JP2021507679A - 細胞内標的化のための環状gmpキレートペプチド - Google Patents
細胞内標的化のための環状gmpキレートペプチド Download PDFInfo
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Abstract
Description
−ホモ二量体化、cGMPの不在下でのキナーゼ活性の抑制、及びタンパク質基質を含む他のタンパク質との相互作用を仲介するN末端ドメイン;
−同一でない2つのcGMP結合部位を含む調節ドメイン;
−ATPから標的タンパク質のセリン/スレオニン側鎖のヒドロキシル基へのリン酸転移を触媒するキナーゼドメイン、で構成されている。
−配列番号1の配列及び配列番号2の配列に由来するキメラペプチドと;
−配列番号6の配列を含む又はそれからなるcGMP結合ドメインとを含み;
−当該ポリペプチドは、任意の触媒ドメイン
及びその機能変異体を欠いている。
本発明の文脈において、「配列番号1の配列及び配列番号2の配列に由来するキメラペプチド」という用語は、少なくとも配列番号1に対応する配列又はそのフラグメント、及び少なくとも配列番号2に対応する配列又はそのフラグメントを含む又はそれからなるペプチドを指す。
−配列番号2の残基94〜104に融合した、配列番号1の残基1〜78に対応する配列番号3のペプチド。
−配列番号2の残基94〜104に融合し、配列番号1の残基51〜78に融合した、配列番号2の残基1〜65に対応する配列番号4のペプチド;及び
−配列番号2の残基94〜104に融合し、配列番号1の残基69〜78に融合し、配列番号2の残基66〜83に融合した、配列番号1の残基1〜50に対応する配列番号5のペプチド。
コンストラクト:
以後、cGMPSp、cGMPSponge、SponGee又はcGMPSp/SponGeeと指定される本発明によるポリペプチドの例をコードするポリヌクレオチドであって、これらの用語はいずれも以下を使用して調製される5’にFLAGタグを更に含む非常に等しいポリペプチドを表す。
−ATGAGCGAGCTGGAGGAAGACTTTGCCAAGATTCTCATGCTCAAGGAGGAGAGGATCAAAGAGCTGGAGAAGCGGCTGTCAGAGAAGGAGGAAGAAATCCAGGAGCTGAAGAGGAAACTCCATAAATGCCAGTCAGTGCTGCCCGTGCCCTCGACCCACATCGGCCCCCGGACCACCCGGGCACAGGGCATCTCGGCCGAGCCGCAGACCTACAGGTCCTTCCACGACCTCCGAGTGACCCTGCCCTTCTACCCCAAGAGTCCACAGTCCAAGATCGATCTCATAAAGGAGGCCATCCTTGACAATGACTTTATGAAGAACTTGGAGCTGTCACAGATCCAAGAGATTGTGGATTGTATGTACCCAGTGGAGTACGGCAAAGACAGCTGCATCATCAAAGAAGGAGATGTGGGGTCACTGGTGTATGTCATGGAAGATGGTAAGGTTGAAGTTACAAAAGAAGGCGTGAAGCTGTGCACAATGGGTCCTGGTAAAGTGTTTGGAGAGTTGGCTATCCTTTACAACTGTACCCGGACGGCGACCGTCAAAACTCTTGTAAATGTGAAACTCTGGGCCATTGATCGACAATGTTTTCAGACGATAATGATGAGGACAGGACTTATCAAGCATACCGAGTATATGGAATTTTTAAAAAGCGTTCCAACATTCCAGAGCCTTCCTGAAGAGATCCTCAGTAAACTTGCTGACGTCCTTGAAGAGACCCACTATGAAAATGGGGAATATATCATCAGGCAAGGTGCAAGAGGGGACACCTTCTTTATCATCAGTAAAGGAAAGGTTAATGTCACTCGTGAAGACTCGCCCAATGAAGACCCAGTCTTTCTTAGAACCTTAGGAAAAGGAGATTGGTTTGGAGAGAAAGCCTTGCAGGGGGAAGATGTGAGAACAGCGAATGTAATTGCGGCAGAAGCTGTAACCTGCCTTGTGATCGACAGAGACTCTTTCAAACATTTGATTGGAGGATTAGATGATGTTTCTAAAAAGCATATGAAGATGCAGAAGCTAAG。
