JP2004537037A - Methods for identifying small molecules that bind to specific RNA structural motifs - Google Patents

Abstract

The present invention relates to methods for screening and identifying test compounds that bind to a preselected target ribonucleic acid ("RNA"). A direct non-competitive binding assay is advantageously used to screen a library of compounds for compounds that selectively bind to a preselected target RNA. Binding of the target RNA molecule to a particular test compound may be detected using any physical method that measures a change in physical properties of the target RNA bound to the test compound. The structure of the test compound bound to the labeled RNA is also determined. The method used may depend in part on the nature of the library being screened. The method of the present invention provides a simple and sensitive assay for high-throughput screening of libraries of compounds to identify pharmaceutical lead compounds.

Description

（配列番号６）
【０１６４】
一般標的領域：
(1) 5'非翻訳領域-nts 1-152
(2) 3'非翻訳領域-nts 852-1643
初期特異的標的モチーフ：
3'非翻訳領域のグループＩ AU-リッチエレメント（ARE）クラスター
5' AUUUAUUUAUUUAUUUAUUUA 3'（配列番号1）
6.2. 顆粒球 - マクロファージコロニー刺激因子（「 GM-CSF 」）
GenBankアクセッション番号NM_000758：

（配列番号７）
【０１６５】
GenBankアクセッション番号XM_003751：

（配列番号８）
【０１６６】
一般標的領域：
(1) 5'非翻訳領域-nts 1-32
(2) 3'非翻訳領域-nts 468-78
初期特異的標的モチーフ：
3'非翻訳領域のグループＩAU-リッチエレメント（ARE）クラスター
5' AUUUAUUUAUUUAUUUAUUUA 3'（配列番号1）
6.3. インターロイキン２（「 IL-2 」）
GenBankアクセッション番号U25676：

（配列番号９）
【０１６７】
一般標的領域：
(1) 5'非翻訳領域-nts 1-47
(2) 3'非翻訳領域-nts 519-825
初期特異的標的モチーフ：
3'非翻訳領域のグループIII AU-リッチエレメント（ARE）クラスター
5'NAUUUAUUUAUUUAN 3' (配列番号１０)
6.4. インターロイキン６（「 IL-6 」）
GenBankアクセッション番号NM_000600：

（配列番号１１）
【０１６８】
一般標的領域：
(1) 5'非翻訳領域-nts 1-62
(2) 3'非翻訳領域-nts 699-1125
初期特異的標的モチーフ：
3'非翻訳領域のグループIIIAU-リッチエレメント（ARE）クラスター
5'NAUUUAUUUAUUUAN 3' (配列番号１０)
6.5. 血管内皮増殖因子（「 VEGF 」）
GenBankアクセッション番号AF022375:

（配列番号１２）
【０１６９】
一般標的領域：
(1) 5'非翻訳領域-nts 1-701
(2) 3'非翻訳領域-nts 1275-3166
初期特異的標的モチーフ：
(1) 5'非翻訳領域 nts 513-704中の内部リボソームエントリー部位（IRES）

（配列番号１３）
【０１７０】
(2) 3'非翻訳領域のグループIII AU-リッチエレメント（ARE）クラスター
5'NAUUUAUUUAUUUAN 3' (配列番号１０)
6.6. ヒト免疫不全ウイルス（「 HIV-1 」）
GenBankアクセッション番号NC_001802：

（配列番号１４）
【０１７１】
初期特異的標的モチーフ：
(1) トランス活性化応答領域／Tatタンパク質結合部位-TAR RNA-nts 1-60
「最小限」TAR RNA エレメント
5' GGCAGAUCUGAGCCUGGGAGCUCUCUGCC 3'（配列番号１５）
(2) Gag/Polフレームシフト部位-「最小限」フレームシフト エレメント
5' UUUUUUAGGGAAGAUCUGGCCUUCCUACAAGGGAAGGCCAGGGAAUUUUCUU 3'（配列番号１６）
6.7. Ｃ型肝炎ウイルス（「 HCV 」 - 遺伝子型 la および lb ）
GenBankアクセッション番号NC_001433：

（配列番号１７）
【０１７２】
一般標的領域：
5'非翻訳領域nts 1-328 - 内部リボソームエントリー部位（IRES）

（配列番号１８）
【０１７３】
初期特異的標的モチーフ：
(1) HCV IRES内のサブドメインIIIc-nts 213-226
5'AUUUGGGCGUGCCC3'（配列番号１９）
(2) HCV IRES内のサブドメインIIId-nts 241-267
5'GCCGAGUAGUGUUGGGUCGCGAAAGGC3'（配列番号２０）
6.8. リボヌクレアーゼＰ RNA （「 RN アーゼ P 」）
GenBankアクセッション番号
X15624 ホモサピエンス（Homo sapiens）RNアーゼP H1 RNA:

（配列番号２１）
【０１７４】
U64885 黄色ブドウ球菌（Staphylococcus aureus）RNアーゼP（rrnB）RNA：

（配列番号２２）
【０１７５】
M17569 大腸菌（Escherichia coli）リボヌクレアーゼＰ（rnpB）遺伝子のRNA成分（M1 RNA）：

（配列番号２３）
【０１７６】
Z70692 ヒト結核菌（Mycobacterium tuberculosis）RNアーゼP（rnpB）RNA:

（配列番号２４）
【０１７７】
6.9. アポトーシスタンパク質のＸ連鎖インヒビター（「 XIAP 」）
GenBankアクセッション番号U45880:

（配列番号２５）
【０１７８】
一般標的領域：
5'非翻訳領域の内部リボソームエントリー部位（IRES）：

（配列番号２６）
【０１７９】
初期特異的標的モチーフ：
XIAP IRES内のRNPコア結合部位
5'GGAUUUCCUAAUAUAAUGUUCUCUUUUU3'（配列番号２７）
6.10. サービビン（ Survivin ）
GenBankアクセッション番号NM_001168：

