現性を有することを示している。1つの臓器のみにおいて同定されたタンパク質は21〜46%の範囲にあり、S−ニトロシル化が臓器特異的な役割もまた務めること示す。in vivoのS−ニトロシル化のeNOSによって生成される一酸化窒素への依存度はeNOSヌルマウス(eNOS−/−)の同じ臓器における内在性S化部位を解析することによって調べた。同定されたタンパク質の47〜87%のS−ニトロシル化にeNOSの存在が必要であったことから、eNOSは内在性のS−ニトロソシステインプロテオームに大きく貢献している。前記脳および前記心臓は他の臓器と比べてeNOS活性への依存が最も少なく、これらの臓器におけるタンパク質のS−ニトロシル化への神経型一酸化窒素合成酵素などの他のアイソフォームの貢献を示唆している。eNOS−/−マウスの臓器における相当数のS−ニトロソシステインペプチドの欠如は、S−ニトロソシステインプロテオームを同定する本方法の正確性をさらに強固なものにしている。
脂肪酸代謝に関わるタンパク質のクラスタリングが本解析において明確であった。前記マウス肝臓において、S−ニトロシル化タンパク質はレプチンホルモンに対する肝臓の反応を取り巻くネットワーク内にクラスター化していた(Doulias et al. (2010) Proc. Natl. Acad. Sci., 107:16958−16963)。本実験で、脂肪酸β酸化の律速段階を触媒するVLCADは、野生型マウスの肝臓ではS−ニトロシル化されていたがレプチン欠損マウス(ob/ob)、またはeNOS−/−マウスではされていなかった(図1)。S−ニトロシル化のVLCAD活性および主要代謝器官である、前記肝臓における脂肪酸代謝に対する生物学的な影響を調べた。
Claims (6)
- 極長鎖アシルコエンザイムA脱水素酵素欠損症(VLCADD)を治療するためのS−ニトロソ−N−アセチルシステイン(SNO−NAC)を有する組成物。
- 請求項1記載の組成物において、前記組成物は、少なくとも1つの薬学的に許容可能な担体、及びVLCADDの治療のための少なくとも一つの別の治療剤を含む組成物。
- 請求項2記載の組成物において、前記別の治療剤はトリヘプタノインまたはベザフィブラートである、組成物。
- 請求項1記載の組成物において、前記SNO−NACは、少なくとも1つの薬学的に許容可能な担体と共に製剤化される、組成物。
- 請求項1記載の組成物において、前記VLCADDは、極長鎖アシルコエンザイムA脱水素酵素(VLCAD)中のミスセンス変異またはヌル変異によって特徴づけられる、組成物。
- 請求項5記載の組成物において、前記VLCADDは、極長鎖アシルコエンザイムA脱水素酵素(VLCAD)中のミスセンス変異によって特徴づけられる、組成物。
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