網膜外植片、又は標的化バージョンのSponGee及びGFPを共発現するHEK細胞を、PB中の4%PFAで30分間固定し、PBS中の1%Triton及び3%BSAで透過処理及びブロックを行い、次いで、DsRedに対する抗体(Clontech、ロット#1306037、以前に同様のアッセイで使用された抗体(Averaimo et al.,Nat Commun.,7:12896,2016)に続いて、AlexaFluor594(Life Technologies)及びGFP(Life Technologies、ロット番号1789911、以前にNicol et al.,Nat.Neurosci:,10:340−347,2007で検証済み)に結合した二次抗体、又はα−チューブリン(Sigma、ロット番号T6199、以前にNicol et al.,Nat.Neurosci:,10:340−347,2007で検証済み)に続いてAlexaFluor 488(Life Technologies)に結合した二次抗体と共にインキュベートした。
画像は、40倍の油浸対物レンズ(N.A.1.3)及びソフトウェアMetamorph(Molecular Devices)を連結させた倒立DMI6000B落射蛍光顕微鏡(Leica)で取得した。ライブイメージング実験では、0.2mM又は2mMのCaClを含むHBSバッファーで細胞を灌流した。タプシガルギンを1mMで使用した)。CFP(483/32nm)及びYFP(542/27)チャネルの画像を20秒ごとに同時に取得した。画像をImageJで加工し、バックグラウンド及びブリードスルー(bleedthrough)を補正した後、CFP/YFPの比率を計算した。共焦点画像を63倍の油浸対物レンズ(N.A.1.45)で取得し、標本全体を含むZスタックをナイキスト周波数でサンプリングした。画像をImageJ及びPhotoshopで描画した。
妊娠中のC57BL6/J及びRjOrl:SWISSマウス、並びにSprague−DawleyラットをJanvier Labsから購入した。動物の処理はいずれも、施設のガイドラインに従って行われ、地域の倫理委員会(C57BL6/Jマウス、C2EA−05:Comite d’ethique en experimentation animale Charles Darwin;Sprague−Dawleyラット、ethics committee C2EA−59:Comite d’ethique en matiere d’experimentation animale Paris Centre et Sud)によって承認された。動物を12時間の明/12時間の暗サイクルの下で飼育した。日付を付けた交配からの胚(個々の実験を説明する各欄に記載されている発生段階)は実験中には性別が決定されておらず、雌雄の比率は1に近いと予想される。
HEK293細胞を、ポリリジン被覆カバーガラスにプレーティングし、翌日、製造元の指示に従ってLipofectamine 2000(Thermo Fisher)を使用して、pCX−mRFP又はpCX−SponGeeベクターでトランスフェクトした。プレーティングの3日後、細胞を4%パラホルムアルデヒドで固定し、切断型カスパーゼ3(Asp175;Cell Signaling;ロット#0043)及びα−チューブリン(Sigma)に対する抗体で免疫細胞化学用に加工するか、又はCellEvent カスパーゼ3/7グリーン検出試薬(Thermo Fisher)で30分間処理した後、固定してα−チューブリン抗体で標識した。各実験で、mRFP又はSponGeeの陽性細胞集団における非標識細胞に対するカスパーゼ3陽性の割合を、DM6000顕微鏡(Leica Microsystems)の20倍の空気対物レンズで取得された10個のランダムに選択されたフィールドから計算した。