（配列番号２８）
【０１８０】
７. 実施例：低分子量化合物と結合した染料標識標的 RNA の同定
本実施例に報じた結果は、ゲル移動度シフトアッセイ（gel mobility shift assay）を利用してTatペプチドおよびゲンタマイシンなどの小分子とそれらのそれぞれの標的RNAとの結合を検出できるのを示す。
【０１８１】
7.1. 材料および方法
7.1.1. バッファー
トリス-塩化カリウム（TK）バッファーは、50mM トリス-HCl pH7.4、20mM KC1、0.1％Triton X-100、および0.5mM MgCl₂からなる。トリス-ホウ酸-EDTA（TBE）バッファーは、45mM トリス-ホウ酸 pH 8.0、および1mM EDTAからなる。トリス-塩化カリウムマグネシウム（TKM）バッファーは、50mM トリス-HCl pH7.4、20mM KC1、0.1％ Triton X-100および5mM MgCl₂からなる。
【０１８２】
7.1.1. ゲルシフト分析（ gel retardation analysis ）
RNAオリゴヌクレオチドは、Dharmacon Inc.（Lafayette, CO）から購入した。5'フルオレセイン標識した16S rRNA Ａ部位に対応するオリゴヌクレオチド（5'-GGCGUCACACCUUCGGGUGAAGUCGCC-3'（配列番号２９）；Moazed & Noller, 1987, Nature 327:389-394；Woodcockら, 1991, EMBO J. 10:3099-3103；Yoshizawaら, 1998, EMBO J. 17:6437-6448）、または5'フルオレセイン標識したHIV-1 TARエレメントTAR RNAに対応するオリゴヌクレオチド（5'-GGCGUCACACCUUCGGGUGAAGUCGCC-3'（配列番号３０）；Huqら, 1999, Nucleic Acids Research. 27:1084-1093；Hwangら, 1999, Proc. Natl. Acad. Sci. USA 96:12997-13002）のいずれかの500 pmolを、5'-³²Ｐシチジン3',5'-二リン酸（NEN）およびT4 RNAリガーゼ（NEBiolabs）を用いて10％DMSO中で、製造業者の指示の通り、3'標識した。標識したオリゴヌクレオチドを、G-25 セファデックス（Sephadex）カラム（Boehringer Mannheim）を用いて精製した。Tat-TARゲルシフト反応（gel retardation reaction）については、Huqらの方法（Nucleic Acids Research, 1999,27:1084-1093）を利用し、0.5mM MgCl₂および12量体のTatペプチド（YGRKKRRQRRRP（配列番号３１）；一文字アミノ酸コード）を含有するTKバッファーを用いた。16S rRNA-ゲンタマイシン反応については、Huqらの方法を利用し、TKMバッファーを用いた。20μl反応容積内で、³²Ｐシチジン標識したオリゴヌクレオチド50pmolと硫酸ゲンタマイシン（Sigma）または短かいTatペプチド（Tat_47-58）をTKまたはTKMバッファー中で90℃にて2分間加熱し、そして室温（ほぼ24℃）にて2時間にわたって冷却させた。次いで、30％グリセロール10μlをそれぞれの反応チューブに加え、そして全サンプルをTBE非変性ポリアクリルアミドゲル上に供給し、1200-1600ボルト-時間、4℃にて電気泳動した。ゲルを増感スクリーンに曝し、放射能をTyphoonホスホルイメージャー（Molecular Dynamics）を用いて定量した。
【０１８３】
7.2. 背景
小分子とリボソームなどの天然RNA構造との相互作用を実証するために利用される１つの方法は、化学フットプリントまたはトウプリント（toe printing）と呼ばれる方法である（Moazed & Noller, 1987, Nature 327:389-394；Woodcockら, 1991, EMBO J. 10:3099-3103；Yoshizawaら, 1998, EMBO J. 17:6437-6448）。本明細書では、RNA-小分子相互作用をモニターするための、ゲル移動度シフトアッセイの利用を記載する。この手法は、RNA単独と小分子-RNA複合体とを対比してその移動度の差に基づいて、小分子-RNA相互作用の高速可視化を可能にする。この手法を確証するために、よく特性決定されている16S rRNA上のゲンタマイシン結合部位に対応するRNAオリゴヌクレオチド（Moazed & Noller, 1987, Nature 327 :389-394）および同じくよく特性決定されているHIV-1 TAR エレメント上のHIV-1 TAT タンパク質結合部位（Huqら, 1999, Nucleic Acids Res. 27:1084-1093）を選んだ。これらの実験の目的は、薬物発見のためにマイクロキャピラリー電気泳動などのハイスループット方式のクロマトグラフィ技術を利用する基礎を築くことにある。
【０１８４】
7.3. 結果
ゲルシフトアッセイを、Tat_47-58ペプチドおよびTAR RNAオリゴヌクレオチドを用いて実施した。図１に示したように、生成物を12％非変性ポリアクリルアミドゲル上で分離すると、Tatペプチドの存在のもとでは明確なシフトが見られる。ペプチドを欠く反応では、遊離RNAだけが見られる。これらの観察は、他のTatペプチドを用いて行われた今までの報告を確認するものである（Hamyら, 1997, Proc. Natl. Acad. Sci. USA 94:3548-3553；Huqら, 1999, Nucleic Acids Res. 27:1084-1093）。
【０１８５】
図１の結果に基づいて、小分子とのRNA相互作用も、この方法を用いて可視化しうるという仮説をたてた。図２に示したように、様々な濃度のゲンタマイシンを16S rRNA Ａ部位に対応するRNAオリゴヌクレオチドに添加すると、移動度シフトを生じる。これらの結果は、小分子ゲンタマイシンの溶液中の規定された構造を有するRNAオリゴヌクレオチドとの結合は、この手法を利用してモニターできることを実証する。さらに、図２に示したように、10ng/mlの低いゲンタマイシンの濃度が移動度シフトを生じる。
【０１８６】
さらに低い濃度のゲンタマイシンでもゲルシフトを生じるのに十分であるかどうかを確認するために、ゲンタマイシンの濃度が100ng/ml〜10pg/mlの範囲であることを除くと図２に示したのと同様である実験を実施した。図３に示したように、ゲル移動度シフトは、ゲンタマイシン濃度が10pg/mlまで低いときでも生じる。さらに、図３に示した結果はシフトが16S rRNAオリゴヌクレオチドに特異的であることを実証し、HIV TAR RNAエレメントに対応する無関係なオリゴヌクレオチドを使用すると10pg/mlゲンタマイシンとインキュベートしてもゲル移動度シフトを生じない。さらに、もし10pg/mlの低濃度のゲンタマイシンがゲル移動度シフトを生じるのであれば、少量の化合物を多様な化合物のライブラリーからこの方式でスクリーニングすると、RNA構造モチーフに対する変化を検出することが可能であるに違いない。
【０１８７】
ゲンタマイシン-RNA相互作用のさらなる解析は、相互作用がMgおよび温度に依存性であることを示した。図４に示したように、MgCl₂が存在しないとき（TK バッファー）、ゲルシフトを生じるためには、反応液に1mg/mlのゲンタマイシンを加えなければならない。
【０１８８】
同様に、ゲンタマイシンを加えるときの反応温度も重要である。変性／再生サイクル全体においてゲンタマイシンが反応液に存在すると、すなわち、ゲンタマイシンを90℃または85℃に加えると、ゲルシフトが見られる（データは示してない）。対照的に、ゲンタマイシンを再生ステップが75℃まで進行した後に加えると、移動度シフトは生じない。これらの結果は、ゲンタマイシンが再生プロセスの初期に形成されるRNA構造を認識しかつそれと相互作用しうるという考えと一致する。
【０１８９】
８. 実施例：キャピラリー電気泳動による、小分子化合物と結合した染料標識標的 RNA の同定
本実施例に報じた結果は、TatペプチドとTAR RNAのようなペプチドとその標的RNAの間の相互作用は、自動キャピラリー電気泳動システムのゲルシフトアッセイによりモニターすることができることを示す。
【０１９０】
8.1. 材料と方法
8.1.1. バッファー
トリス-塩化カリウム（TK）バッファーは、50mM トリス-HCl pH7.4、20mM KC1、0.1％ Triton X-100、および0.5mM MgCl₂からなる。トリス-ホウ酸-EDTA（TBE）バッファーは、45mM トリス-ホウ酸 pH 8.0、および1mM EDTAからなる。トリス-塩化カリウムマグネシウム（TKM）バッファーは、50mM トリス-HCl pH7.4、20mM KC1、0.1％ Triton X-100および5mM MgCl₂からなる。
【０１９１】
8.1.1. キャピラリー電気泳動を用いるゲルシフト分析
RNAオリゴヌクレオチドは、Dharmacon Inc.（Lafayette, CO）から購入した。5'フルオレセイン標識したHIV-1 TARエレメントTAR RNA（5'-GGCGUCACACCUUCGGGUGAAGUCGCC-3'（配列番号３０）；Huqら, 1999, Nucleic Acids Research. 27:1084-1093；Hwangら, 1999, Proc. Natl. Acad. Sci. USA 96:12997-13002）に対応するオリゴヌクレオチド500pmolを用いた。Tat-TARゲルシフト反応については、Huqらの方法（Nucleic Acids Research, 1999,27:1084-1093）を利用し、0.5mM MgCl₂および12量体のTatペプチド（YGRKKRRQRRRP（配列番号31）；一文字アミノ酸コード）を含有するTKバッファーを用いた。20μl反応容積内で、標識したオリゴヌクレオチド50pmolと短かいTatペプチド（Tat_47-58）とをTKまたはTKMバッファー中で90℃にて2分間加熱し、そして室温（ほぼ24℃）に2時間にわたって冷却させた。反応液をSCE9610自動キャピラリー電気泳動装置（SpectruMedix; State College, Pennsylvania）上に供給した。
【０１９２】
8.2. 結果
先の第７節の実施例において報じたように、ペプチドとRNAとの間の相互作用は、ゲルシフトアッセイによりモニターすることができる。ペプチドとRNAとの間の相互作用は、ゲルシフトアッセイにより自動キャピラリー電気泳動システムによりモニターしうるという仮説をたてた。この仮説を試験するために、自動キャピラリー電気泳動システムによるゲルシフトアッセイを、Tat_47-58ペプチドとTAR RNAオリゴヌクレオチドを用いて実施した。図５に示したように、Tatペプチドの存在のもとでキャピラリー電気泳動システムを用いて、添加するTatペプチドの濃度が増加すると明確なシフトが見られる。ペプチドを欠く反応物においては、遊離RNAに対応するピークだけが観察される。これらの観察は、Tatペプチドを用いて行われた今までの報告を確証した（Hamyら, 1997, Proc. Natl. Acad. Sci. USA 94:3548-3553；Huqら, 1999, Nucleic Acids Res. 27:1084-1093）。
【０１９３】
本発明は本明細書に記載の特定の実施例により範囲が限定されるものでない。実際、当業者には、これらの記載に加えて上記の説明と添付図面から、本発明の様々な改変が明らかであろう。かかる改変は添付した請求の範囲内に入ると意図している。
【０１９４】
様々な出版物を本明細書に引用したが、これらの開示は参照によりその全文が組み入れられる
本発明は以下の番号を付したパラグラフに挙げられた次の実施形態により説明することができる。
【０１９５】
１. 標的RNA分子と結合する試験化合物を同定する方法であって、(a)検出可能に標識された標的RNA分子と試験化合物のライブラリーを、標識された標的RNAと試験化合物のライブラリーのメンバーとの直接結合を可能にする条件下で接触させて、検出可能に標識された標的RNA：試験化合物複合体を形成するステップ；(b)ステップ(a)において形成した検出可能に標識された標的RNA：試験化合物複合体を、複合体形成していない標的RNA分子および試験化合物から分離するステップ；ならびに(c)RNA:試験化合物複合体中のRNAと結合した試験化合物の構造を決定するステップを含んでなる前記方法。
【０１９６】
２. 標的RNA分子がHIV TARエレメント、内部リボソームエントリー部位、「スリッパリー部位」、不安定性エレメント、またはアデニル酸ウリジル酸リッチエレメントを含有する、パラグラフ１の方法。
【０１９７】
３. RNA分子が、腫瘍壊死因子α（「TNF-α」）、顆粒球-マクロファージコロニー刺激因子（「GM-CSF」）、インターロイキン２（「IL-2」）、インターロイキン６（「IL-6」）、血管内皮増殖因子（「VEGF」）、ヒト免疫不全ウイルスI（「HIV-1」）、Ｃ型肝炎ウイルス（「HCV」-遺伝子型1aおよび1b）、リボヌクレアーゼＰ RNA（「RNアーゼP」）、アポトーシスタンパク質のＸ連鎖インヒビター（「XIAP」）、またはサービビンに対するmRNAから誘導されたエレメントである、パラグラフ１の方法。
【０１９８】
４. 検出可能に標識されたRNAが蛍光染料、リン光染料、紫外染料、赤外染料、可視染料、放射能標識、酵素、分光比色標識、アフィニティタグ、またはナノ粒子を用いて標識された、パラグラフ１の方法。
【０１９９】
５. 試験化合物がペプトイド；ランダムバイオオリゴマー；ヒダントイン、ベンゾジアゼピンおよびジペプチドなどのダイバーソマー（diversomer）；ビニログポリペプチド；非ペプチド性ペプチドミメチック；オリゴカーバメート；ペプチジルホスホネート；ペプチド核酸ライブラリー；抗体ライブラリー；または炭水化物ライブラリー；ならびに、限定されるものでないが、ベンゾジアゼピン、イソプレノイド、チアゾリジノン、メタチアザノン、ピロリジン、モルホリノ化合物、またはジアゼピンジオンなどのライブラリーを含む小有機分子ライブラリーを含んでなるコンビナトリアルライブラリーから選択される、パラグラフ１の方法。
【０２００】
６. 試験化合物のライブラリーのスクリーニングが、試験化合物を、好ましくは生理条件を近似するかまたは模倣するバッファーと塩類の組み合わせを含む水溶液の存在のもとで標的核酸と接触させることを含んでなる、パラグラフ１の方法。
【０２０１】
７. 前記水溶液が場合によってはさらに、DNA、酵母tRNA、サケ精子DNA、ホモリボポリマー、および非特異的RNAを含む非特異的核酸を含んでなる、パラグラフ６の方法。
【０２０２】
８. 前記水溶液がさらにバッファー、塩類の組み合わせ、および場合によっては、洗剤または界面活性剤を含んでなる、パラグラフ６の方法。他の実施形態においては、前記水溶液はさらに、約0mM〜約100mM KCl、約0mM〜約1M NaCl、および約0mM〜約200mM MgCl₂の塩類の組み合わせを含んでなる。好ましい実施形態においては、塩類の組み合わせは約100mM KCl、約500mM NaCl、および約10mM MgCl₂である。他の実施形態においては、前記溶液は、場合によっては、約0.01％〜約0.5％（w/v）の洗剤または界面活性剤を含んでなる。
【０２０３】
９. 試験化合物と複合体形成した標的核酸の物理的特性の非結合標的核酸からの改変を検出するいずれの方法を利用して、パラグラフ１の方法における複合体形成した標的核酸と複合体形成していない標的核酸の分離を行ってもよい。好ましい実施形態においては、電気泳動を利用して複合体形成した標的核酸と複合体形成していない標的核酸の分離を行う。好ましい実施形態においては、電気泳動は、キャピラリー電気泳動である。他の実施形態においては、蛍光分光法、表面プラズモン共鳴、質量分析、シンチレーション近接アッセイ、NMR分光法による構造活性相関（「SAR」）、サイズ排除クロマトグラフィ、アフィニティクロマトグラフィ、およびナノ粒子凝集を利用して複合体形成した標的核酸と複合体形成していない標的核酸の分離を行う。
【０２０４】
10. パラグラフ１のRNA:試験化合物複合体の試験化合物の構造を、部分的に、試験化合物のライブラリーのタイプにより決定する。コンビナトリアルライブラリーが小有機分子ライブラリーである好ましい実施形態においては、質量分析、NMR、または振動分光法を利用して試験化合物の構造を決定する。
【図面の簡単な説明】
【０２０５】
【図１】図１は、ペプチド-RNA相互作用を検出するためのゲルシフト分析を示す。TKバッファー中で、増加する濃度（0.1μM、0.2μM、0.4μM、0.8μM、1.6μM）のTat_47-58ペプチドを含有する20μl反応液に、50pmol TAR RNAオリゴヌクレオチドを加えた。次いで反応混合物を90℃にて2分間加熱し、徐々に24℃まで冷却した。30％グリセロール10mlをそれぞれのサンプルに加え、12％無変性ポリアクリルアミドゲルに適用した。ゲルをTBEバッファー中で4℃にて1200ボルト-時間の条件で電気泳動した。電気泳動の後に、ゲルを乾燥しかつ放射能をホスホルイメージャーを用いて定量した。加えたペプチドの濃度をそれぞれのレーンの上方に示した。
【図２】図２は、ゲンタマイシンが16S rRNAに対応するオリゴヌクレオチドと相互作用することを示す。TKMバッファー中で、増加する濃度（1ng/ml、10ng/ml、100ng/ml、1μg/ml、10μg/ml、50μg/ml、500μg/ml）のゲンタマイシンを含有する20μl反応液を、RNAオリゴヌクレオチド50pmolに加え、90℃にて2分間加熱し、徐々に24℃まで冷却した。次いで30％グリセロール10mlをそれぞれのサンプルに加え、13.5％無変性ポリアクリルアミドゲルに適用した。ゲルをTBEバッファー中で4℃にて1200ボルト-時間の条件で電気泳動した。電気泳動の後に、ゲルを乾燥しかつ放射能をホスホルイメージャーを用いて定量した。加えたゲンタマイシンの濃度をそれぞれのレーンの上方に示した。
【図３】図３は、ゲンタマイシン10pg/mlの存在が16S rRNAオリゴヌクレオチドの存在でのゲル移動度シフトを生じることを示す。増加する濃度（100ng/ml、10ng/ml、1ng/ml、100pg/ml、および10pg/ml）のゲンタマイシンを含有する20μl反応液を、TKMバッファー中のRNAオリゴヌクレオチド50pmolに加え、図２に記載のように処理した。
【図４】図４は、MgCl₂の不在のもとでは、ゲンタマイシンと16S rRNAオリゴヌクレオチドとの結合が弱いことを示す。ゲンタマイシン（1mg/ml、100μg/ml、10μg/ml、1μg/ml、0.1μg/ml、および10ng/ml）を含有する反応混合物を、TKMバッファーがMgCl₂を含有しないことを除いて、図２に記載したように処理した。
【図５】図５は、ペプチド-RNA相互作用を検出するためのゲルシフト分析である。TKバッファー中で、増加する濃度（0.1μM、0.2μM、0.4μM、0.8μM、1.6μM）のTat_47-58ペプチドを含有する反応液に、TAR RNAオリゴヌクレオチド50pmolを加えた。次いで反応混合物を90℃にて2分間加熱し、徐々に24℃まで冷却した。反応液をSCE9610自動キャピラリー電気泳動装置（SpectruMedix；State College、Pennsylvania）に供給した。ピークは遊離TAR RNA（「TAR」）またはTat-TAR複合体（「Tat-TAR」）の量に対応する。加えたペプチドの濃度をそれぞれのレーンの下方に示した。【Technical field】
[0001]
1.Foreword
The present invention relates to methods for screening and identifying test compounds that bind to a preselected target ribonucleic acid ("RNA"). A direct non-competitive binding assay is advantageously used to screen a library of compounds for compounds that selectively bind to a preselected target RNA. Binding of the target RNA molecule to a particular test compound is detected using any physical method that measures a change in the physical properties of the target RNA bound to the test compound. The method of the present invention provides a simple and sensitive assay for identifying drug lead compounds by high-throughput screening of a library of compounds.
[Background Art]
[0002]
2.Background of the Invention
Protein-nucleic acid interactions are involved in a number of cellular functions, including transcription, RNA splicing, mRNA decay, and mRNA translation. Easily accessible synthetic molecules that can bind with high affinity to specific sequences of single- or double-stranded nucleic acids have the potential to interfere with these interactions in a controllable manner, and Attractive as a scientific and medical tool. Successful methods of blocking the function of target nucleic acids include duplex-forming antisense oligonucleotides (Miller, 1996, Progress in Nucl. Acid Res. & Mol. Biol. 52: 261-291; Ojwang & Rando, 1999, “Using type I tumor necrosis factor receptor, N₇Achieving antisense inhibition by oligodeoxynucleotides containing N₇ modified 2'-deoxyguanosine using tumor necrosis factor receptor type 1, METHODS): A Companion to Methods in Enzymology 18: 244-251) and peptide nucleic acids that bind to nucleic acids via Watson-Crick base pairing ("PNA") ) (Nielsen, 1999, Current Opinion in Bioteclmology 10: 71-75). Triplex forming anti-gene oligonucleotides (Ping et al., 1997, RNA 3: 850-860; Aggarwal et al., 1996, Cancer Res. 56: 5156-5164; U.S. Patent No. 5,650,316); Designing pyrrole-imidazole polyamide oligomers (Gottesfeld et al., 1997, Nature 387: 202-205; White et al., 1998, Nature 391: 468-471) that are each specific for major and minor grooves. You can also.
[0003]
In addition to synthetic nucleic acids (ie, antisense, ribozymes, and triple helix-forming molecules), there are examples of deoxyribonucleic acids ("DNA"), such as transcription or translation, or natural products that interfere with RNA processes. For example, calicheamicin oligosaccharide, a specific carbohydrate-based host cell factor, inhibits in vivo transcription by interfering with sequence-specific binding of transcription factors to DNA (Ho et al., 1994, Proc. Natl. Acad. Sci. USA 91: 9203-9207; Liu et al., 1996, Proc. Natl. Acad. Sci. USA 93: 940-944). Certain antibiotic classes have been characterized and found to interact with RNA. For example, the antibiotic thiostreptone binds tightly to 60-mers derived from ribosomal RNA (Cundliffe et al., 1990, in "The Ribosome: Structure, Function & Evolution"). Schlessinger et al., Eds.) American Society for Microbiology, Washington, DC pp.479-490). Bacterial resistance to various antibiotics often involves methylation at specific rRNA sites (Cundliffe, 1989, Ann. Rev. Microbiol. 43: 207-233). Aminoglycosidic aminocyclitol (aminoglycoside) and peptide antibiotics are known to suppress group I intron splicing by binding to specific regions of RNA (von Ahsen et al., 1991, Nature (London) 353). : 368-370). Several of these aminoglycosides have also been found to inhibit hammerhead ribozyme function (Stage et al., 1995, RNA 1: 95-101). In addition, certain aminoglycosides and other protein synthesis inhibitors have been found to interact with specific bases of 16S rRNA (Woodcock et al., 1991, EMBO J. 10: 3099-3103). Oligonucleotide analogs of 16S rRNA have also been shown to interact with certain aminoglycosides (Purohit et al., 1994, Nature 370: 659-662). One molecular basis of hypersensitivity to aminoglycosides has been found to be located at single base changes in mitochondrial rRNA (Hutchin et al., 1993, Nucleic Acids Res. 21: 4174-4179). Aminoglycosides have also been shown to suppress the interaction between certain structural RNA motifs and their corresponding RNA binding proteins. Zapp et al. (Cell, 1993, 74: 969-978) report that the aminoglycosides neomycin B, lividomycin A, and tobramycin are compatible with Rev (a viral regulatory protein required for viral gene expression) and HIV RNA IIB (or RRE). ) Demonstrates that the region can block binding to viral recognition elements. This block appears to be the result of direct competitive binding of the antibiotic with the RRE RNA structural motif.
[0004]
Single-stranded sections of RNA can fold into complex tertiary structures consisting of local motifs such as loops, bulges, pseudoknots, guanosine quartets, and turns (Chastain & Tinoco, 1991, Progress in Nucleic Acid Res. & Mol. Biol. 41: 131-177; Chow & Bogdan, 1997, Chemical Reviews 97: 1489-1514; Rando & Hogan, 1998, "Oligo Forming Guanosine Quadruple. "Biologic activity of guanosine quartet forming oligonucleotides" in "Applied Antisense Oligonucleotide Technology" Stein. & Krieg (ed.) John Wiley and Sons, New York, pp. 335-352 ). Such a structure is crucial to the activity of the nucleic acid and can affect functions such as controlling mRNA transcription, stability, or translation (Weeks & Crothers, 1993, Science 261: 1574-1577). Because these functions depend on the natural three-dimensional structural motif of single-stranded nucleic acids, the general and easy-to-use sequence specificity of double and triple helix nucleic acid formation used in the design of antisense and ribozyme-type molecules It is difficult to identify or design synthetic drugs that bind to these motifs using statistical recognition rules. Screening techniques generally involve identifying compounds that disrupt the interaction between the target RNA and a physiological host cell factor that has been previously identified to specifically interact with a particular target RNA. Involved in the designed competition assay. Generally, such assays require the identification and characterization of host cell factors that may be required for the function of the target RNA. In the assay, both the target RNA and its preselected host cell binding partner are used in a competitive format to identify compounds that disrupt or interfere with these two components.
[0005]
Citation or confirmation of any reference in Section 2 of this application is not an admission that such reference is available as prior art to the present invention.
DISCLOSURE OF THE INVENTION
[0006]
3.Summary of the Invention
The present invention relates to a method for identifying a compound that binds to a target element of a preselected nucleic acid, including but not limited to a particular RNA sequence, RNA structural motif, and / or RNA structural element. Small molecules are screened using specific target RNA sequences, RNA structural motifs and / or RNA structural elements as targets, and small molecules that bind to these specific sequences, motifs and / or structural elements are identified. For example, a method of screening a library of test compounds using a preselected target RNA having a detectable label, preferably under physiological conditions, is described. Any complexes formed between the target RNA and members of the library are identified using a physical method that detects a change in the physical properties of the target RNA bound to the test compound. In particular, the present invention relates to a method of screening a library of test compounds released in solution, in labeled test tubes or in microtiter plates, or in microarrays, using target RNA having a detectable label. . Compounds in the library that bind to the labeled target RNA will form a detectable complex. The detectably labeled conjugate can then be converted from uncomplexed, unlabeled test compounds in the library and from uncomplexed, labeled target RNA to, but not limited to, It can be identified and removed by a variety of methods, including electrophoresis, chromatography, or methods of identifying changes in thermostable properties. Such methods include, but are not limited to, electrophoresis, fluorescence spectroscopy, surface plasmon resonance, mass spectrometry, scintillation proximity assay, structure activity relationship by NMR spectroscopy ("SAR"), size exclusion chromatography, affinity chromatography, and Particle aggregation. The structure of the test compound bound to the labeled RNA is then determined. The method may depend, in part, on the nature of the library being screened. For example, an array or microarray of test compounds, each having an address or identifier, can be ascertained, for example, by referencing the original compound list in which positive samples were applied to each test assay. Other methods of identifying test compounds include de novo structure determination of the test compound using mass spectrometry or nuclear magnetic resonance ("NMR"). The identified test compound is useful for any purpose that can utilize the binding reactant as an inhibitor of target molecule function, as a probe, as a sequestrant, etc., for example, in assay methods, diagnostic methods, and cell sorting. . In addition, small organic molecules that specifically interact with target RNA molecules may be useful as lead compounds in therapeutic drug development.
[0007]
The methods described herein for identifying compounds that bind directly to a particular preselected target RNA are suitable for high-throughput screening. The direct binding method of the present invention provides advantages over natural RNA binding proteins: drug screening systems for competitors that suppress target RNA complex formation; ie, competitive assays. The direct binding method of the present invention is rapid and can be easily prepared for implementation, for example by a technician, by adapting it to high-throughput screening. The methods of the present invention also eliminate the bias inherent in competitive drug screening systems that require the use of preselected host cell factors that may not have a physiological relevance to the activity of the target RNA. Instead, the methods of the present invention are utilized to identify any compound that can directly bind to a particular target RNA sequence, RNA structural motif and / or RNA structural element, preferably under physiological conditions. . As a result, the compounds thus identified can inhibit the interaction of the target RNA with any one or more natural (known or unknown) host cell factors required for RNA activity in vivo. .
[0008]
The present invention will be more fully understood by reference to the detailed description and examples which are intended to illustrate non-limiting embodiments of the invention.
[0009]
3.1.Definition
As used herein, the term “target nucleic acid” refers to RNA, DNA, or a chemically modified variant thereof. In a preferred embodiment, the target nucleic acid is RNA. Target nucleic acid also refers to the tertiary structure of nucleic acids, such as, but not limited to, loops, bulges, pseudoknots, guanosine quads, and turns. Target nucleic acids also include, but are not limited to, HIV TAR elements, internal ribosome entry sites, "slippery sites", instability elements, and adenylate uridylic acid-rich elements, such as Also means RNA elements, which are described in section 5.1. Non-limiting examples of target nucleic acids are reported in Sections 5.1 and 6.
[0010]
As used herein, the term "library" refers to a plurality of test compounds that are contacted with a target nucleic acid molecule. The library may be a combinatorial library, for example, a collection of test compounds synthesized using combinatorial chemistry techniques, or a collection of unique chemicals of low molecular weight (less than 1000 daltons) each occupying a unique three-dimensional space. .
[0011]
As used herein, the term "label" or "detectable label" refers to a composition that is directly or indirectly detectable by spectroscopy, photochemistry, biochemistry, immunochemistry, or chemical methods. I do. For example, useful labels include radioisotopes (eg,³²P,³⁵S, and^ThreeH), dyes, fluorescent dyes, electron-dense reagents, enzymes and their substrates (eg, alkaline phosphatase and horseradish peroxidase commonly used in enzyme-linked immunoassays), biotin-streptavidin, Haptens and proteins for which digoxigenin, or antisera or monoclonal antibodies thereto, are available. In addition, as a label or detectable moiety, an "affinity tag" that, when coupled with a target nucleic acid and incubated with a test compound or compound library, allows the affinity capture of molecules bound to the target nucleic acid along with the target nucleic acid Is mentioned. One skilled in the art will appreciate that an affinity tag attached to a target nucleic acid, by definition, has a complementary ligand coupled to a solid support that allows its capture. For example, useful affinity tags and complementary partners include, but are not limited to, biotin-streptavidin, complementary nucleic acid fragments (eg, oligo dT-oligo dA, oligo T-oligo A, oligo dG-oligo dC, Oligo G-oligo C), aptamers, or haptens and proteins for which antisera or monoclonal antibodies are available. The label or detectable moiety is typically attached to the molecule to be detected, either covalently through a linker or chemical bond or through ionic, van der Waals or hydrogen bonds.
[0012]
As used herein, the term "dye" refers to a molecule that, upon exposure to radiation, emits radiation at a detectable level, either visually or via conventional spectroscopic means. As used herein, the term "visible dye" has a chromophore that absorbs radiation in the visible region of the spectrum (i.e., at a wavelength of about 400 nm to about 700 nm), such that irradiation of the transmitted visible region is visual or normal. Means a molecule that can be detected by spectroscopy. As used herein, the term "ultraviolet dye" refers to a molecule having a chromophore that absorbs radiation in the ultraviolet region of the spectrum (i.e., at a wavelength of about 30 nm to about 400 nm). As used herein, the term "infrared dye" refers to a molecule having a chromophore that absorbs radiation in the infrared region of the spectrum (i.e., at a wavelength of about 700 nm to about 3,000 nm). A "chromophore" is a network of dye atoms that, upon exposure to radiation, emits radiation at levels detectable either visually or via conventional spectroscopy. Those skilled in the art will readily appreciate that dyes absorb radiation in one region of the spectrum, but may emit radiation in other regions of the spectrum. For example, ultraviolet dyes can emit radiation in the visible region of the spectrum. Those skilled in the art will also readily appreciate that dyes can emit radiation via fluorescence or phosphorescence.
[0013]
The phrase “pharmaceutically acceptable salt” as used herein includes, but is not limited to, salts of acidic or basic groups that may be present in a test compound identified using the methods of the present invention. . Test compounds that are basic in nature are capable of forming various salts with various inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., those that contain a pharmaceutically acceptable anion, and The salts of, but are not limited to, sulfuric acid, citric acid, maleic acid, acetic acid, oxalic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, hydrogen sulfate, phosphoric acid, acidic phosphoric acid, Isonicotine, acetic acid, lactic acid, salicylic acid, citric acid, acidic citric acid, tartaric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, succinic acid, maleic acid, genicic acid, fumaric acid, gluconic acid, glucaronate ( glucaronate), saccharate, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfone And pamoate (pamoate) (i.e., 1,1'-methylene - (2-hydroxy-3-naphthoate) - bis) comprises a salt. Test compounds that contain an amino moiety may form pharmaceutically or cosmetically acceptable salts with various amino acids, in addition to the acids mentioned above. Test compounds that are acidic in nature are capable of forming salts with various pharmaceutically or cosmetically acceptable cations. Examples of such salts include the alkali metal or alkaline earth metal salts, and especially the calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.
[0014]
As used herein, "substantially one type of test compound" refers to a situation in which an assay requires, at some point, only one compound in each reaction, if the result is that the target RNA molecule and the test compound Demonstrating that a binding event occurs between and means that the test compound can be performed in a readily identifiable manner.
[0015]
4.Description of the drawings
See below for a brief description of the figures.
BEST MODE FOR CARRYING OUT THE INVENTION
[0016]
5.DETAILED DESCRIPTION OF THE INVENTION
The present invention is not limited to compounds that bind to a preselected nucleic acid, including, but not limited to, a sequencing structural motif of a preselected target RNA, or a structural element, particularly a target element of RNA. And a method for identifying A method for screening a library of test compounds using a preselected target RNA having a detectable label is described. Any complexes formed between the target RNA and members of the library are identified using a physical method that detects a change in the physical properties of the target RNA bound to the test compound. Changes in physical properties of the RNA-test compound complex relative to the target RNA or test compound include, but are not limited to, changes in mobility, changes in charge, or changes in thermal stability due to changes in mass Can be measured by a method such as Such methods include, but are not limited to, electrophoresis, fluorescence spectroscopy, surface plasmon resonance, mass spectrometry, scintillation proximity assay, structure activity relationship by NMR spectroscopy ("SAR"), size exclusion chromatography, affinity chromatography, And nanoparticle aggregation. In particular, the present invention utilizes target RNAs having a detectable label to screen libraries of test compounds released in solution in labeled tubes or in microtiter plates or in microarrays. About the method. Compounds in the library that bind to the labeled target RNA will produce a detectably labeled complex. The detectably labeled complex is then identified by various methods that can identify changes in the physical properties of the complexed target RNA and the unlabeled uncomplexed test compound in the library. Can be taken from The structure of the test compound bound to the labeled RNA is also determined. The method used may depend in part on the nature of the library being screened. For example, an array or microarray of test compounds, each having an address or identifier, can be elucidated, for example, by cross-referencing a positive sample with the original list of compounds applied to the individual test assay. Other methods of identifying test compounds include de novo structure determination of the test compound using mass spectrometry or nuclear magnetic resonance ("NMR").
[0017]
Accordingly, the method of the present invention provides a method for enhancing a library of test compounds, wherein the library of test compounds is characterized by readily identifying test compounds of the library that specifically bind to a preselected target nucleic acid from unbound members of the library. Provides a simple and sensitive assay for throughput screening. The structure of the binding molecule is decoded from the input library in a manner that depends on the type of library used. The test compound thus identified is useful for any purpose utilizing a binding reaction. For example, in assay methods, diagnostic methods, cell sorting, as an inhibitor of target molecule function, as a probe, as a blocking agent, It is also useful as a lead compound for therapeutic drug development. Small organic compounds identified to interact specifically with target RNA molecules are particularly attractive candidate compounds as lead compounds for therapeutic drug development.
[0018]
The assays of the present invention reduce the bias introduced by competitive binding assays that require the identification and use of host cell factors (perhaps essential for modulating RNA function) as binding partners for target RNA. The assays of the invention are designed to detect any compound or agent that binds to the target RNA, preferably under physiological conditions. Such agents can then be tested for biological activity without establishing or estimating which host cell factors are required to modulate the function and / or activity of the target RNA.
[0019]