海馬神経細胞培養を、本質的に以前に記載されたように行った(Leterrier et al.,.Neurosci.,26:3141−3153,2006)。簡潔に言えば、ラット胚の海馬を胎生18日目に解剖した。トリプシン処理後、火仕上げした(fire−polished)パスツールピペットで分離を行った。細胞を計数し、ポリ−D−リジンで被覆した直径18mmのガラス製のカバーガラス上に300〜400細胞・mm−2の密度でプレーティングした。プレーティング培地は、2%B27(Life Technologies)で補完し、安定化グルタミン(0.5mM)及びペニシリンG(10U.ml−1)/ストレプトマイシン(10g.ml−1)を含むNeurobasal(Life Technologies)であった。プレーティングの4時間後、カバーガラスを、80%コンフルエントなグリア層で24時間順化した補完Neurobasal培地を含むペトリ皿に移した。製造元の指示に従い、リポフェクタミン2000(Life Technologies)を使用して、in vitroで6日後に(DIV)ニューロンにトランスフェクトした。
少なくとも5日間の適応を可能にするため、手術の1週間前に交配の日が明らかな(timed−pregnant)雌性動物(Janvier Labs)を動物施設に配送した。子宮内(in utero)エレクトロポレーションを以前に記載されたように行った(Loulier et al.,PLoS Biol.,7:e1000176,2009)。E14.5の雌性動物にケタミン/キシラジンで麻酔をかけ、正中線開腹術を行って子宮角を露出させ、傾斜照明のもとで胚の可視化を可能にした。滅菌Fast Green色素(Sigma)と3:1で組み合わされた2つのプラスミドベクターを含む1μLのDNAを、INJECT+MATIC(INJECT+MATIC)マイクロインジェクターによって駆動されるガラスキャピラリーピペット(先端が面取りされている75μm〜125μmの外径)を各胚の側脳室に注入した。2つの異なるプラスミドベクターを同時に注入した。1つ目は、エレクトロポレーションの対照として使用される、ニワトリベータアクチンプロモータ(pCX−eGFP)の制御下で緑色蛍光タンパク質をコードするプラスミドであった。2つ目は、赤色蛍光タンパク質(mRFP)の対照コンストラクト又はSponGee−mRFPをコードするプラスミドであった。直径5mmのピンセット電極(Sonidel Limited)の陽極を背側終脳の上に置き、50ミリ秒の持続時間の4回の35Vパルスを子宮嚢に印加した。子宮内手術後、切開部位を縫合糸(4−0、Ethicon)で閉じ、マウスを清潔なケージで回復させた。マウスを手術の4日後に安楽死させてE18.5胚の脳を採取するか、又は出生後P10で分析するために出産させた。胚の脳を解剖して取り出し、Antigenfix(Diapath)固定液に一晩浸漬し、切片化する前にPBSですすいだ。P10マウスをペントバルビタールナトリウム(150mg・kg−1)で深く麻酔し、Antigenfix(Diapath)で経心臓的に灌流し、脳を解剖して取り出し、同じ溶液で一晩、後固定した。胎児及び出生後の脳のサンプルを、振動するブレードミクロトーム(Leica VT 1000S)で200μmの厚さに切片化した。最後に、切片をVectashield+Dapi(Vector laboratory)にマウントするか、10μg・mL−1ビス−ベンズイミド(Sigma Aldrich)で2時間インキュベートし、Mowiol 4−88(Sigma Aldrich)にマウントした。共焦点画像を10倍の対物レンズ(NA 0.4)で取得し、標本全体を含むZスタックをナイキスト周波数でサンプリングした。画像をImageJ及びPhotoshopで描画した。
画像を、40倍の油浸対物レンズ(N.A.1.3)及びソフトウェアMetamorph(Molecular Devices)を連結させた倒立DMI6000B落射蛍光顕微鏡(Leica)で取得した。ライブイメージング実験では、ThPDE5VV又はH147でトランスフェクトされたmRFP又はSponGeeを共発現する細胞を、1mM CaCl2、0.3μMMgCl2、0.5mM Na2HPO4、0.5mM NaH2PO4、0.4μMMgSO4、4.25mM KCl、14μMNaHCO3、120mM NaCl、0.0004%CuSO4、1.24μMFe(NO3)3、1.5μMFeSO4、1.5μMチミジン、0.51mMリポ酸、1.5mM ZnSO4、0.5μMピルビン酸ナトリウム(いずれもSigma製)、1×MEMアミノ酸(Life Technologies)、1×非必須アミノ酸(Life Technologies)、25mM HEPES(Sigma)、0.5mMプトレシン(Sigma)、0.01%BSA(Sigma)、0.46%グルコース(Sigma)、1mMグルタミン(Life Technologies)、2%ペニシリンストレプトマイシン(Life Technologies)を用いて灌流した。ビタミンB12及びリボフラビンはそれらの自家蛍光のため省略された。スペルミンNONOateは50μM、ODQ(Tocris)は10μM、Forskolin(Sigma)は10μMで使用した。CFP(483/32nm)及びYFP(542/27)チャネルの画像を20秒ごとに同時に取得した。画像をImageJで加工し、バックグラウンド及びブリードスルーを補正した後、CFP/YGPの比率をコンピュータで計算した。共焦点画像を63倍の油浸対物レンズ(N.A.1.45)で取得し、標本全体を含むZスタックをナイキスト周波数でサンプリングした。画像をImageJ及びPhotoshopで描画した。
ThPDE5VVプローブでトランスフェクトしたニューロンを、油浸CFI Plan APO VC60倍、NA1.4対物(Nikon)を使用して、37℃の恒温チャンバー内のPerfect Focus System(PFS)を備えた電動Nikon Eclipse Ti−E/B倒立顕微鏡でDIV8及びDIV11の間のビデオ顕微鏡によって撮像した。取得をCFPの励起波長(434nm+/−15nm)で、Intensilight(Nikon)を使用して行った。放射光は、FF509−FDi01ダイクロイックミラー、FF01−483/32−25CFPフィルター及びFF01−542/27−25YFPフィルターを備えたOptosplit IIビームスプリッター(Cairn Research)を通過し、2倍の拡大レンズの後ろに取り付けられたEM−CCDカメラ(Evolve512、Photometrics)によって収集された。Metamorph7.7(Molecular Devices)による設定を試験的に行うことで取得を実施した。全てのフィルターセットをSemrockから購入した。18−mmカバーガラス上の培養ニューロンを、撮像培地:120mM NaCl、3mM KCl、10mM HEPES、2mM CaCl2、2mM MgCl2、10mM D−グルコース、2%B27、0,001%BSAを含む閉じた撮像チャンバーに入れた。取得は70分間継続し、2分ごとに1つの画像を登録し、同じカバーガラス上に4個〜6個のニューロンを並行して登録した。取得開始から30分後、ビヒクル溶液又はODQ100μM(R&D Systems)を取得開始から40分後に適用し、DEA NONOate(Sigma)50μM又はForskolin(Sigma)10μMを適用した。
ImageJで画像を2つの部分に分割し、CFPチャネル及びYFPチャネルを分離した。次いで、1つ又は幾つかの目的の領域(ROI:Region Of Interest)の各時間点でのFRET比を計算することにより、Matlabでデータを分析した。ユーザーは、各位置のROIを定義した。各画像についてFRET比の値は(IY−BY)/(IC−BC)に対応し、式中、IYはYFPチャネルのROIの平均強度であり;BYは、YFPチャネルのバックグラウンドの平均強度であり;ICは、CFPチャネルのROIの平均強度であり;BCは、CFPチャネルのバックグラウンドの値である。各ROIについて、FRET比はベースライン平均によって正規化され、最初の治療注入前の7つの時間点として定義された。FRET比=100*(Rc−Ro)/Ro、式中、Rcは粗FRET比の値であり、Roはベースラインの平均である。各ニューロンコンパートメントで得られた定量結果を共にグループ化し、ベースライン及びSEMに対して正規化された平均FRET比を各時点で計算した。Matlab上の体細胞及び樹状突起の偏差を補正した。処理を追加する前の最後の7つの時間点で、体細胞及び樹状突起の全てのニューロンについて平均勾配をそれぞれ計算し、全てのFRET比率の時間点から差し引いた。
分析から除外したデータはない。サンプルサイズの計算は行わなかった。数匹の動物、カバーガラス、又は生化学的アッセイが同じ実験条件で分析されることが多いため、サンプルサイズは3つの再現性のある独立した実験の後に十分であると見なされ、n≧3に至った。動物又は培養物は同等であり、処理前は互いに区別できなかったため、形式的な無作為化プロセスを必要とせずに事実上サンプルを無作為化した。多くの場合、顕微鏡写真はその実験条件を目で容易に追跡できるため、データの盲検分析を達成することは困難である。注意深い盲検法が行われた場合、実験は、同一の実験条件での非盲検実験で得られた結果を再現した。Matlabを使用したThPDE5VVセンサーの検証を除いて、ImageJを使用して画像の計算及び分析を行った。統計学的検定を、GraphPad Prism(GraphPad Software Inc.)を使用して計算した。