Section 5.1 describes examples of protein-RNA interactions that are important for various cellular functions and multiple target RNA elements that can be used to identify test compounds. Compounds that suppress these interactions by binding to RNA and that successfully compete with native proteins or host cell factors that bind endogenously to RNA include, for example, diseases or diseases such as infection or unchecked growth. It may be important in treating or preventing abnormal symptoms. Section 5.2 describes detectable labels for target nucleic acids that are useful in the methods of the invention. Section 5.3 describes a library of test compounds. Section 5.4 provides conditions for binding a labeled target RNA to a test compound and detecting RNA that binds to the test compound using the method of the present invention. Section 5.5 provides a method for separating complexes of target RNA bound to a test compound from unbound RNA. Section 5.6 describes a method for identifying a test compound that has bound to a target RNA. Section 5.7 describes the secondary, biological screening of test compounds identified by the methods of the present invention for testing the effects of test compounds in vivo. Section 5.8 describes the use of a test compound identified according to the methods of the present invention to treat or prevent a disease or condition in a mammal.
[0020]
5.1.Biologically important RNA- Host cell factor interaction
Nucleic acids, especially RNA, can fold into complex tertiary structures, including bulges, loops, triple helices, and pseudoknots, providing binding sites for host cell factors such as proteins and other RNA. Can. RNA-protein and RNA-RNA interactions are important for a variety of cellular functions, including transcription, RNA splicing, RNA stability and translation. In addition, binding of such host cell factors to RNA can alter the stability and translation efficiency of such RNA, and thus affect subsequent translation. For example, several diseases are associated with protein overproduction or reduced protein function. In this case, the identification of compounds that modulate RNA stability and translation efficiency may be useful for treating and preventing such diseases.
[0021]
The methods of the invention are useful for identifying test compounds that bind to a target RNA element in a high-throughput screening assay of a library of test compounds in solution. In particular, the methods of the present invention are useful for identifying test compounds that bind to a target RNA element in vivo and inhibit the interaction of that RNA with one or more host cell factors. Molecules identified by the methods of the invention are useful for inhibiting the formation of specific bound RNA: host cell factor complexes in vivo.
[0022]
In some embodiments, the test compound identified by the method of the present invention binds to a messenger RNA by binding to one or more regulatory elements in the 5 'untranslated region, 3' untranslated region, or coding region of the mRNA. ("MRNA") is useful for translation, eg, to increase or decrease protein production. Compounds that bind to mRNA, among others, increase or decrease the rate of mRNA processing, alter the transport of mRNA through cells, block or enhance the binding of mRNA to ribosomes, suppressor proteins or enhancer proteins, or maintain mRNA stability. Can be modified. Thus, compounds that increase or decrease mRNA translation can be used to treat or prevent disease. For example, diseases associated with overproduction of a protein, such as amyloidosis, or associated with the production of a mutant protein, such as Ras, may result in reduced translation of the mRNA encoding the overproduced protein to suppress protein production. Can be treated or prevented. Conversely, the symptoms of diseases associated with impaired protein function, such as hemophilia, may be due to increased translation of impaired proteins, e.g., mRNA encoding factor IX in multiple forms of hemophilia. Can be treated.
[0023]
The methods of the present invention can be used to identify compounds that bind to mRNAs encoding various proteins involved in the progression of disease in mammals. These mRNAs include, but are not limited to, amyloid protein and amyloid precursor protein; anti-angiogenic proteins such as angiostatin, endostatin, METH-1 and METH-2; survivin ), Blood coagulation factors such as factor IX, factor VIII and other factors of the blood coagulation cascade; collagen; cyclin-dependent kinase, cyclin D1, cyclin E, WAF1, cdk4 inhibitor, and MTS1. Cyclin and cyclin inhibitor; cystic fibrosis transmembrane conductance regulator (CFTR); IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL -7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17 and other Cytokines such as interleukin; hematopoietic growth factors such as erythropoietin (Epo); colony-stimulating factors such as G-CSF, GM-CSF, M-CSF, SCF and thrombopoietin; BNDF, BMP, GGRP, EGF, FGF Growth factors such as GDNF, GGF, HGF, IGF-1, IGF-2, KGF, myotrophin, NGF, OSM, PDGF, somatotrophin, TGF-β, TGF-α and VEGF; interferon Antiviral cytokines such as interferon-induced antiviral proteins, TNF-α, and TNF-β; cathepsin K, cytochrome P-450 and other cytochromes, farnesyltransferase, glutathione-S transferase, heparanase, HMG CoA Synthase, N-acetyltransferase, phenylaniline hydroxylase, phosphodiesterase, ras carb Enzymes such as syl-terminal proteases, telomerase and TNF converting enzymes; cadherins, for example glycoproteins such as N-cadherin and E-cadherin; cell adhesion molecules; selectins; transmembrane glycoproteins such as CD40; heat shock proteins; Reductase, atrial natriuretic factor, calcitonin, corticotropin releasing factor, diuretic hormone, glucagon, gonadotropin, gonadotropin releasing hormone, growth hormone, growth hormone releasing factor, somatotropin, insulin, leptin, luteinizing hormone, luteinizing hormone Hormones such as releasing hormone, parathyroid hormone, thyroid hormone, and thyroid stimulating hormone; respond to immune responses including antibodies, CTLA4, hemagglutinin, MHC protein, VLA-4, and kallikrein-kininogen-kinin system Related proteins; ligands such as CD4; sis, hst, protein tyrosine kinase receptor, ras, abl, mos, myc, fos, jun, H-ras, ki-ras, c-fms, bcl-2, L-myc, Oncogene products such as c-myc, gip, gsp, and HER-2; growth factor receptors including bombesin receptor, estrogen receptor, GABA receptor, EGFR, PDGFR, FGFR, and NGFR, GTP-binding regulatory protein , Interleukin receptor, ion channel receptor, leukotriene receptor antagonist, lipoprotein receptor, opioid pain receptor, substance P receptor, retinoic acid and retinoid receptor, steroid receptor, T cell receptor, thyroid hormone receptor Tissue plasminogen activator; transmembrane receptor; calcium pump, proton pump, Na / Ca exchanger, MRP1, MRP2, P170, LRP, Transmembrane transport system, such as fine cMOAT; transferrin; and APC, brca1, brca2, DCC, MCC, MTS1, NF1, NF2, nm23, tumor suppressor gene products, such as p53 and Rb, include mRNA encoding. In addition to the eukaryotic genes listed above, the present invention is used to define molecules that interfere with the transcription or translation efficiency of viruses, bacteria, or fungi, as described, and thus are novel anti-infectious disease therapeutics. You can make the basis of. Other target genes include, but are not limited to, the genes disclosed in Sections 5.1 and 6.
[0024]
The methods of the present invention can be used to identify mRNA binding test compounds that increase or decrease protein production, thereby treating or preventing a disease associated with decreased or increased protein production, respectively. The methods of the invention can be useful for identifying test compounds that treat or prevent disease in mammals, including cats, dogs, pigs, horses, goats, sheep, cows, primates, and humans. Such diseases include, but are not limited to, amyloidosis, hemophilia, Alzheimer's disease, atherosclerosis, cancer, giant disease, dwarfism, hypothyroidism, hyperthyroidism, inflammation, cystic fibrosis Disease, autoimmune disorders, diabetes, aging, obesity, neurodegenerative disorders, and Parkinson's disease. Other diseases include, but are not limited to, those described in Section 5.1 and those caused by abnormal expression of the genes disclosed in Example 6. In addition to the eukaryotic genes listed above, the present invention is used to define molecules that interfere with the transcription or translation efficiency of viruses, bacteria, or fungi, as described, and thus are novel anti-infectious disease therapeutics. You can make the basis of.
[0025]
In other embodiments, the test compound identified by the method of the invention is capable of interacting RNA, such as transfer RNA ("tRNA"), enzymatic RNA or ribosomal RNA ("rRNA"), with a protein or with other RNA. Useful for preventing action and thus, for example, preventing assembly of protein-RNA or RNA-RNA complexes that are essential for cell survival. As used herein, the term "enzyme RNA" refers to an enzyme that self-splices or associates with one or more proteins, for example, as in RNase P, telomerase or small nuclear ribonucleoprotein particles. Means the RNA molecule that forms. For example, suppression of the interaction between rRNA and one or more ribosomal proteins may suppress ribosome assembly and result in cells unable to synthesize the protein. In addition, suppression of the interaction of the precursor rRNA with a ribonuclease or ribonucleoprotein complex that processes the precursor rRNA (such as RNase P) prevents rRNA maturation and its assembly into the ribosome. Similarly, the tRNA: tRNA synthetase complex may be suppressed by the test compound identified by the method of the invention, such that the tRNA molecule does not carry amino acids. Such interactions include, but are not limited to, the interaction between rRNA and ribosomal proteins, the interaction between tRNA and tRNA synthetase, the interaction between RNase P proteins and RNase P RNA, and the interaction between telomerase proteins. Interaction with telomerase RNA.
[0026]
In another embodiment, the test compound identified by the method of the invention is useful for treating or preventing a viral, bacterial, protozoan, or fungal infection. For example, transcriptional up-regulation of the human immunodeficiency virus type 1 ("HIV-1") gene requires binding of the HIV Tat protein to HIV transactivation response region RNA ("TAR RNA"). HIV TAR RNA is a 59 base stem-loop structure located at the 5 'end of all nascent HIV-1 transcripts (Jones & Peterlin, 1994, Annu. Rev. Biochem. 63: 717-43). The Tat protein is known to interact with uracil 23 in the bulge region of the TAR RNA stem. Thus, TAR RNA is a potential candidate for test compounds, such as small peptides and peptide analogs, that bind to the bulge region of TAR RNA and inhibit the formation of the Tat-TAR RNA complex involved in HIV-1 up-regulation. It is a binding target (see Hwang et al., 1999 Proc. Natl. Acad. Sci. USA 96: 12997-13002). Thus, test compounds that bind to TAR RNA are useful as anti-HIV therapeutics (Hamy et al., 1997, Proc. Natl. Acad. Sci. USA 94: 3548-3553; Hamy et al., 1998, Biochemistry 37: 5086-). 5095; Mei et al., 1998, Biochemistry 37: 14204-14212), and thus are useful for treating or preventing AIDS.
[0027]
The methods of the present invention can be used to identify test compounds that treat or prevent a viral, bacterial, protozoan or fungal infection in a patient. In some embodiments, the methods of the invention are essential for identifying compounds that reduce translation of a microbial gene by interacting with mRNA, as described above, or for the survival of a virus or microorganism. It is useful to identify compounds that inhibit the interaction of certain microbial RNAs with proteins or other ligands. Examples of microbial target RNAs useful for identifying antiviral, antibacterial, antiprotozoal and antifungal compounds in the present invention include, but are not limited to, INFα, INFγ, RNase L, RNase L inhibitor protein Common antiviral and anti-inflammatory targets, such as mRNA for PKR, tumor necrosis factor, interleukin 1-15, and IMP dehydrogenase; internal ribosome entry sites; HIV-1 CT-rich domain and RNase H mRNA; (HCV mRNA HCV internal ribosome entry site (required to direct translation of HBV) and the 3'-untranslated tail of the HCV genome; rotavirus NSP3 binding site that binds to protein NSP3 required for rotavirus mRNA translation; HBV-ε Domains; 5 'and 3' untranslated regions including dengue virus IRES; INFα, IFNβ and INFγ; Plasmodium falciparum mRNA; 16S ribosomal subunit ribosomal RNA and RNA components of bacterial RNase P; and telomerase RNA components of fungal and cancer cells. Other target virus and bacterial mRNAs include, but are not limited to, those disclosed in Section 6.
[0028]
One skilled in the art will appreciate that while such target RNAs are functionally conserved in various species (eg, from yeast to humans), they exhibit nucleotide sequence and structural diversity. Thus, for example, inhibition of yeast telomerase by antifungal compounds identified by the methods of the present invention may not interfere with human telomerase and normal human cell growth.
[0029]
Thus, assays that use the methods of the invention to interfere with the interaction of host cell factors with one or more target RNAs that are important for cell growth or survival or that are essential for the life cycle of a virus, bacterium, protozoa or fungus. The compound may be identified. Such test compounds and / or congeners that exhibit the desired biological and pharmacological activity are required for the treatment or prevention of diseases caused by viral, bacterial, protozoal, or fungal infections. Can be administered to patients. Such diseases include, but are not limited to, HIV infection, AIDS, human T-cell leukemia, SIV infection, FIV infection, feline leukemia, hepatitis A, hepatitis B, hepatitis C, dengue, malaria, rotavirus infection , Severe acute gastroenteritis, diarrhea, encephalitis, hemorrhagic fever, syphilis, legionella, pertussis, gonorrhea, sepsis, influenza, pneumonia, ringworm infection, candida infection, and meningitis.
[0030]
Examples of RNA elements involved in regulation of gene expression, i.e., mRNA stability, translation efficiency via translation initiation, and ribosome assembly include, but are not limited to, HIV TAR elements, internal ribosomes, discussed below. Entry sites, "slippery sites", instability elements, and uridylic acid adenylate-rich elements.
[0031]
5.1.1.HIV TAR element
Transcriptional up-regulation of the human immunodeficiency virus type 1 ("HIV-1") gene is an HIV Tat protein and a 59-base stem-loop structure located at the 5 'end of all nascent HIV-1 transcripts. Requires binding to the HIV transactivation response region RNA ("TAR RNA") (Jones & Peterlin, 1994, Annu. Rev. Biochem. 63: 717-43). The Tat protein is known to interact with uracil 23 in the bulge region of the TAR RNA stem. Thus, TAR RNA binds to the bulge region of TAR RNA and inhibits the formation of the Tat-TAR RNA complex involved in HIV-1 up-regulation, useful binding to test compounds such as small peptides and peptide analogs. Target (see Hwang et al., 1999 Proc. Natl. Acad. Sci. USA 96: 12997-13002). Thus, test compounds that bind to TAR RNA may be useful as anti-HIV therapeutics (Hamy et al., 1997, Proc. Natl. Acad. Sci. USA 94: 3548-3553; Hamy et al., 1998, Biochemistry 37: 5086). -5095; Mei et al., 1998, Biochemistry 37: 14204-14212), and are therefore useful for treating or preventing AIDS.
[0032]
5.1.2.Internal ribosome entry site (" IRES ")
Internal ribosome entry sites ("IRES") are found in the 5 'untranslated regions ("5' UTRs") of multiple mRNAs and are thought to be involved in regulating translation efficiency. When the IRES element is located mRNA downstream of the translation stop codon, it directs ribosome reentry (Ghattas et al., 1991, Mol. Cell. Biol. 11: 5848-5959), at the start of the second open reading frame. Enables translation to start.
[0033]
According to the review of Jang et al., A large segment of the 5 'untranslated region, approximately 400 nucleotides in length, is composed of the uncapped 5' of the picornavirus mRNA, a mammalian positive-strand RNA virus whose genome acts as an mRNA. Facilitates internal entry of the ribosome, independent of the ends. This 400 nucleotide segment (IRES) maps approximately 200 nt downstream from the 5 'end and is highly structured. The IRES elements of various picornaviruses are functionally similar in vitro and in vivo, but not identical in sequence or structure. However, on the one hand, the IRES elements of entero and rhinoviruses and on the other hand, the IRES elements of cardio and aphthoviruses show similarities corresponding to systematic relatives. All IRES elements contain a conserved Yn-Xm-AUG unit (Y, pyrimidine; X, nucleotide), which appears to be essential for IRES function. The IRES element of cardio, entero and aphthoviruses binds to a cellular protein, p57. In the case of cardioviruses, interactions between specific stem-loops of the IRES are essential for translation in vitro. The entero and cardiovirus IRES elements also bind to the cellular protein p52, but the significance of this interaction is not yet known. The function of p57 or p52 in cellular metabolism is unknown. Since the Picornavirus IRES element functions in vivo in the absence of any viral gene product, it is speculated that the IRES-like element is also present in certain cellular mRNAs, releasing them from cap structure-dependent translation (Jang et al., 1990, Enzyme 44 (1-4): 292-309).
[0034]
5.1.3."Slippery site"
When the ribosome shifts from one translational reading frame to another to synthesize two viral proteins from a single viral mRNA, the programmed or directed ribosome frameshift is referred to as a "slippery site." Directed by a unique site in the called viral mRNA. The slippery site directs the ribosome frameshift in the -1 or +1 direction to slip the ribosome by one base in the 5 'direction, thereby placing the ribosome in a new reading frame and producing a new protein.
[0035]
Programmed or directed ribosome frameshifting is particularly valuable for viruses that package their positive strand, thereby eliminating the need to splice their mRNAs, reducing the risk of packaging defective genomes and synthesizing Adjust the ratio of viral proteins. Examples of programmed translational frame shifts (both +1 and -1 shifts) include the ScV system (Lopinski et al., 2000, Mol. Cell. Biol. 20 (4): 1095-103), retrovirus (Falk et al., 1993). 67: 273-6277; Jacks & Varmus, 1985, Science 230: 1237-1242; Morikawa & Bishop, 1992, Virology 186: 389-397; Nam et al., 1993, J. Virol. 67: 196- 203); Coronavirus (Brierley et al., 1987, EMBO J. 6: 3779-3785; Herold & Siddell, 1993, Nucleic Acids Res. 21: 5838-5842); Giardia virus which is also a member of the family Totiviridae (Totiviridae) giardiavirus) (Wang et al., 1993, Proc. Natl. Acad. Sci. USA 90: 8595-8599); two bacterial genes (Blinkowa & Walker, 1990, Nucleic Acids Res., 18: 1725-1729; Craigen & Caskey) 1986, Nature 322: 273); bacteriophage genes (Condron et al., 1991, Nucleic Acids Res. 19: 5607-5612); astroviruses (Marczinke et al., 1994, J. Virol. 68: 5588-5595). ; Yeast EST3 (Lundblad & Morris, 1997, Curr. Biol. 7: 969-976); and rat, mouse, Xenopus, and Drosophila ornithine decarboxylase antienzymes (Matsufuji et al., 1995, Cell 80: 51-60); and It has been identified in a number of cellular genes (Herold & Siddell, 1993, Nucleic Acids Res. 21: 5838-5842).
[0036]
Drugs targeting ribosome frameshift minimize the problem of viral drug resistance. Because this strategy targets host cell processes rather than being introduced into the cell by the virus, it minimizes the ability of the virus to evolve into drug resistant mutants. Compounds that target RNA elements involved in the regulation of programmed frameshifts should have multiple advantages, including (a) any selective pressure on the host cell translation machinery to accommodate the drug. Exists only on the host evolution timescale, which is on the order of millions of years, (b) ribosomal frameshifting is not used to express any host protein, and (c) modulates host protein activity. Altering the virus's frameshifting efficiency by rate includes minimizing the likelihood that the virus can acquire resistance to such suppression by mutation of its own genome.
[0037]
5.1.4.Instability element
An “Instability element” can be defined as a specific sequence element that promotes the recognition of an unstable mRNA by a cell turnover mechanism. Instability elements have been found in mRNA protein coding as well as in untranslated regions.
[0038]
Altering the regulation of normal mRNA stability can lead to disease. Alterations in mRNA stability have been suggested in diseases such as, but not limited to, cancer, immune disorders, heart disease, and fibrosis disorders.
[0039]
There are several mutations that delete the instability element, which in turn leads to mRNA stabilization, and consequently may be involved in the development of cancer. In Burkitt's lymphoma, a portion of the c-myc proto-oncogene translocates to the Ig locus, producing a more than 5-fold more stable form of c-myc mRNA (eg, Kapstein et al., 1996, J. Biol). Chem. 271 (31): 18875-84). Highly carcinogenic v-fos mRNA lacks the 3'UTR adenylate uridylate-rich element ("ARE") found in the more unstable and less carcinogenic c-fos mRNA (eg, Schiavi et al., 1992). , Biochim Biophys Acta. 1114 (2-3): 95-106). The difference between benign cervical lesions caused by unintegrated cyclic human papillomavirus type 16 and its integrated form lacking the 3'UTR ARE and associated with cervical cancer is due to the oncogenic protein encoding E6 / E7 It may be the result of transcript stabilization. Integration of the virus results in the deletion of the ARE labile element, stabilizing the transcript and overexpressing the protein (eg, Jeon & Lambert, 1995, Proc. Natl. Acad. Sci. USA 92 (5) : 1654-8). Deletion of the ARE from the 3'UTR of the IL-2 and IL-3 genes increases the stability of these mRNAs, promotes high expression of these proteins, and leads to the formation of cancer cells (eg, Stoecklin Et al., 2000, Mol. Cell. Biol. 20 (11): 3753-63).
[0040]
Mutations in trans-acting factors involved in mRNA turnover may also promote cancer. In monocyte tumors, lymphokine GM-CSF mRNA is specifically stabilized as a result of oncogenic lesions of trans-acting factors that control mRNA turnover rates. In addition, normally unstable IL-3 transcripts are inappropriately long-lived in mast tumor cells. Similarly, labile GM-CSF mRNA is highly stabilized in bladder cancer cells. See, for example, Bickel et al., 1990, J. Immunol. 145 (3): 840-5.
[0041]
The immune system is regulated by a number of regulatory molecules that activate or suppress the immune response. It is now clearly demonstrated that the stability of the transcripts encoding these proteins is highly regulated. Modification of the regulation of these molecules can lead to misregulation of this process, with profound medical consequences. For example, recent findings using transgenic mice show that misregulation of the stability of key modulators TNFα mRNA leads to diseases such as, but not limited to, rheumatoid arthritis and Crohn's disease-like liver disease. It is shown that. See, for example, Clark, 2000, Arthritis Res. 2 (3): 172-4.
[0042]
Cardiac smooth muscle is modulated by β-adrenergic receptors, which in turn respond to the sympathetic neurotransmitter norepinephrine and the adrenal hormone epinephrine. Chronic heart disease is characterized by impairment of smooth muscle cells, partly resulting from the more rapid breakdown of β-adrenergic receptor mRNA. See, for example, Ellis & Frielle, 1999, Biochem. Biophys. Res. Commun. 258 (3): 552-8.
[0043]
Many diseases result from overexpression of collagen. For example, cirrhosis results from liver damage as a result of cancer, viral infection, or alcohol abuse. Such damage is due to misregulation of collagen expression resulting in the formation of large collagen deposits. Recent findings indicate that a fairly large increase in collagen expression is mostly due to its mRNA stabilization. See, for example, Lindquist et al., 2000, Am. J. Physiol. Gastrointest. Liver Physiol. 279 (3): G471-6.
[0044]
5.1.5.Adenylate uridylic acid rich element (" ARE ")
Adenylate uridylate-rich elements ("AREs") are found in the 3 'untranslated regions ("3' UTRs") of multiple mRNAs and include, but are not limited to, transcription factors, cytokines and lymphokines. Involved in mRNA turnover. AREs can function as both stabilizing and destabilizing elements. ARE mRNAs are classified into five groups according to sequence (Bakheet et al., 2001, Nucl. Acids Res. 29 (1): 246-254). Website where the full text is incorporated by referencehttp://rc.kfshrc.edu.sa/aredThe current database contains mRNAs containing AREs and their cluster groups. ARE motifs are classified as follows:
Group I cluster (AUUUAUUUAUUUAUUUAUUUA) SEQ ID NO: 1
Group II cluster (AUUUAUUUAUUUAUUUA) stretch SEQ ID NO: 2
Group III cluster (WAUUUAUUUAUUUAW) stretch SEQ ID NO: 3
Group IV cluster (WWAUUUAUUUAWW) stretch SEQ ID NO: 4
Group V cluster (WWWWAUUUAWWWW) stretch SEQ ID NO: 5
The ARE-mRNA was clustered into five groups containing 5, 4, 3, and 2 pentameric repeats, but the last group contained only one pentamer within the 13 bp ARE pattern. Using a thorough literature search, follow the NCBI-COG functional annotation (Tatusov et al., 2001, Nucleic Acids Research, 29 (1): 22-28) as well as the categories of inflammation, immune response, development / differentiation and , Assigned a functional category.
[0045]
Group I contains a number of secreted proteins including GM-CSF, IL-1, IL-11, IL-12 and Gro-β that affect hematopoiesis and immune cell proliferation (Witsell & Schook, 1992, Proc. Natl Acad. Sci. USA, 89: 4754-4758). Although TNFα is both a pro-inflammatory and anti-tumor protein, there is experimental evidence that it can act as a growth factor in certain leukemias and lymphomas (Liu et al., 2000, J. Biol. Chem. 275: 21086-21093). ).
[0046]
Unlike Group I, Groups II-V include immune responses, cell cycle and proliferation, inflammation and coagulation, angiogenesis, metabolism, energy, DNA binding and transcription, nutrient transport and ionic homeostasis, protein synthesis, cell biogenesis, signaling And a functionally diverse gene family comprising apoptosis (Bakheet et al., 2001, Nucl. Acids Res. 29 (1): 246-254).
[0047]
Several research groups have described ARE-binding proteins that affect ARE-mRNA stability. Among the well-characterized proteins are mammalian homologs of ELAV (embryonic lethal abnormal vision) proteins, including AUF1, HuR and He1-N2 (Zhang et al., 1993, Mol. Cell). Biol. 13: 7652-7665; Levine et al., 1993, Mol. Cell. Biol. 13: 3494-3504; Ma et al., 1996, J. Biol. Chem. 271: 8144-8151). The zinc finger protein tristetraproline has been identified as another ARE binding protein with destabilizing activity on TNFα, IL-3 and GM-CSF mRNA (Stoecklin et al., 2000, Mol. Cell. Biol. 20: 3753 -3763; Carballo et al., 2000, Blood 95: 1891-1899).
[0048]
Genes containing AREs, including but not limited to multiple early response genes that regulate cell growth and response to exogenous drugs, are associated with one or more ARE clusters because they are clearly important in biological systems. And the identification of compounds that potentially modulate the stability of the target RNA may be of potential therapeutic value.
[0049]
5.2.Detectably labeled target RNA
Target nucleic acids, including but not limited to RNA and DNA, useful in the methods of the invention have a label that is detectable via conventional spectroscopic or radiographic means. Preferably, the target nucleic acid is labeled with a covalently linked dye molecule. Useful dye molecule labels include, but are not limited to, fluorescent dyes, phosphorescent dyes, ultraviolet dyes, infrared dyes, and visible dyes. Preferably, the dye is a visible dye.
[0050]
Labels useful in the present invention include, but are not limited to, spectroscopic labels such as fluorescent dyes (eg, fluorescein and fluorescein isothiocyanate (FITC) and Oregon Green)^TMDerivatives, such as rhodamine and derivatives (eg, Texas Red, tetramethylrhodamine isothiocyanate (TRITC), bora-3a, 4a-diaza-s-indacene (BODIPY®) and derivatives, etc.), digoxigenin, biotin, Phycoerythrin, AMCA, CyDye^TM, Etc.), radioactive labels (eg,^ThreeH,¹²⁵I,³⁵S,¹⁴C,³²P,³³P, etc.), enzymes (eg, horseradish peroxidase, alkaline phosphatase, etc.), spectroscopic colorimetric labels such as colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, or 0.1-1000 nm And nanoclusters of inorganic ions having a specified dimension. Useful affinity tags and complementary partners include, but are not limited to, biotin-streptavidin, complementary nucleic acid fragments (eg, oligo dT-oligo dA, oligo T-oligo A, oligo dG-oligo dC, oligo G -Oligo-C), aptamer-streptavidin, or haptens and proteins against which antisera or monoclonal antibodies may be utilized. A label can be directly or indirectly coupled to a component of a detection assay (eg, a detection reagent) by methods well known in the art. A variety of labels can be utilized, with the required sensitivity, ease of conjugation to the compound, stability requirements, available instruments, and labels depending on the disposal provision.
[0051]