本発明によるポリペプチドの例(本明細書で設計されたcGMPSp、cGMP Sponge、SponGee又はcGMPSp/SponGee)は、PKG−Iα及びPKG−Iβの一部を含むcGMP依存性プロテインキナーゼ(PKG)の高親和性キメラ変異体に基づいて設計された。cGMPSp/SponGeeは、結合部位とキメラ親和性ドメインを含み、キナーゼドメインを除外して下流のエフェクターの活性化を防ぐ(図1)。このコンストラクトを、cGMPSp/SponGee発現細胞の同定を容易にするために、蛍光タンパク質mRFPに更に融合させた。
Claims (15)
- ポリペプチドであって:
−配列番号1の配列及び配列番号2の配列に由来するキメラペプチドと;
−配列番号6の配列を含む又はそれからなるcGMP結合ドメインと;を含み、
−前記ポリペプチドは任意の触媒ドメイン及びその機能的変異体を欠いている、
ポリペプチド。 - 前記キメラペプチドが、配列番号3、配列番号4又は配列番号5、好ましくは配列番号3の配列、及びその機能的変異体からなるか又はそれらを含む、請求項1に記載のポリペプチド。
- 前記cGMP結合ドメインが、配列番号7の配列又は配列番号8の配列、及びその機能的変異体を含む又はそれからなる、請求項1又は2に記載のポリペプチド。
- 前記cGMP結合ドメインが配列番号9の配列又は配列番号10の配列、及びその機能的変異体を含む又はそれからなる、請求項1又は2に記載のポリペプチド。
- 前記キメラペプチド及び前記cGMP結合ドメインが隣接配列を形成し、更に好ましくは前記キメラペプチドのC末端が前記cGMP結合ドメインのN末端に融合している、請求項1〜4のいずれかに記載のポリペプチド。
- ペプチドシグナルを含む、請求項1〜5のいずれかに記載のポリペプチド。
- 配列番号7の配列又はその機能的変異体のタンデムリピートを含むペプチドシグナルを含む、請求項1〜6のいずれかに記載のポリペプチド。
- 好ましくは緑色蛍光タンパク質(GFP)、シアン蛍光タンパク質(CFP)、黄色蛍光タンパク質(YGP)、赤色蛍光タンパク質(RFP)及びそれらの変異体からなる一覧から選択される蛍光ペプチドを含む、請求項1〜6のいずれかに記載のポリペプチド。
- 請求項1〜8のいずれかに記載のポリペプチドをコードするポリヌクレオチド。
- 請求項9に記載のポリヌクレオチドを含む組み換えベクター。
- 請求項9に記載のポリヌクレオチド又は請求項10に記載の組み換えベクターで形質転換された宿主細胞又は非ヒト生物。
- 請求項1〜8のいずれかに記載の少なくとも1つのポリペプチド、請求項9に記載のポリヌクレオチド、請求項10に記載のベクター、及び/又は請求項11に記載の宿主細胞、並びに薬学的に許容される担体を含む医薬組成物。
- in vivo及び/又はin vitroにおける、好ましくはcGMP濃度の安定化及び/又はcGMPシグナル伝達の阻害のためのcGMPキレータとしての請求項1〜8に記載のいずれかのポリペプチド、請求項9に記載のポリヌクレオチド、又は請求項10に記載のベクターの非治療的使用。
- 医薬品として使用するための、請求項1〜8のいずれかに記載のポリペプチド、請求項9に記載のポリヌクレオチド、請求項10に記載のベクター、請求項11に記載の宿主細胞、又は請求項12に記載の組成物。
- 細胞内cGMPシグナル伝達機能障害に関連する病態、好ましくは網膜色素変性;好ましくは脳卒中、静脈血栓症又は動脈血栓症からなる一覧から選択される心血管疾患、;統合失調症;ハンチントン病;色覚異常;好ましくは結腸直腸癌、肺癌、肺疾患からなる一覧から選択される癌;勃起不全及び薬物乱用の予防及び/又は治療における使用のための請求項1〜8のいずれかに記載のポリペプチド、請求項9に記載のポリヌクレオチド、請求項10に記載のベクター、請求項11に記載の宿主細胞、又は請求項12に記載の組成物。
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