In one embodiment, the nucleic acid labeled at one or more specific locations is chemically synthesized using phosphoramidites or other solution or solid phase methods. Details of the chemistry utilized to make polynucleotides by the phosphoramidite method are well known (eg, Caruthers et al., US Pat. Nos. 4,458,066 and 4,415,732; Caruthers et al., 1982, Genetic Engineering 4: 1-17; Users Manual, "Model 392 and 394 Polynucleotide Synthesizers"), 1990, pages 6-1 to 6-22, Applied Biosystems, Part No. 901237; Ojwang et al., 1997, Biochemistry, 36: 6033-6045). The phosphoramidite method of polynucleotide synthesis is the preferred method because of the efficient and rapid coupling and stability of the starting materials. Synthesis is performed by growth of a polynucleotide chain attached to a solid support, and generally excess reagents in the liquid phase can be removed by washing, decanting, and / or filtration, so that during the synthesis cycle, There is no need for a purification step.
[0052]
The following briefly describes the steps that describe a typical polynucleotide synthesis cycle using the phosphoramidite method. First, the solid support to which the 3 'end of the protected nucleoside monomer is attached is treated with an acid, for example, trichloroacetic acid, to remove the 5'-hydroxyl protecting group to give a free hydroxyl group, followed by a subsequent coupling reaction. Prepare for. After the coupling reaction is completed by contacting the support-bound nucleoside with a protected nucleoside phosphoramidite monomer and a weak acid such as tetrazole, an activated intermediate is formed. Weak acids protonate the nitrogen atom of the phosphoramidite to form a reactive intermediate. Nucleoside addition is generally completed within 30 seconds. Next, a capping step is performed to terminate the ends of the polynucleotide chain where no nucleoside addition has occurred. Capping is preferably performed with acetic anhydride and 1-methylimidazole. The phosphite group of the internucleotide linkage is then converted to a more stable phosphotriester by oxidation using iodine as the preferred oxidizing agent and water as the oxygen donor. After oxidation, the hydroxyl protecting group of the newly added nucleoside is removed using a protic acid such as trichloroacetic acid or dichloroacetic acid. This cycle is repeated one or more times until the elongation of the chain is completed. After synthesis, the polynucleotide strand is cleaved from the support using a base, such as ammonium hydroxide or t-butylamine. The cleavage reaction also removes any phosphate protecting groups, for example, cyanoethyl. Finally, any protecting groups on the exocyclic amine of the base and any protecting groups on the dye are removed by treating the polynucleotide solution in a base at an elevated temperature, eg, about 55 ° C. Preferably, various protecting groups are removed using ammonium hydroxide or t-butylamine.
[0053]
Any of the nucleoside phosphoramidite monomers can be labeled using standard phosphoramidite chemistry methods (Hwang et al., 1999, Proc. Natl. Acad. Sci. USA 96 (23): 12997-13002; Ojwang). Et al., 1997, Biochemistry. 36: 6033-6045 and references cited therein). Dye molecules useful for covalently linking to the phosphoramidite preferably contain a primary hydroxy group that is not part of the chromophore of the dye. Examples of dye molecules include, but are not limited to, Disperse Dye CAS 4439-31-0, Disperse Dye CAS 6054-58-6, Disperse Dye CAS 4392-69-2 (Sigma-Aldrich, St. Louis, MO ), Dispersed Red, and 1-pyrene butanol (Molecular Probes, Eugene, OR). Other dyes useful for binding phosphoramidites, such as fluorescein, cy3, and cy5 fluorescent dyes, will be apparent to those of skill in the art, for example, Sigma-Aldrich, St. Louis, MO or Molecular Probes, Inc., Can be purchased from Eugene, OR.
[0054]
In other embodiments, dye-labeled target RNA molecules are enzymatically synthesized using in vitro transcription (Hwang et al., 1999, Proc. Natl. Acad. Sci. USA 96 (23): 12997-13002 and References cited therein). In this embodiment, the template DNA is denatured by heating at about 90 ° C., and the oligonucleotide primers are denatured, for example, by gradually cooling the mixture of denatured template and primer from about 90 ° C. to room temperature. Anneal to template DNA. A mixture of ribonucleoside-5'-triphosphates containing one or more dye-labeled ribonucleotides (Sigma-Aldrich, St. Louis, MO) capable of supporting template-directed enzymatic extension of the primed template ( For example, a mixture comprising GTP, ATP, CTP, and UTP) is added to the primed template. Next, a polymerase enzyme is added to the mixture under conditions under which the polymerase enzyme is active, as is well known to those skilled in the art. During polymerase-mediated strand synthesis, incorporation of labeled ribonucleotides creates a labeled polynucleotide.
[0055]
In yet another embodiment of the present invention, the nucleic acid molecule is end-labeled after synthesis. The method of labeling the 5 'end of the oligonucleotide is not limited, but (i) periodate oxidation of 5' to 5 'linked ribonucleotides followed by a reaction using an amine-reactive label (Heller & Morisson, 1985, in "Rapid Detection and Identification of Infectious Agents", DT Kingsbury and S. Falkow, eds., Pp.245-256, Academic Press); (ii) Ethylenediamine and Condensation with a 5'-phosphorylated polynucleotide followed by reaction with an amine-reactive label (Morrison, European Patent Application 232 967); (iii) Aliphatic amine substitution using aminohexyl phosphite reagent in solid-phase DNA synthesis Introduction of the group and subsequent reaction with an amine-reactive label (Cardullo et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8790-8794); and (iv) phosphatase treatment followed by ATP-? S and kinase. By end-labeling with The 5 'terminus of the nucleic acid, the introduction of maleimide-labeled fluorescent dye and specifically and efficiently react to thiophosphate group (Czworkowski et, 1991, Biochem 30:. 4821-4830) and the like.
[0056]
A detectable label must not be incorporated into the target nucleic acid at the specific binding site where the test compound will bind, since the presence of the covalently attached label may sterically or chemically hinder the binding of the test compound at this site. Thus, if the region of the target nucleic acid that binds the host cell factor is known, the detectable label is preferably incorporated into the nucleic acid molecule at one or more locations spatially or sequencely separated from the binding region.
[0057]
Post-synthesis labeled target nucleic acids can be purified using standard techniques known to those skilled in the art (Hwang et al., 1999, Proc. Natl. Acad. Sci. USA 96 (23): 12997-13002 and See references cited). Depending on the length of the target nucleic acid and its method of synthesis, such purification techniques include, but are not limited to, reversed-phase high-performance liquid chromatography ("reverse-phase HPLC"), high-performance liquid chromatography ("FPLC"), And gel purification. After purification, the target RNA is heated in a buffer, e.g., a buffer containing about 50 mM Tris-HCI, pH 8 and 100 mM NaCl, preferably to approximately 85-95 ° C. and gradually cooled to room temperature, Refold to its original conformation.
[0058]
In other embodiments, the target nucleic acid can be radiolabeled. A radioactive label, such as, but not limited to, a phosphorus, sulfur, or hydrogen isotope, added after or during the synthesis of the target nucleic acid, may be incorporated into the nucleotide. Methods for the synthesis and purification of radiolabeled nucleic acids are well known to those skilled in the art. See, for example, Sambrook et al., 1989, in "Molecular Cloning: A Laboratory Manual", pp 10.2-10.70, Cold Spring Harbor Laboratory Press, and the like, the entire contents of which are incorporated herein by reference. See the references cited in
[0059]
In other embodiments, the target nucleic acids can be bound to inorganic nanoparticles. Nanoparticles include, but are not limited to, Ag_TwoS, ZnS, CdS, CdTe, Au, or TiO_TwoIs a cluster of ions having a controlled size of 0.1 to 1000 nm, consisting of a metal, metal oxide, or semiconductor containing Nanoparticles have unique optical, electronic and catalytic properties compared to bulk materials, which properties can be adjusted by the size of the particles. Methods for attaching nucleic acids are well known to those of skill in the art (eg, Niemeyer, 2001, Angew. Chem. Int. Ed. 40: 4129-4158, International Patent, the disclosure of which is incorporated herein by reference in its entirety). See published WO / 0218643 and references cited therein).
[0060]
5.3.Library of small molecules
Libraries to be screened using the methods of the present invention may include various types of test compounds. In some embodiments, the test compound is a nucleic acid or peptide molecule. In a non-limiting example, the peptide molecule may be present in a phage display library. In other embodiments, the type of test compound includes, but is not limited to, a non-natural amino acid, eg, a phosphorus analog of an amino acid such as a D-amino acid, α-aminophosphate and α-aminophosphate, or Peptide analogs (including peptides comprising amino acids having peptide bonds), nucleic acid analogs such as phosphorothioates and PNAs, hormones, antigens, synthetic or natural drugs, opiates, dopamine, serotonin, catecholamines, thrombin, acetylcholine, Prostaglandins, organic molecules, pheromones, adenosine, sucrose, glucose, lactose and galactose. Libraries of polypeptides or proteins can also be utilized.
[0061]
In a preferred embodiment, the combinatorial library is a library of small organic molecules such as, but not limited to, benzodiazepines, isoprenoids, thiazolidinones, metathiazanones, pyrrolidines, morpholino compounds, and diazepindione. In other embodiments, the combinatorial library is a peptoid; a random bio-oligomer; a diversomer such as hydantoin, benzodiazepine and dipeptide; a vinylog polypeptide; a non-peptidic peptidomimetics; an oligocarbamate; A phosphonate; a peptide nucleic acid library; an antibody library; or a carbohydrate library. Combinatorial libraries are themselves commercially available (eg, Advanced ChemTech Europe Ltd., Cambridgeshire, UK; ASINEX, Moscow Russia; BioFocus plc, Sittingbourne, UK; Bionet Research (a division of Key Organics Limited), Camelford, UK ChemBridge Corporation, San Diego, California; ChemDiv Inc, San Diego, California; ChemRx Advanced Technologies, South San Francisco, California; ComGenex Inc., Budapest, Hungary; Evotec OAI Ltd, Abingdon, UK; IF LAB Ltd., Kiev, Ukraine; Maybridge plc, Cornwall, UK; PharmaCore, Inc., North Carolina; SIDDCO Inc, Tucson, Arizona; TimTec Inc, Newark, Delaware; Tripos Receptor Research Ltd, Bude, UK; Toslab, Ekaterinburg, Russia)
In one embodiment, a combinatorial compound library for the methods of the invention may be synthesized. Synthetic methods directed to the generation of large collections or libraries of small organic compounds that can be screened for pharmacological, biological or other activities are very important (Dolle, 2001, J. Comb. Chem. 3: 477-517; Hall et al., 2001, J. Comb. Chem. 3: 125-150; Dolle, 2000, J. Comb. Chem. 2: 383-433; Dolle, 1999, J. Comb. Chem. 1 : 235-282). Synthetic methods applied to generate large combinatorial libraries are performed in solution or on a solid phase, ie, on a solid support. Solid phase synthesis makes it easier to perform multi-step reactions and drive the reactions to high yield to completion because the excess reagents can be easily added and washed off after each reaction step. Solid phase combinatorial synthesis also often improves isolation, purification and screening. However, more traditional solution phase chemistry supports a wider variety of organic reactions than solid phase chemistry. Methods and strategies for the synthesis of combinatorial libraries are described in "A Practical Guide to Combinatorial Chemistry", AW Czarnik and SH Dewitt, eds., American Chemical Society, 1997; "The Combinatorial Index." BA Bunin, Academic Press, 1998; "Organic Synthesis on Solid Phase", FZ Dorwald, Wiley-VCH, 2000; and "Solid-Phase Organic Syntheses", Vol. 1, AW Czarnik, Ed., Wiley Interscience, 2001.
[0062]
The combinatorial compound library of the present invention is disclosed in U.S. Pat.No. 6,358,479 to Frisina et al., U.S. Pat.No. 6,190,619 to Kilcoin et al., U.S. Pat.No. 6,132,686 to Gallup et al. U.S. Patent No. 6,126,904 to Zuellig et al., U.S. Patent No. 6,074,613 to Harness et al., U.S. Patent No. 6,054,100 to Stanchfield et al., U.S. Patent No. 5,746,982 to Saneii et al. be able to. These patents describe synthesizers that can accommodate multiple reaction vessels for parallel synthesis of a large number of individual compounds or for combinatorial libraries of compounds.
[0063]
In one embodiment, a combinatorial compound library can be synthesized in solution. The method disclosed in US Pat. No. 6,194,612 to Boger et al., Which is incorporated herein by reference in its entirety, features compounds that are useful as templates for solution phase synthesis of combinatorial libraries. The template is designed so that the reaction products can be easily purified from unreacted reactants using liquid / liquid or solid / liquid extraction. Compounds produced by combinatorial synthesis using a template will preferably be small organic molecules. Multiple compounds in the library may mimic the action of a non-peptide or peptide. In contrast to solid phase synthesis of combinatorial compound libraries, solution phase synthesis does not require the use of special protocols to monitor the individual steps of a multi-step solid phase synthesis (Egner et al., 1995, J. Org. Chem. 60: 2652; Anderson et al., 1995, J. Org. Chem. 60: 2650; Fitch et al., 1994, J. Org. Chem. 59: 7955; Look et al., 1994, J. Org. Chem. 49: 7588. Metzger et al., 1993, Angew. Chem., Int. Ed. Engl. 32: 894; Youngquist et al., 1994, Rapid Commun. Mass Spect. 8:77; Chu et al., 1995, J. Am. Chem. Soc. 117. Brummel et al., 1994, Science 264: 399; Stevanovic et al., 1993, Bioorg. Med. Chem. Lett. 3: 431).
[0064]
Combinatorial compound libraries useful in the methods of the invention can be synthesized on a solid support. In one embodiment, a library of compounds is synthesized on a solid support using a split synthesis method (a protocol that separates and mixes the solid support during synthesis) (Lam et al., 1997, Chem. Rev. 97: 41-448; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10922-10926 and references cited therein). Each solid support in the final library has substantially one type of test compound attached to its surface. Other methods of synthesizing combinatorial libraries on solid supports, where one product binds to the respective support, will be known to those of skill in the art (eg, herein incorporated by reference in its entirety, Nefzi et al., 1997, Chem. Rev. 97: 449-472 and U.S. Patent No. 6,087,186 to Cargill et al.).
[0065]
The term "solid support" as used herein is not limited to a particular type of solid support. A rather large number of supports are available and known to those skilled in the art. Solid supports include silica gel, resins, derivatized plastic films, glass beads, cotton, plastic beads, polystyrene beads, alumina gel, and polysaccharides. Suitable solid supports can be selected based on the desired end use and suitability for various synthesis protocols. For example, solid supports for peptide synthesis include polystyrene, including p-methylbenzhydrylamine (pMBHA) resin (Peptides International, Louisville, Ky.), Chloromethyl polystyrene, hydroxymethyl polystyrene and aminomethyl polystyrene (eg, Bachem Inc.). , PAM-resin obtained from Peninsula Laboratories), poly (dimethylacrylamide) -grafted styrene co-divinyl-benzene (eg, POLYHIPE resin obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories) , Polystyrene resins grafted with polyethylene glycol (eg, TENTAGEL or ARGOGEL, Bayer, Tubingen, Germany), polydimethylacrylamide resins (obtained from Milligen / Biosearch, California), or resins such as Sepharose (Pharmacia, Sweden) It may be.
[0066]
In one embodiment, a solid support is suitable for in vivo applications, ie, may serve as a carrier or support for administering a test compound to a patient (eg, TENTAGEL, Bayer, Tubingen, Germany). In certain embodiments, the solid support is tasty and / or ingestible.
[0067]
In embodiments of the present invention, compounds may be attached to a solid support via a linker. The linker may be integral with and part of the solid support, or may be non-integral and synthesized on the solid support or attached after synthesis. The linker not only provides a point at which the test compound attaches to the solid support, but also allows different groups of molecules to be cleaved from the solid support under different conditions, depending on the nature of the linker. Is also useful. For example, a linker is, inter alia, electrophilicly cleaved, nucleophilically cleaved, photocleavable, enzymatically cleaved, cleaved by a metal, cleaved under reducing conditions, or under oxidizing conditions. Can be cut.
[0068]
In other embodiments, combinatorial compound libraries are disclosed in European Patent Application 1,118,359 Al to Lehn, the entirety of which is incorporated by reference; Huc & Nguyen, 2001, Comb. Chem. High Throughput. Screen. 4: 53-74; Lehn and Eliseev, 2001, Science 291: 2331-2332; Cousins et al. 2000, Curr. Opin. Chem. Biol. 4: 270-279; and Karan & Miller, 2000, Drug. Disc. Today 5: 67-75. And can be constructed in situ using dynamic combinatorial chemistry.
[0069]
Dynamic combinatorial chemistry is a non-covalent interaction with a target biomolecule, including but not limited to proteins, RNA, or DNA, in which the constituent subunits exist as a mixture in the presence of the biomolecule. It takes advantage of the combination to favor the assembly of the most strongly binding molecules. According to the laws of thermodynamics, when a collection of molecules binds and recombines in equilibrium through reversible chemical reactions in solution, the molecule that binds most strongly to the biological molecule of the template, preferably one molecule, , May be present in greater amounts than all other possible combinations. Reversible chemical reactions include, but are not limited to, imine, acyl-hydrazone, amide, acetal, and ester formation between a carbonyl-containing compound and an amine, hydrazine, or alcohol; thiol exchange between disulfides; boric acid Alcohol exchange in esters; Diels-Alder reaction; heat or light induced sigmatropy or ring electron rearrangement; or Michael reaction.
[0070]
In a preferred embodiment of this technique, the components of the dynamic combinatorial compound library are combined in the absence of the target RNA to reach equilibrium, and then, in the presence of the target RNA, preferably under physiological conditions. Incubate until an equilibrium of 2 is reached. The second perturbed equilibrium (the so-called "templated mixture") is not necessary, but is not limited to, further chemical transformations including reduction, oxidation, hydrolysis, acidification, or basification. To prevent the restoration of the original equilibrium when the dynamic combinatorial compound library is separated from the target RNA.
[0071]
In a preferred embodiment of this technique, the major products of the templated dynamic combinatorial library can be separated and directly identified from the minor products. In another embodiment, the identity of the major products can be identified by elucidation strategies involving the preparation of derivative dynamic combinatorial libraries described in European Patent Application 1,118,359 Al, which is incorporated by reference in its entirety. Here, each component of the mixture may be taken as a group, but preferably one at a time, and the ability of the derivative library mixture to bind to the target RNA at chemical equilibrium is determined. The component that, when removed, will most likely reduce the ability of the derivative dynamic combinatorial library to bind to the target RNA is probably the major product component in the original dynamic combinatorial library.
[0072]
5.4.Library screening
After labeling a target nucleic acid such as, but not limited to, RNA or DNA and synthesizing or purchasing a test compound library or both, screening the library with the labeled target nucleic acid and binding to the nucleic acid The test compound to be tested is identified. Screening comprises contacting a labeled target nucleic acid with individual or small groups of components of a compound library. Contacting is preferably performed in an aqueous solution, and most preferably under physiological conditions. The aqueous solution preferably stabilizes the labeled target nucleic acid and prevents denaturation or degradation of the nucleic acid without interfering with the binding of the test compound. The aqueous solution may be similar to a solution that forms a complex between the target RNA and its corresponding host cell factor (if known) in vitro. For example, a TK buffer commonly used to form a Tat protein-TAR RNA complex in vitro can be used in the methods of the invention as an aqueous solution to screen a test compound library for TAR RNA binding compounds. .
[0073]
The method of the invention for screening a library of test compounds preferably comprises contacting the test compound with a target nucleic acid in the presence of an aqueous solution, wherein the aqueous solution is preferably subjected to physiological conditions. Comprising a combination of buffers and salts that approximate or mimic. The aqueous solution optionally further comprises, but is not limited to, DNA; yeast tRNA; salmon sperm DNA; homoribopolymers such as, but not limited to, poly IC, poly A, poly U, and poly C; Contains non-specific nucleic acids, such as non-specific RNA. A non-specific RNA may be an unlabeled target nucleic acid having a mutation at its binding site, such that it cannot interact with a test compound at that site. For example, if the library is screened using dye-labeled TAR RNA, unlabeled TAR RNA having a mutation in the uracil 23 / cytosine 24 bulge region may be present in the aqueous solution. Without being bound by any theory, the addition of unlabeled RNA, which is essentially identical to the dye-labeled target RNA except for a mutation at the binding site, can be combined with other regions of the dye-labeled target RNA. Interaction with the test compound or solid support can be minimized to prevent false positive results.
[0074]
The solution further contains a buffer, a combination of salts, and, optionally, a detergent or surfactant. The pH of the solution typically ranges from about 5 to about 8, preferably from about 6 to about 8, most preferably from about 6.5 to about 8. Various buffers may be used to achieve the desired pH. Suitable buffers include, but are not limited to, Tris, Mes, Bis-Tris, Ada, Aces, Pipes, Mopso, Bis-Tris propane, Bes, Mops, Tes, Hepes, Dipso, Mobs, Tapso, Trizma, Heppso, Popso, TEA, Epps, Tricine, Gly-Gly, Bicine, and sodium-potassium phosphate. The buffering agent is one that contains about 10 mM to about 100 mM, preferably about 25 mM to about 75 mM, and most preferably about 40 mM to about 60 mM buffer. The pH of the aqueous solution depends on the type of target RNA used and the test compound in the library and can be optimized for various screening reactions; therefore, the type and amount of buffer used in the solution will Can vary from one to another. In a preferred embodiment, the aqueous solution has a pH of about 7.4, which can be achieved using about 50 mM Tris buffer.
[0075]
In addition to a suitable buffer, the aqueous solution may further comprise about 0 mM to about 100 mM KCl, about 0 mM to about 1 M NaCl, and about 0 mM to about 200 mM MgCl._TwoOf salts. In a preferred embodiment, the salt combination is about 100 mM KCl, 500 mM NaCl, and 10 mM MgCl_TwoIt is. Without being bound by any theory, Applicants believe that KCl, NaCl, and MgCl_TwoWas found to stabilize the target RNA so that most RNA was not denatured or digested. The optimal concentration of each salt used in the aqueous solution depends, inter alia, on the target RNA used and can be determined using routine experimentation.
[0076]
The solution optionally contains from about 0.01% to about 0.5% (w / v) of a detergent or surfactant. Without being bound by any theory, small amounts of detergents or detergents in solution reduce non-specific binding of target RNA to the solid support, control aggregation of target RNA molecules and improve stability. May increase. Typical detergents useful in the method of the present invention include, but are not limited to, deoxycholic acid, 1-heptanesulfonic acid, N-laurylsarcosine, lauryl sulfate, 1-octanesulfonic acid and salts of taurocholic acid and the like. Anionic detergents; cationic detergents such as benzalkonium chloride, cetylpyridinium chloride, methylbenzethonium chloride and decamethonium bromide; CHAPS, CHAPSO, alkyl betaine, alkylamidoalkyl betaine, N-dodecyl-N, N-dimethyl-3 Zwitterionic detergents of -ammonio-1-propanesulfonate and phosphatidylcholine; and n-decyl aD-glucopyranoside, n-decyl β-D-maltopyranoside, n-dodecyl β-D-maltoside, n-octyl β-D- Glucopyranosides, sorbitan esters, n-tetradecyl β-D-maltoside, octylphenoxy Nonionic detergents such as polyethoxyethanol (Nonidet P-40), nonylphenoxy polyethoxyethanol (NP-40), and triton. Preferably, the detergent, if present, is a non-ionic detergent. Typical surfactants useful in the methods of the invention include, but are not limited to, ammonium lauryl sulfate, polyethylene glycol, butyl glucoside, decyl glucoside, polysorbate 80, lauric acid, myristic acid, palmitic acid. Acids, potassium palmitate, undecanoic acid, lauryl betaine, and lauryl alcohol. More preferably, the detergent, if present, is Triton X-100 and is present in an amount of about 0.1% (w / v).
[0077]
Non-specific binding between a labeled target nucleic acid and a test compound can be further minimized by treating the binding reaction with one or more blocking agents. In one embodiment, the binding reaction is treated with a blocking agent, such as bovine serum albumin ("BSA"), and then contacted with a labeled target nucleic acid. In other embodiments, the conjugation reactants are treated sequentially with at least two different blocking agents. This blocking step is preferably performed at room temperature for about 0.5 to about 3 hours. In the next step, the reaction mixture is further treated with unlabeled RNA having a mutation at the binding site. This blocking step is preferably performed at about 4 ° C. for about 12 to about 36 hours before adding the dye-labeled target RNA. Preferably, the solution used in the one or more blocking steps is substantially similar to the aqueous solution used to screen the library with the dye-labeled target RNA, for example, in pH and salt concentration.
[0078]
Upon contacting the mixture of the labeled target nucleic acid and the test compound, the mixture is maintained under constant agitation, preferably at 4 ° C., for about 1 day to about 5 days, preferably about 2 days to about 3 days. After the incubation period, the bound compound can be released from the released compound by the following electrophoresis technique (see Section 5.5.1), or to confirm the reactants that have bound to the labeled target nucleic acid, or Determined using any of the methods disclosed in Section 5.5 below. In other embodiments, if a technique is used to distinguish bound and unbound target nucleic acid (ie, spectroscopy), it is not necessary to separate the complexed target nucleic acid from the free target nucleic acid. Absent.
[0079]
The method of identifying a small molecule bound to a labeled nucleic acid can vary with the type of label on the target nucleic acid. For example, if the target RNA is labeled with a visible fluorescent dye, the target RNA complex separates the bound target from the free target by electrophoresis or size differential techniques using individual reactants. Preferably, the identification is performed using chromatography techniques. A reactant corresponding to a change in the movement of the complexed RNA can be cross-referenced to the small molecule compound added to the reactant. Alternatively, the complexed target RNA may be screened en masse, then separated from the free target RNA using electrophoresis or size differential techniques, and the resulting complexed target is then mass spectrometrically analyzed. Analyze using technology. In this way, the bound small molecule can be identified based on its molecular weight. In this reaction, the exact molecular weights of all compounds in the library are known in advance. In other embodiments, without limitation, the use of techniques such as spectroscopy may not require that the test compound bound to the target nucleic acid be separated from the unbound target nucleic acid.
[0080]
5.5.Separation methods for screening test compounds
Any method that detects changes in the physical properties of the target nucleic acid complexed with the test compound from the unbound target nucleic acid can be used to separate the target nucleic acid that is not complexed with the complexed target nucleic acid May be. Methods that can be used to physically separate complexed target RNA from unbound target RNA include, but are not limited to, electrophoresis, fluorescence spectroscopy, surface plasmon resonance, mass spectrometry, scintillation proximity Assays, structure-activity relationships by NMR spectroscopy ("SAR"), size exclusion chromatography, affinity chromatography, and nanoparticle aggregation.
[0081]
5.5.1.Electrophoresis
The method of separating the target RNA complex bound to the test compound from the unbound RNA may include any electrophoretic separation method, and the method is not limited, but includes denaturation. And non-denaturing polyacrylamide gel electrophoresis, urea gel electrophoresis, gel filtration, pulsed field gel electrophoresis, two-dimensional gel electrophoresis, continuous flow electrophoresis, zone electrophoresis, agarose gel electrophoresis, and capillary electrophoresis .
[0082]
In a preferred embodiment, high-throughput screening of test compounds bound to target RNA is performed using an automated electrophoresis system comprising a capillary cartridge having a plurality of capillary tubes. Such devices for performing automated capillary gel electrophoresis are disclosed in U.S. Patent Nos. 5,885,430; 5,916,428; 6,027,627; and 6,063,251, the disclosures of which are incorporated by reference in their entireties.
[0083]
Using the device disclosed in US Pat. No. 5,885,430, which is incorporated by reference in its entirety, a sample can be co-introduced into multiple capillary tubes directly from a microtiter tray having a standard size. US Patent No. 5,885,430 discloses a disposable capillary cartridge that can be washed between electrophoresis runs, the cartridge having a plurality of capillary tubes. The first end of each capillary tube is held in a mounting plate, and the first ends collectively form an array on the mounting plate. The first end-to-end spacing corresponds to the center-to-center spacing of wells of a microtiter tray having a standard size. Thus, the first end of the capillary tube can be simultaneously dipped into the sample present in the well of the tray. A second mounting plate that holds the second end of the capillary tube is placed on the cartridge. The second ends of the capillary tubes are arranged in an array corresponding to the wells in the microtiter tray, such that each capillary tube is isolated from its adjacent tube, so that each end is placed in an individual well. No cross contamination when immersed in water.
[0084]
Plate holes may be provided in each mount plate and capillary tubes may be inserted through these plate holes. In such a case, the surface of the mount plate having the exposed capillary end can be pressurized by hermetically sealing the plate hole. Applying a positive pressure near the capillary opening of this mounting plate allows the introduction of air and liquid during the electrophoresis operation, and uses this to remove gels and other substances from the capillary tubes during condition adjustment. It can be forcibly discharged. The capillary tubes may be protected from damage by using a cannula containing a needle and / or a plastic tube when placed in these plate holes. If metal cannulas or the like are used, they can act as electrical contacts for current during electrophoresis. If a second mounting plate is present, the second mounting plate is provided with a plate hole through which the second end of the capillary tube protrudes. In this case, the second mounting plate serves as the pressure containment member of the pressure cell, and the second end of the capillary tube communicates with the internal cavity of the pressure cell. The pressure cell may also have an inlet and an outlet. Gels, buffer solutions, detergents, etc. are introduced into the interior cavity through the inlet, and each can simultaneously enter the second end of the capillary.
[0085]
In another preferred embodiment, the automated electrophoresis system may include a chip system consisting of complexly designed interconnected channels, the system comprising a tiny, continuously operating electrophoresis device. In this section, the reaction with one sample is carried out in one region of the chip while the enzymatic reaction is carried out and analyzed using the part of the channel designed as Electrophoretic separation is performed on different parts of the chip. No. 5,699,157; No. 5,842,787; No. 5,869,004; No. 5,876,675; No. 5,942,443; No. 5,948,227; No. 6,042,709; No. 6,042,710; No. 6,046,056; No. 6,048,498; Nos. 6,132,685; 6,150,119; 6,150,180; 6,153,073; 6,167,910; 6,171,850; 6,186,660, the disclosures of which are incorporated by reference in their entireties.
[0086]
The system disclosed in U.S. Patent No. 5,699,157, which is hereby incorporated by reference in its entirety, provides a high speed target material for application in the fields of chemistry, biochemistry, biotechnology, molecular biology and many other areas. A microfluidic system for electrophoretic analysis is provided. The system has a channel in the equipment, a light source and a receiver. As the channel holds the substance of interest in the liquid in the electric field, the substance moves through the channel and separates into bands depending on the species. The light source excites the fluorescence in the species band, and the receiver is positioned to receive the fluorescence from the band. Since the system further comprises means for masking the channel, the receiver can receive fluorescence only in periodically spaced regions along the channel. The system also has a unit connected to analyze the modulation frequency of the light intensity received by the receiver, so as to determine the speed of the band along the channel and thereby enable the analysis of the substance.
[0087]
The system disclosed in U.S. Pat. No. 5,699,157 is also a method for performing a high speed electrophoretic analysis of a substance of interest, the step of retaining the substance of interest in a solution in a channel of a microfluidic system; Subjecting the substance of interest to travel through the channel and separate into species bands; directing light toward the channel; simultaneously receiving light from periodically spaced regions along the channel; Analyzing the frequency of the light intensity of the received light to determine the velocity of the band along the channel and analyzing the material. By determining the velocity of the species band, the electrophoretic mobility of the species is determined and the species is identified.
[0088]
U.S. Pat. No. 5,842,787, which is hereby incorporated by reference in its entirety, has at least a portion that has a greatly varied depth than those previously described (such as the device disclosed in U.S. Pat. No. 5,699,157). It is generally related to devices and systems that use channels, where the depth of the channel is not limited, but reduces sample perturbation, reduces non-specific sample mixing due to diffusion, and resolution. Provide a number of beneficial and unexpected results, such as improved performance.
[0089]
In another embodiment, the electrophoretic separation method comprises polyacrylamide gel electrophoresis. In a preferred embodiment, polyacrylamide gel electrophoresis is non-denaturing and allows the mobility of target RNA bound to a test compound to be distinguished from free target RNA. If the polyacrylamide gel electrophoresis is denatured, the target RNA: test compound complex must be cross-linked before electrophoresis to prevent dissociation of the target RNA during electrophoresis from the test compound. Such techniques are well-known to those skilled in the art.
[0090]
In one embodiment of the method of the present invention, the binding of the test compound to the target nucleic acid can be detected by gel electrophoresis analysis of interference footprinting, preferably in an automated manner. RNA can be degraded at specific base sites, including ribonuclease A, U_Two, CL_Three, T₁, Phy M, and enzymatic methods such as B. cereus or diethylpyrocarbonate, sodium hydroxide, hydrazine, piperidine formate, dimethyl sulfate, [2,12-dimethyl-3,7,11,17 -Tetraazacyclo [l1.3.1] heptadeca-1 (17), 2,11,13,15-pentaenato] nickel (II) (NiCR), cobalt (II) chloride, or iron (II) ethylenediaminetetraacetate (Fe -EDTA), which are described, for example, in Zheng et al., 1999, Biochem. 37: 2207-2214; Latham & Cech, 1989, Science 245: which is hereby incorporated by reference in its entirety. 276-282; and described in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, pp 12.61-12.73, Cold Spring Harbor Laboratory Press, and references cited therein. Have been. The specific pattern of the cleavage site is determined by the accessibility of the particular base to the reagent used to cause the cleavage, and thus, for example, by the three-dimensional structure of the RNA.
[0091]
The interaction of the small molecule with the target nucleic acid can alter the accessibility of the base to these cleavage reagents, either by causing a conformational change in the target nucleic acid or by covering the base at the binding interface. it can. As the test compound binds to the nucleic acid and the accessibility of the base to the cleavage reagent changes, the observed cleavage pattern changes. Using this method, small molecules and RNA can be used, for example, as described in Prudent et al., 1995, J. Am. Chem. Soc. 117: 10145-10146 and Mei et al., 1998, Biochem. 37: 14204-14212. Can be identified and characterized.
[0092]
In a preferred embodiment of this technique, a detectably labeled target nucleic acid is incubated with an individual test compound and then subjected to treatment with an enzymatic or chemical cleavage reagent. The reaction mixture may preferably be tested directly or may be further treated to isolate and concentrate nucleic acids. The generated fragments are separated by electrophoresis and the cleavage pattern can be compared to a cleavage reaction performed in the absence of the test compound. A change in the cleavage pattern directly indicates that the test compound binds to the target nucleic acid. Multiple test compounds can be tested both in parallel and sequentially.
[0093]
Other embodiments of electrophoretic separation include, but are not limited to, urea gel electrophoresis, gel filtration, pulsed field gel electrophoresis, two-dimensional gel electrophoresis, continuous flow electrophoresis, zone electrophoresis and agarose gel electrophoresis. Electrophoresis.
[0094]
5.5.2.Fluorescence spectroscopy
In a preferred embodiment, fluorescence polarization spectroscopy, ie, optical optics that can identify the percentage of either bound or unbound fluorescent molecules in solution (eg, a labeled target nucleic acid of the invention). Using the detection method, the reaction results can be read without electrophoretic separation of the sample. No. 5,842,787; 5,869,004; 5,876,675; 5,942,443; 5,948,227; 6,042,709; 6,042,710; 6,046,056; 6,048,498; 6,150,180; 6,153,073; 6,167,910; 6,171,850; 6,186,660 (the disclosures of which are incorporated by reference in their entirety) utilizing fluorescence polarization spectroscopy. And read the reaction result. The application of fluorescence polarization spectroscopy to the chip systems disclosed in the above-listed US patents is rapid, efficient, and well suited for high-throughput screening.
[0095]
In other embodiments, compounds that have affinity for the target nucleic acid of interest can be labeled with a fluorophore to screen for test compounds that bind to the target nucleic acid. For example, in a screen utilizing fluorescence quench technology in a 96-well plate format, pyrene-containing aminoglycoside analogs are used to identify antagonists that bind to the prokaryotic 16S rRNA A site, which contains the natural target of the aminoglycoside antibiotic. , Accurately monitored (Hamasaki & Rando, 1998, Anal. Biochem. 261 (2): 183-90).
[0096]
In another embodiment, test compounds that bind to a target nucleic acid can be screened using fluorescence resonance energy transfer (FRET). FRET, a characteristic change in fluorescence, occurs when two fluorophores with overlapping emission and excitation wavelength bands are held together close together, for example, by a binding event. In a preferred embodiment, the fluorophore on the target nucleic acid and the fluorophore on the test compound have overlapping excitation and emission spectra so that one fluorophore (donor) transfers its emission energy. To excite the other fluorophore (acceptor). The acceptor preferably emits light of a different wavelength upon relaxation to the ground state, or non-radiatively relaxes to quench fluorescence. FRET is very sensitive to the distance between two fluorophores and can measure molecular distances less than 10 nm. For example, U.S. Pat.No. 6,337,183 to Arenas et al., Which is incorporated by reference in its entirety, describes screening for compounds that bind RNA, using FRET to determine the effect of a test compound on the stability of a target RNA molecule. In the screening, the target RNA is labeled using both a fluorescent acceptor molecule and a donor molecule, and the fold structure of the RNA is measured by the distance between two fluorophores determined by FRET. Matsumoto et al. (2000, Bioorg. Med. Chem. Lett. 10: 1857-1861) label one end of a peptide that binds to HIV-1 TAR RNA with a fluorescein fluorophore and use the other end with tetramethyl A system for labeling with rhodamine is described. Changes in the conformation of the peptide upon binding to RNA provided a FRET signal to screen for compounds that bound to TAR RNA.
[0097]
In a preferred embodiment, both the target nucleic acid and the compound having affinity for the target nucleic acid of interest are labeled with fluorophores (donor and acceptor) having overlapping emission and excitation spectra, but the fluorescence Groups include, but are not limited to, fluorescein and derivatives, rhodamine and derivatives, cyanine dyes and derivatives, bora-3a, 4a-diaza-s-indacene (BODIPY®) and derivatives, pyrene, nanoparticles, Or non-fluorescent quenching molecules. Binding of the labeled test compound to the target nucleic acid can be identified by a change in fluorescence observable as a result of FRET.
[0098]
If the target nucleic acid is labeled with a donor fluorophore, the test compound is labeled with an acceptor fluorophore. Conversely, if the target nucleic acid is labeled with an acceptor fluorophore, the test compound will be labeled with a donor fluorophore. Various labels can be used, with the choice of label depending on the sensitivity required, ease of binding to the compound, stability requirements, available instruments, and disposal regulations. The fluorophore on the target nucleic acid must be close to the binding site of the test compound, but not incorporated into the specific binding site of the target nucleic acid where the test compound is expected to bind. This is because the presence of the covalently attached label can hinder the binding of the test compound at this site sterically or chemically.
[0099]
In yet another embodiment, a homogeneous time-resolved fluorescence ("HTRF") technique based on time-resolved energy transfer from a lanthanide ion complex to a suitable acceptor species is adapted for high-throughput screening for inhibitors of RNA protein complexes. (Hemmila, 1999, J. Biomol. Screening 4: 303-307; Mathis, 1999, J. Biomol. Screening 4: 309-313). HTRF is similar to fluorescence resonance energy transfer utilizing ordinary organic dye pairs, but has several advantages, such as increased sensitivity and efficiency, and background elimination (Xavier et al., 2000, Trends Biotechnol. 18 (8): 349-356).
[0100]
Fluorescence spectroscopy has traditionally been used to characterize DNA-protein and protein-protein interactions, whereas fluorescence spectroscopy has been less used to characterize RNA-protein interactions. The reason is that the absorption of RNA nucleotides interferes with the endogenous tryptophan fluorescence of the protein (Xavier et al., 2000, Trends Biotechnol. 18 (8): 349-356). However, fluorescence spectroscopy has been used to study single tryptophan residues within the arginine-rich RNA binding domain of the Rev protein and its interaction with RRE in time-resolved fluorescence studies (Kwon & Carson, 1998, Anal. Biochem. 264: 133-140). Thus, in the present invention, fluorescence spectroscopy is less preferred if the test compound or peptide or protein has endogenous tryptophan fluorescence. However, fluorescence spectroscopy can be used for test compounds that have no intrinsic fluorescence.
[0101]
5.5.3.Surface plasmon resonance (" SPR ")
By using surface plasmon resonance ("SPR") to track the association project in "real time", the kinetic and equilibrium constants of polymer interactions can be determined (Schuck, 1997). , Annu. Rev. Biophys. Biomol. Struct. 26: 541-566).
[0102]
The principle of SPR is summarized by Xavier et al. (Trends Biotechnol., 2000, 18 (8): 349-356) as follows. Total internal reflection occurs at the interface between two materials having different refractive indices. The electromagnetic field of the incident light seeps out across the interface as an evanescent wave and spreads over the surface and into the medium by hundreds of nanometers. Insertion of a thin foil of gold at the interface causes SPR due to the absorption of energy from evanescent waves by the free electron cloud (plasmon) of the metal. As a result of this absorption, there is a reduction in the intensity of the reflected light at a particular angle of incidence. The evanescent wave profile is precisely dependent on the refractive index of the medium being probed. Therefore, the angle at which absorption occurs is very sensitive to changes in the refractive index of the external medium. All proteins and nucleic acids are known to change the refractive index of water by the same amount per unit mass, regardless of their amino acid or nucleotide composition (the change in refractive index is different for proteins and nucleic acids). As the protein or nucleic acid content of the layer in the sensor changes, so does the refractive index. Typically, either in a flow cell (Biacore AB, Uppsala, Sweden) or in a stirred cuvette (Affinity Sensors, Santa Fe, New Mexico), immobilize one member of the complex to the dextran layer and then transfer the other member. Introduce into the solution. It has been confirmed that there is a linear correlation between the surface concentration of the protein or nucleic acid and the shift of the resonance angle, and this can be used to measure the kinetic rate constant and / or the equilibrium constant.
[0103]
In the present invention, the target RNA may be immobilized on the sensor surface by a streptavidin-biotin bond, the bond being disclosed by Crouch et al. (Methods Mol. Biol., 1999, 118: 143-160). Biotinylation of RNA can occur during or after synthesis, via conversion of the 3'-terminal ribonucleoside of the RNA to a reactive free amino group or at the 5'-terminal guanosine monophosphorothioate incorporated by T7 polymerase. Perform using. Using SPR, between the HIV Rev protein and RRE (Van Ryk & Venkatesan, 1999, J. Biol. Chem. 274: 17452-17463) and aminoglycoside antibiotics with RRE and model RNA from 16S ribosomal A site (Hendrix et al., 1997, J. Am. Chem. Soc. 119: 3641-3648; Wong et al., 1998, Chem. Biol. 5: 397-406) have stoichiometry and affinity of each interaction. Has been determined.
[0104]
In one embodiment of the present invention, the target nucleic acid is immobilized on the sensor surface (eg, by streptavidin-biotin binding) and SPR is utilized to determine (a) whether the target RNA binds to the test compound. And (b) the properties of the binding between the target nucleic acid of the present invention and the test compound can be further determined.
[0105]
5.5.4.Mass spectrometry
An automated method for analyzing mass spectrometer data that can analyze complex mixtures containing thousands of components and calibrate background noise, multiply charged peaks and atomic isotope peaks, No. 6,147,344, which is incorporated herein by reference in its entirety. The system disclosed in US Pat. No. 6,147,344 is a method of analyzing mass spectrometer data, in which a control sample measurement is performed to perform a background noise check. The values of peak height and width at each m / z ratio are stored in memory as a function of time. A mass spectrometer operation is performed on the material to be analyzed, and the peak height and width values at each m / z ratio versus time are stored in a second memory location. The mass spectrometer operation of the material to be analyzed is repeated a certain number of times, and the stored control sample value at each m / z ratio level at each time increment is subtracted from the value of each corresponding run. , Thus obtaining the difference value in the respective mass ratio for each of the plurality of runs at the respective time increments. If the MS value minus background noise does not exceed a preset value, no m / z ratio data points will be recorded, thus eliminating background noise, chemical noise and false positive peaks from the mass spectrometer data. You. The data stored for each of the multiple runs is then compared to a predetermined value at each m / z ratio, and the resulting set of peaks (already confirmed to exceed background) is Is stored at the m / z point where is significant.
[0106]
One possibility of using mass spectrometry in high-throughput screening is the integration of SPR and mass spectrometry. Attempted research techniques are direct analysis of analytes retained on a sensor chip and mass spectrometry of eluted analytes (Sonksen et al., 1998, Anal. Chem. 70: 2731-2736; Nelson & Krone, 1999, J. Mol. Recog. 12: 77-93). Interfacing the sensor chip with the mass spectrometer is particularly important for establishing high-throughput methods for analyzing biomolecular interactions and screening targets for small molecule inhibitors using SPR in combination with a mass spectrometer. And further development is needed in the reuse of sensor chips (Xavier et al., 2000, Trends Biotechnol. 18 (8): 349-356).
[0107]
In one embodiment of the present invention, the target nucleic acid complexed with the test compound can be measured by any of the mass spectrometry methods described above. In addition, mass spectrometry can be used to elucidate the structure of the test compound.
[0108]
5.5.5.Scintillation proximity assay (" SPA ")
The scintillation proximity assay ("SPA") is a method that can be used to screen for small molecules that bind to target RNA. SPA involves radiolabeling either the target RNA or the test compound and then quantifying binding to scintillant-impregnated beads or other members on the surface (Cook, 1996, Drug Discov. Today 1: 287. -294). Currently, fluorescence-based techniques are preferred for high-throughput screening (Pope et al., 1999, Drug Discov. Today 4: 350-362).
[0109]
Screening for small molecules that inhibit the Tat peptide: TAR RNA interaction has been performed using SPA, and inhibitors of that interaction have been isolated and characterized (Mei et al., 1997, Bioorg. Med. Chem. 5: 1173-1184; Mei et al., 1998, Biochemistry 37: 14204-14212). Similar techniques can be used to identify small molecules that directly bind to a preselected target RNA element and can be used in the present invention.
[0110]
If microplates are available where the scintillant is incorporated directly into the microtiter well plastic, SPA can be adapted for high-throughput screening (Nakayama et al., 1998, J. Biomol. Screening 3: 43-48). Thus, one embodiment of the present invention comprises (a) labeling a target nucleic acid with a radioactive or fluorescent label; (b) placing each test compound in a microtiter well coated with a scintillant. And contacting the labeled nucleic acid with the test compound under the conditions of being connected to the microtiter well; and (c) identifying the test compound by its position in the microplate. And identifying and quantifying the test compound bound to the target nucleic acid.
[0111]
5.5.6.NMR Structure-activity relationship by spectroscopy ( SAR )
NMR spectroscopy is a useful technique to identify complexed target nucleic acids by qualitatively determining changes in chemical shifts, specifically from distances measured using relaxation effects. Based techniques have been used to identify small molecule conjugates that are protein drug targets (Xavier et al., 2000, Trends Biotechnol. 18 (8): 349-356). Determination of the structure-activity relationship ("SAR") by NMR is the first technique described for NMR, in which small molecules that bind to adjacent subsites bind to the two-dimensional target protein.¹H-¹⁵It was identified from the N spectrum (Shuker et al., 1996, Science 274: 1531-1534). The signal from the bound molecule is monitored using line broadening, transition NOE, and pulsed field gradient diffusion measurements (Moore, 1999, Curr. Opin. Biotechnol. 10: 54-58). A strategy for generating leads by NMR using a library of small molecules has recently been reported (Fejzo et al., 1999, Chem. Biol. 6: 755-769).
[0112]
In one embodiment of the present invention, the target nucleic acid complexed with the test compound can be confirmed by SAR by NMR. Further, the structure of the test compound can be elucidated using SAR by NMR.
[0113]
5.5.7.Size exclusion chromatography
In another embodiment of the invention, a test compound bound to a target nucleic acid can be purified from a complex mixture of compounds using size exclusion chromatography. Size exclusion chromatography separates molecules based on their size and uses a gel-based medium consisting of beads with a particular size distribution. When this medium is applied to the column, it sediments into a densely packed matrix, forming an array of complex pores. Separation is achieved on the basis of molecular size by the uptake or exclusion of molecules by these pores. Small molecules are entrapped in the pores, such that their movement through the matrix is slowed by increasing the distance they must travel before eluting. When applied to the matrix, large molecules are locked out of the pores and move with the void volume. In the present invention, the target nucleic acid is incubated with the mixture of test compounds released in solution to reach equilibrium. When applied to a size exclusion column, free test compound in solution is retained by the column and test compound bound to the target nucleic acid passes through the column. In a preferred embodiment, bound test compounds are separated from unbound test compounds using spin columns commonly used for "desalting" nucleic acids (eg, Bio-Spin columns manufactured by BIO-RAD). . In other embodiments, the size exclusion matrix is loaded into a multi-well plate to allow for high-throughput separation of the mixture (eg, a PLASMID 96-well SEC plate manufactured by Millipore).
[0114]
5.5.8.Affinity chromatography
In one embodiment of the invention, affinity capture is used to purify test compounds bound to a target nucleic acid labeled with an affinity tag from a complex mixture of compounds. To do this, the target nucleic acid labeled with the affinity tag is incubated with a mixture of the free test compound in solution and then once equilibrium has been established, is captured on a solid support, or The target nucleic acid labeled by the affinity tag is captured on a solid support and then allowed to reach equilibrium with the mixture of test compounds.
[0115]
Solid supports typically consist of, but are not limited to, cross-linked agarose beads having attached a ligand for an affinity tag. Alternatively, the solid support has a self-assembled monolayer (SAM) having a covalently bound ligand for an affinity tag on the target nucleic acid or having a unique affinity for said tag, or It may be a solid glass, silicon, metal, or carbon, plastic (polystyrene, polypropylene) surface.
[0116]
Once the complex between the target nucleic acid and the test compound has reached equilibrium and has been captured, the solid support is washed with a large excess of binding buffer to retain the bound compound and retain unbound One skilled in the art will appreciate that removing the compound can be readily accomplished. In addition, retention of high affinity compounds and removal of low affinity compounds can be performed by various methods that increase the stringency of the wash. Such methods include, but are not limited to, increasing the number and time of washes, increasing the salt concentration of the wash buffer, adding detergents or detergents to the wash buffer, and washing nonspecific competitors. Addition to a buffer.
[0117]
In one embodiment, the test compound itself is detectably labeled with a fluorescent dye, radioisotope, or nanoparticle. The test compound is applied to the captured target nucleic acids in a spatially addressed manner (eg, in separate wells of a 96-well microplate), and binding between the test compound and the target nucleic acid is determined on the test compound. Can be confirmed using fluorescence by the presence of a detectable label.
[0118]
After removing unbound compounds, compounds that bind with high affinity to the target nucleic acid can be eluted from the immobilized target nucleic acid and analyzed. Elution of the test compound may be achieved by any method that disrupts the non-covalent interaction between the target nucleic acid and the compound. Methods of elution include, but are not limited to, changing the pH, changing the salt concentration, applying organic solvents, and applying molecules that compete with the bound ligand. In a preferred embodiment, the method used for elution releases the compound from the target RNA, but does not affect the interaction between the affinity tag and the solid support, thereby achieving selective elution of the test compound Can. Further, in a preferred embodiment, an elution buffer (eg, 0 M to 5 M ammonium acetate) is used that is volatile and allows subsequent concentration of the eluted compound by lyophilization.
[0119]
5.5.9.Nanoparticle aggregation
In one embodiment of the invention, both the target nucleic acid and the test compound are labeled with nanoparticles. Nanoparticles are clusters of ions with a controlled size of 0.1-1000 nm, including but not limited to Ag_TwoIt is made of a metal, a metal oxide, or a semiconductor containing S, ZnS, CdS, CdTe, Au, or TiO2. Methods for attaching nucleic acids and small molecules to nanoparticles are well known to those skilled in the art (reviewed in Niemeyer, 2001, Angew. Chem. Int. Ed. 40: 4129-4158. References cited therein). The literature is hereby incorporated by reference in its entirety). In particular, for example, as described by Mitchel et al. 1999, J. Am. Chem. Soc. 121: 8122-8123, multiple copies of a target nucleic acid are bound to a single nanoparticle and multiple copies of a test compound are When bound to another nanoparticle, the interaction between the test compound and the target compound induces aggregation of the nanoparticle. Aggregates can be detected by changes in absorbance or fluorescence spectra and physically separated from unbound components by filtration or centrifugation.
[0120]
5.6.Method for identifying or characterizing a test compound bound to a target nucleic acid
If the library comprises an array or microarray of test compounds, and each test compound has an address or identifier, the test compound is compared to the original list of compounds that applied the positive sample to each test assay. Can be analyzed by cross-reference.
[0121]
If the library is a peptide or nucleic acid library, the sequence of the test compound can be determined by direct peptide or nucleic acid sequencing. Such methods are well-known to those skilled in the art.
[0122]
Various physicochemical techniques can be used for de novo characterization of the test compound bound to the target.
[0123]
5.6.1.Mass spectrometry
Mass spectroscopy (eg, electrospray ionization (“ESI”) and matrix-assisted laser desorption ionization (“MALDI”), Fourier transform ion cyclotron resonance (“FT-ICR”)) can be used to identify high levels of test compounds that bind to target RNA. It can be used for both throughput screening and elucidation of the structure of a test compound. And in one example of mass spectroscopy, the separation of bound and unbound complexes and elucidation of the structure of the test compound can be performed in a single step.
[0124]
MALDI uses a pulsed laser and a time-of-flight analyzer for ion desorption and has been used to detect the tRNA: amino-acyl-tRNA synthetase complex in a non-covalent complex (Gruic-Sovulj et al.). , 1997, J. Biol. Chem. 272: 32084-32091). However, detection requires a covalent crosslink between the target nucleic acid and the test compound because non-covalent bonds can dissociate during the MALDI process.
[0125]
ESI mass spectrometry ("ESI-MS") has been very useful for studying non-covalent molecular interactions. This is because, unlike the MALDI method, ESI-MS generates molecular ions with little or no fragmentation (Xavier et al., 2000, Trends Biotechnol. 18 (8): 349-356). Complexes formed by HIV Tat peptides and proteins and TAR RNA have been studied using ESI-MS (Sannes-Lowery et al., 1997, Anal. Chem. 69: 5130-5135).
Fourier transform ion cyclotron resonance ("FT-ICR") mass spectrometry provides high resolution spectra, isotope-resolved precursor ion selection, and accurate mass assignment (Xavier et al., 2000, Trends Biotechnol. 18 (8 ): 349-356). The interaction of aminoglycoside antibiotics with cognate and non-cognate RNAs has been studied using FT-ICR (Hofstadler et al., 1999, Anal. Chem. 71: 3436-3440; Griffey et al., 1999, Proc. Natl. Acad. Sci. USA 96: 10129-10133). As with all of the mass spectrometry methods discussed herein, FT-ICR does not require labeling of the target RNA or test compound.
[0126]
The advantage of mass spectroscopy is that not only can the structure of the test compound be elucidated, but also the structure of the test compound bound to a preselected target RNA. Such information allows for the discovery of a consensus structure of a test compound that specifically binds to a preselected target RNA.
[0127]
5.6.2.NMR Spectroscopy
As described above, NMR spectroscopy is a technique for identifying a binding site in a target nucleic acid by qualitatively determining a change in a chemical shift from a distance measured by using a relaxation effect. Examples of NMR that can be used in the present invention include, but are not limited to, one-dimensional NMR, two-dimensional NMR, correlation spectroscopy (COSY), and nuclear Overhauser effect (“NOE”) spectroscopy. Such methods for determining the structure of a test compound are well known to those skilled in the art.
[0128]
As with mass spectroscopy, the advantage of NMR is that not only can the structure of the test compound be elucidated, but also the structure of the test compound bound to a preselected target RNA. Such information may allow for the discovery of a consensus structure of a test compound that specifically binds to a preselected target RNA.
[0129]
5.6.3.Vibrational spectroscopy
Vibrational spectroscopy (eg, infrared (IR) or Raman spectroscopy) can be used to elucidate the structure of the test compound on the isolated beads.
[0130]
Infrared spectroscopy is an infrared light (wavelength) that is absorbed by a test compound as a result of excitation of a vibrational mode that obeys the quantum mechanical selection rules that require that absorption of light cause a change in the electric dipole moment of the molecule. A frequency of 100 to 10,000 nm) is measured. The infrared spectrum of any molecule has a unique pattern of absorption wavelengths of various intensities, which can be viewed as molecular fingerprints to identify compounds.
[0131]
Infrared spectra were measured in standard mode (double beam instrument) or pre-measured in a scanning mode for the absorption by a test compound of individual frequency light provided by a grating that separated the frequency from a mixed frequency infrared light source. ("Blank") can be measured by measuring relative to intensity (single beam instrument). In a preferred embodiment, the infrared spectrum is measured in pulsed mode (FT-IR), wherein a mixed beam of all infrared light frequencies generated by the interferometer is passed or reflected by the test compound. The resulting interferogram (which may or may not include the interferogram from subsequent pulses in order to increase the signal strength when averaging random noise in the electronic signal) Converts mathematically to a spectrum using a Fourier transform or a fast Fourier transform algorithm.
[0132]
Raman spectroscopy measures the difference in frequency of visible scattered light or ultraviolet light due to absorption of infrared frequencies as compared to the incident beam. An incident monochromatic light beam, usually at a single laser frequency, is not actually absorbed by the test compound but temporarily interacts with the electric field. Although most of the light scattered from the sample remains unchanged (Rayleigh scattering), a portion of the scattered light may have a frequency that is the sum or difference between the incident frequency and the molecular vibration frequency. The selection rule for Raman (inelastic) scattering requires a change in the polarizability of the molecule. Some vibrational transitions are observable in both infrared and Raman spectroscopy, but some should only be observable using either technique. The Raman spectra of any molecule have absorption wavelengths of various intensities in unique patterns, which can be viewed as molecular fingerprints to identify compounds.
[0133]
Raman spectra are measured by illuminating monochromatic light through or preferably reflecting the sample, filtering Rayleigh scattered light, and detecting the frequency of the Raman scattered light. An improved Raman spectrometer is described in US Pat. No. 5,786,893 to Fink et al., Which is incorporated herein by reference.
[0134]
In vibration microscopy analysis, the integration of a visible microscope and a spectrometer allows measurements to be made in a spatially partitioned fashion, addressing single beads. Microscopic infrared spectrometers are described in US Pat. No. 5,581,085 to Reffner et al., Which is incorporated herein by reference in its entirety. An instrument for simultaneously performing micro-infrared and micro-Raman analysis of a sample is described in US Pat. No. 5,841,139 to Sostek et al., Which is incorporated herein by reference in its entirety.
[0135]
In a preferred embodiment, the test compound is identified by matching the IR or Raman spectrum of the test compound with a previously acquired vibrational (IR or Raman) spectrum data set for each compound in the combinatorial library. be able to. In this way, spectra of compounds with known structures are recorded so that when isolated from RNA binding experiments, the compounds can be re-identified by comparison to these spectra.
[0136]
5.7.Secondary biological screening
A test compound identified in a binding assay using a host cell that contains or has been engineered to contain a target RNA element associated with a functional radout system (for convenience, "lead (" lead) "compounds) can be tested for biological activity. For example, a lead compound can be tested in a host cell that has been genetically engineered to contain a target RNA element that controls the expression of a reporter gene. In this example, the lead compound is assayed in the presence or absence of the target RNA. Alternatively, a phenotypic readout or a physiological readout can be used to assess the activity of a target RNA in the presence and absence of a lead compound.
[0137]
In one embodiment, regulates the expression of reporter genes such as, but not limited to, β-galactosidase, green fluorescent protein, red fluorescent protein, luciferase, chloramphenicol acetyltransferase, alkaline phosphatase, and β-lactamase. Testing can be performed on host cells that have been genetically engineered to contain the target RNA element. In a preferred embodiment, the cDNA encoding the target element is fused upstream of the reporter gene, in which case, when the lead compound binds to the target RNA, translation of the reporter gene is suppressed. In other words, steric hindrance caused by the binding between the lead compound and the target RNA suppresses the translation of the reporter gene. This method, called the translation suppression assay ("TRAP"), has been demonstrated in E. coli and S. cerevisiae (Jain & Belasco, 1996, Cell 87 (1): 115-). 25; Huang & Schreiber, 1997, Proc. Natl. Acad. Sci. USA 94: 13396-13401).
[0138]
In other embodiments, phenotypic readouts or physiological readouts can be used to assess the activity of a target RNA in the presence and absence of a lead compound. For example, the target RNA may be overexpressed in cells that express the target RNA endogenously. If the target RNA controls the expression of a gene product involved in cell growth or viability, the in vivo effect of the lead compound can be assayed by measuring the cell growth or viability of the target cells. Alternatively, the reporter gene may be fused downstream of the target RNA sequence, and the effect of the lead compound on reporter gene expression can be assayed.
[0139]
Alternatively, lead compounds identified in a binding assay can be tested for biological activity using animal models for the disease, disease state, or symptom of interest. These animal models include animals that have been genetically engineered to contain a target RNA element associated with a functional readout system, such as a transgenic mouse. Animal model systems can also be used to demonstrate safety and efficacy.
[0140]
Compounds that exhibit the desired biological activity are considered to be lead compounds and can be used to design congeners or analogs that have useful pharmacological activity and physiological profiles. After the lead compound has been identified, variants of the lead that can be more effective can be designed using molecular modeling techniques that have proven useful in conjunction with synthetic studies. These uses include, but are not limited to, Pharmacophore Modeling (Lamothe, et al. 1997, J. Med. Chem. 40: 3542; Mottola et al. 1996, J. Med. Chem. 39: 285). Beusen et al. 1995, Biopolymers 36: 181; see P. Fossa et al. 1998, Comput. Aided Mol. Des. 12: 361), QSAR development (Siddiqui et al. 1999, J. Med. Chem. 42: 4122; Barreca et al. 1999). Bioorg. Med. Chem. 7: 2283; Kroemer et al. 1995, J. Med. Chem. 38: 4917; Schaal et al. 2001, J. Med. Chem. 44: 155; Buolamwini & Assefa 2002, J. Mol. Chem. 45 : 84), Virtual docking and screening / scoring (Anzini et al. 2001, J. Med. Chem. 44: 1134; Faaland et al. 2000, Biochem. Cell. Biol. 78: 415; Silvestri et al. 2000, Bioorg. Med. Chem. 8: 2305; see J. Lee et al. 2001, Bioorg. Med. Chem. 9:19) and, but not limited to, mFold (Zuker et al. Algorithm for structure prediction Thermodynamics: Practical Guide to RNA Secondary Structure Prediction: RNA Practical Guide in RNA Biochemistry and Biotechnology, pp.11-43, J. Barciszewski & BFC Clark, eds. (NATO ASI Series , Kluwer Academic Publishers, 1999) and Mathews et al., 1999 J. Mol. Biol. 288: 911-940); RNA motifs (Macmot et al., 2001, Nucleic Acids Res. 29: 4724-4735; and Vienna RNA package) (Hofacker et al. 1994, Monatsh. Chem. 125: 167-188).
[0141]
Further examples of the use of such techniques can be found in several review articles. These articles include Rotivinen et al., 1988, Acta Pharmaceutical Fennica 97: 159-166; Ripka, 1998, New Scientist 54-57; McKinaly & Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29: 111-122; Perry & Davies, "QSAR: Quantitative Structure-Activity Relationships in Drug Design (QSAR)" pp.189-193 (Alan R. Liss, Inc. 1989); Lewis & Dean, 1989, Proc. R. Soc. Lond. 236: 125-140 and 141-162; Askew et al., 1989, J. Am. Chem. Soc. 111: 1082-1090 and the like. Examples of molecular modeling tools used include Tripos, Inc., St. Louis, Missouri (for example, Sybyl / UNITY, CONCORD, DiverseSolutions), Accelerys, San Diego, California (for example, Catalyst, Wisconsin Package {BLAST and others}), Schrodinger, Portland, Oregon (eg, QikProp, QikFit, Jaguar) or other distributors (eg, BioDesign, Inc. (Pasadena, California), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge) , Ontario, Canada) and privately designed and / or "academic" software (eg, RNAMotif, mFOLD). These application suits and programs include tools for atomistic construction and analysis of drug-like molecules, proteins, and structural models for DNA or RNA and their potential interactions. These also include solubility calculations, permeability measurements, and "drugability" of molecules (eg, "Lipinski Rule of 5" in Lipinski et al. 1997, Adv. Drug Delivery Rev. 23: 3-25). It also provides calculations of important physical properties, such as experimental measurements of. Most importantly, they improve the desired physical properties as determined by the results obtained from the secondary screening protocols described above, while guiding the synthesis of more effective clinical development candidates. Assays and statistical modeling capabilities suitable for developing quantitative structure-activity relationships (QSARs) that are utilized in the patented Sylbyl CoMFA described in U.S. Patent Nos. 6,240,374 and 6,185,506 Technology).
[0142]
5.8.Identified, RNA Use of a compound that binds to the treatment / prevention of a disease
A biologically active compound or a pharmaceutically acceptable salt thereof identified using the method of the present invention may be a patient, preferably a mammal, suffering from a disease whose progress is associated with a target RNA: host cell factor in vivo interaction. It can be administered to animals, more preferably humans. In certain embodiments, such compounds, or pharmaceutically acceptable salts thereof, are used to treat a patient, preferably a mammal, more preferably a human, as a prophylactic treatment against a disease associated with RNA: host cell factor in vivo interaction. To be administered.
[0143]
In one embodiment, "treatment" or "treat" means amelioration of a disease, or amelioration of at least one identifiable symptom thereof. In other embodiments, "treatment" or "treat" refers to an improvement in at least one measurable physical parameter, which is not necessarily recognizable to the patient. In yet other embodiments, "treatment" or "treating" physically inhibits (e.g., stabilizes an identifiable condition) or physiologically inhibits (e.g., physical Parameter stabilization) or to suppress in both. In yet other embodiments, "treatment" or "treat" refers to delaying onset.
[0144]
In certain embodiments, the compound or a pharmaceutically acceptable salt thereof is administered to a patient, preferably a mammal, more preferably a human, as a prophylactic treatment for a disease associated with RNA: host cell factor in vivo interactions. Administer. As used herein, the terms "prevention" or "prevent" mean a reduced risk of developing a disease. In one embodiment, the compound or a pharmaceutically acceptable salt thereof is administered to a patient as a prophylactic treatment. In this embodiment, the patient may have a genetic predisposition to the disease, such as a family history of the disease, or a non-genetic predisposition to the disease. Thus, the compounds and their pharmaceutically acceptable salts may be used for treating one symptom of the disease and preventing another.
[0145]
When administered to a patient, the compound or a pharmaceutically acceptable salt thereof is preferably administered as a component of a composition optionally comprising a pharmaceutically acceptable vehicle. The composition can be administered orally or by any other convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucosal skin linings such as oral, rectal, and intestinal mucosa. It may be administered or together with other biologically active agents. Administration can be systemic or local. Various delivery systems are known, for example, encapsulation in liposomes, microparticles, microcapsules, capsules, and the like, which can be utilized to administer the compound and its pharmaceutically acceptable salts.
[0146]
The method of administration includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, By rectal, by inhalation or topically, especially to the ear, nose, eyes or skin. The mode of administration depends on the judgment of the physician. In most cases, administration will result in the release of the compound or a pharmaceutically acceptable salt thereof into the bloodstream.
[0147]
In certain embodiments, it may be desirable to administer the compound or a pharmaceutically acceptable salt thereof locally. This includes, but is not limited to, local injection during surgery, for example, local application in conjunction with wound dressing after surgery, injection, by use of catheters, by use of suppositories, or This can be achieved by using an implant that is a porous, non-porous or gelatinous material, including membranes or fibers, such as sialastic membranes.
[0148]
In certain embodiments, the compound or a pharmaceutically acceptable salt thereof is introduced into the central nervous system by any suitable route, including intraventricular, intrathecal, and epidural injection. It would be desirable. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a container such as an Ommaya reservoir.
[0149]
Pulmonary administration can also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosol, or by perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the compound and its pharmaceutically acceptable salts can be formulated as a suppository, with traditional binders and vehicles such as triglycerides.
[0150]
In other embodiments, the compounds and pharmaceutically acceptable salts thereof can be delivered in vesicles, particularly liposomes (Langer, 1990, Science 249: 1527-1533; Treat et al., "Infectious Diseases"). And Liposomes in the Therapy of Infectious Disease and Cancer ", Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid. , pp. 317-327; see supra, in general).
[0151]
In still other embodiments, the compounds and pharmaceutically acceptable salts thereof can be delivered in a controlled release system (eg, as described in Goodson, “Medical Applications of Controlled Release”). , Supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review of Langer, 1990, Science 249: 1527-1533 may be utilized. In one embodiment, a pump can be utilized (Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In other embodiments, polymeric materials can be utilized ("Medical Applications of Controlled Release", Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974). "Controlled Drug Bioavailability, Drug Product Design and Performance", Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: See also 105). In yet other embodiments, the controlled release system can be placed in close proximity to the target RNA of the compound or pharmaceutically acceptable salt thereof, requiring less than a systemic dose. .
[0152]
The composition comprising the compound or a pharmaceutically acceptable salt thereof ("the compound composition") is further pharmaceutically acceptable in a suitable amount to provide a dosage form for proper administration to the patient. Vehicle.
[0153]
In certain embodiments, the term “pharmaceutically acceptable” is approved by a federal or state government for use in animals, mammals, and more particularly in humans, or is approved by the United States Pharmacopeia or other general public. Means listed on a recognized pharmacopeia. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is administered. Such pharmaceutical vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. When administered to a patient, the pharmaceutically acceptable vehicle is preferably sterile. Water is a preferred vehicle when a compound of the present invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical vehicles also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene , Glycols, water, ethanol and the like. The compound compositions may, if desired, contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
[0154]
The compound composition may be in the form of a solution, suspension, emulsion, tablet, pill, pellet, capsule, capsule containing liquid, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, or use. Other dosage forms may be appropriate for the present invention. In one embodiment, the pharmaceutically acceptable vehicle is a capsule (see US Pat. No. 5,698,155). Other examples of suitable pharmaceutical vehicles are "Remington's Pharmaceutical Sciences," Alfonso R. Gennaro, eds., Mack Publishing Co. Easton, PA, 19th Edition, 1995, incorporated herein by reference. , pp. 1447-1676.
[0155]
In a preferred embodiment, the compound or a pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition adapted for oral administration to humans according to routine methods. Compositions for oral delivery may be in the form of, for example, tablets, troches, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more agents such as sweeteners, such as fructose, aspartame, or saccharin; flavoring agents, such as peppermint, wintergreen oil, or cherry, to provide a palatable preparation over the mouth. A colorant; and a preservative. In addition, for tablets or pills, the compositions may be coated so as to provide sustained action over a longer period of time by delaying disintegration and absorption in the gastrointestinal tract. Selectively permeable membranes surrounding compounds that are actively driven by osmotic pressure are also suitable for orally administered compositions. In these latter platforms, liquid from the environment surrounding the capsule is absorbed by the propellant compound, which swells and moves the drug or drug composition through the orifice. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profile of a direct release formulation. A time delay material such as glycerol monostearate or glycerol stearate may be employed. Oral compositions include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Preferably, such vehicles are of pharmaceutical quality. Typically, compositions for intravenous administration comprise a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent.
[0156]
In another embodiment, the compound or a pharmaceutically acceptable salt thereof can be formulated for intravenous administration. Compositions for intravenous administration optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the components are supplied separately or mixed together in unit dosage form, such as a dry frozen powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the quantity of active agent. You. When administering the compound or a pharmaceutically acceptable salt thereof by infusion, it may be dispensed, for example, with an infusion bottle containing sterile pharmaceutical quality water or saline. When administering the compound or a pharmaceutically acceptable salt thereof by injection, an ampoule of sterile water for injection or saline can be provided and the components can be administered after mixing.
[0157]
The amount of a compound or a pharmaceutically acceptable salt thereof that may be effective in treating a particular disease will depend on the nature of the disease, and can be determined by standard clinical techniques. In addition, in some cases, in vitro or in vivo assays may be employed to help determine optimal dosage ranges. The exact dose to be employed will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, a suitable dosage range for oral administration is generally from about 0.001 milligram to about 200 milligrams of the compound or a pharmaceutically acceptable salt thereof per kilogram of body weight per day. In certain preferred embodiments of the invention, the oral dose is from about 0.01 milligrams to about 100 milligrams per kilogram of body weight per day, more preferably, from about 0.1 milligrams to about 75 milligrams per kilogram of body weight, and more preferably. About 0.5 milligrams to about 5 milligrams per kilogram of body weight per day. The dosages described herein refer to the total amount administered. That is, when administering one or more compounds, or administering a compound with a therapeutic agent, the preferred dosage corresponds to the total amount administered. Oral compositions preferably contain from about 10% to about 95% by weight of the active ingredient.
[0158]
Suitable dosage ranges for intravenous (iv) administration are from about 0.01 milligrams to about 100 milligrams per kilogram of body weight per day, from about 0.1 milligrams to about 35 milligrams per kilogram of body weight per day, and from about 0.1 milligrams to about 35 milligrams per kilogram of body weight per day. 1 milligram to about 10 milligrams. Suitable dosage ranges for intranasal administration are generally about 0.01 pg to about 1 mg per kilogram of body weight per day. Suppositories generally contain from about 0.01 milligram to about 50 milligrams of the compound of the present invention per kilogram of body weight per day and the active ingredient in the range of about 0.5% to about 10% by weight.
[0159]
The recommended dosage for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, intracerebral, intravaginal, transdermal or inhalation administration is from about 0.001 milligram to about 0.001 milligram per day per kilogram of body weight. The range is 200 milligrams. Suitable doses for topical administration range from about 0.001 milligram to about 1 milligram, depending on the area of administration. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems. Such animal models and systems are well-known in the art.
[0160]
The compounds and their pharmaceutically acceptable salts are assayed for the desired therapeutic or prophylactic activity, preferably in vitro and in vivo, prior to use in humans. For example, in vitro assays may be used to determine whether it is preferable to administer the compound, its pharmaceutically acceptable salts, and / or other therapeutic agents. Animal model systems may be used to demonstrate safety and efficacy.
[0161]
Various compounds can be used to treat or prevent diseases in mammals. Compound types include, but are not limited to, peptides, unnatural amino acids (eg, D-amino acids, phosphorus analogs of amino acids such as α-aminophosphonic acid and α-aminophosphinic acid, or amino acids having non-peptide bonds). Peptide analogs, nucleic acids, nucleic acid analogs such as phosphorothioates or peptide nucleic acids ("PNA"), hormones, antigens, synthetic or natural drugs, opiates, dopamines, serotonin, catecholamines, Thrombin, acetylcholine, prostaglandins, organic molecules, pheromones, adenosine, sucrose, glucose, lactose and galactose.
【Example】
[0162]
6.Example: therapeutic target
The therapeutic targets reported herein are exemplary, and the invention is not limited by the targets described herein. It will be appreciated by those skilled in the art that the therapeutic targets reported herein as DNA sequences can be converted to RNA sequences.
[0163]
6.1.Tumor necrosis factor α (" TNF- α ”)
GenBank accession number X01394:

(SEQ ID NO: 6)
[0164]
General target area:
(1) 5 'untranslated region-nts 1-152
(2) 3 'untranslated region-nts 852-1643
Initial specific target motif:
Group I AU-rich element (ARE) cluster in the 3 'untranslated region
5 'AUUUAUUUAUUUAUUUAUUUA 3' (SEQ ID NO: 1)
6.2.Granulocytes - Macrophage colony stimulating factor (" GM-CSF ")
GenBank accession number NM_000758:

(SEQ ID NO: 7)
[0165]
GenBank accession number XM_003751:

(SEQ ID NO: 8)
[0166]
General target area:
(1) 5 'untranslated region-nts 1-32
(2) 3 'untranslated region-nts 468-78
Initial specific target motif:
Group IAU-rich element (ARE) cluster of 3 'untranslated region
5 'AUUUAUUUAUUUAUUUAUUUA 3' (SEQ ID NO: 1)
6.3.Interleukin 2 (" IL-2 ")
GenBank accession number U25676:

(SEQ ID NO: 9)
[0167]
General target area:
(1) 5 'untranslated region-nts 1-47
(2) 3 'untranslated region-nts 519-825
Initial specific target motif:
Group III AU-rich element (ARE) cluster in the 3 'untranslated region
5 'NAUUUAUUUAUUUAN 3' (SEQ ID NO: 10)
6.4.Interleukin 6 (" IL-6 ")
GenBank accession number NM_000600:

(SEQ ID NO: 11)
[0168]
General target area:
(1) 5 'untranslated region-nts 1-62
(2) 3 'untranslated region-nts 699-1125
Initial specific target motif:
Group IIIAU-rich element (ARE) cluster in the 3 'untranslated region
5 'NAUUUAUUUAUUUAN 3' (SEQ ID NO: 10)
6.5.Vascular endothelial growth factor (" VEGF ")
GenBank accession number AF022375:

(SEQ ID NO: 12)
[0169]
General target area:
(1) 5 'untranslated region-nts 1-701
(2) 3 'untranslated region-nts 1275-3166
Initial specific target motif:
(1) Internal ribosome entry site (IRES) in the 5 'untranslated region nts 513-704

(SEQ ID NO: 13)
[0170]
(2) 3 'untranslated region group III AU-rich element (ARE) cluster
5 'NAUUUAUUUAUUUAN 3' (SEQ ID NO: 10)
6.6.Human immunodeficiency virus (" HIV-1 ")
GenBank accession number NC_001802:

(SEQ ID NO: 14)
[0171]
Initial specific target motif:
(1) Transactivation response region / Tat protein binding site-TAR RNA-nts 1-60
"Minimal" TAR RNA element
5 'GGCAGAUCUGAGCCUGGGAGCUCUCUGCC 3' (SEQ ID NO: 15)
(2) Gag / Pol frameshift site-"minimal" frameshift element
5 'UUUUUUAGGGAAGAUCUGGCCUUCCUACAAGGGAAGGCCAGGGAAUUUUCUU 3' (SEQ ID NO: 16)
6.7.Hepatitis C virus (" HCV " - Genotype la and lb )
GenBank accession number NC_001433:

(SEQ ID NO: 17)
[0172]
General target area:
5 'untranslated region nts 1-328-Internal ribosome entry site (IRES)

(SEQ ID NO: 18)
[0173]
Initial specific target motif:
(1) HCV IRES subdomain IIIc-nts 213-226
5'AUUUGGGCGUGCCC3 '(SEQ ID NO: 19)
(2) Subdomain IIId-nts in HCV IRES 241-267
5'GCCGAGUAGUGUUGGGUCGCGAAAGGC3 '(SEQ ID NO: 20)
6.8.Ribonuclease P RNA (" RN Aase P ")
GenBank accession number
X15624 Homo sapiens RNase P H1 RNA:

(SEQ ID NO: 21)
[0174]
U64885 Staphylococcus aureus RNase P (rrnB) RNA:

(SEQ ID NO: 22)
[0175]
M17569 RNA component (M1 RNA) of Escherichia coli ribonuclease P (rnpB) gene:

(SEQ ID NO: 23)
[0176]
Z70692 Mycobacterium tuberculosis RNase P (rnpB) RNA:

(SEQ ID NO: 24)
[0177]
6.9.X-linked inhibitors of apoptotic proteins (" XIAP ")
GenBank accession number U45880:

(SEQ ID NO: 25)
[0178]
General target area:
Internal ribosome entry site (IRES) in the 5 'untranslated region:

(SEQ ID NO: 26)
[0179]
Initial specific target motif:
RNP core binding site in XIAP IRES
5'GGAUUUCCUAAUAUAAUGUUCUCUUUUU3 '(SEQ ID NO: 27)
6.10.Survivin ( Survivin )
GenBank accession number NM_001168:

(SEQ ID NO: 28)
[0180]
7.Example: Dye-labeled target bound to low molecular weight compound RNA Identification
The results reported in this example show that the binding of small molecules such as the Tat peptide and gentamicin to their respective target RNAs can be detected using a gel mobility shift assay.
[0181]
7.1.Materials and methods
7.1.1.buffer
Tris-potassium chloride (TK) buffer contains 50 mM Tris-HCl pH 7.4, 20 mM KC1, 0.1% Triton X-100, and 0.5 mM MgCl_TwoConsists of Tris-borate-EDTA (TBE) buffer consists of 45 mM Tris-borate pH 8.0, and 1 mM EDTA. Tris-potassium magnesium chloride (TKM) buffer is 50mM Tris-HCl pH 7.4, 20mM KC1, 0.1% Triton X-100 and 5mM MgCl_TwoConsists of
[0182]
7.1.1.Gel shift analysis ( gel retardation analysis )
RNA oligonucleotides were purchased from Dharmacon Inc. (Lafayette, CO). Oligonucleotides corresponding to the 5 'fluorescein-labeled 16S rRNA A site (5'-GGCGUCACACCUUCGGGUGAAGUCGCC-3' (SEQ ID NO: 29); Moazed & Noller, 1987, Nature 327: 389-394; Woodcock et al., 1991, EMBO J. 10 3099-3103; Yoshizawa et al., 1998, EMBO J. 17: 6437-6448), or an oligonucleotide corresponding to the 5 'fluorescein-labeled HIV-1 TAR element TAR RNA (5'-GGCGUCACACACCUUCGGGUGAAGUCGCC-3' (SEQ ID NO: 30) Huq et al., 1999, Nucleic Acids Research. 27: 1084-1093; Hwang et al., 1999, Proc. Natl. Acad. Sci. USA 96: 12997-13002).³²3'-labeled with P-cytidine 3 ', 5'-diphosphate (NEN) and T4 RNA ligase (NEBiolabs) in 10% DMSO according to the manufacturer's instructions. The labeled oligonucleotide was purified using a G-25 Sephadex column (Boehringer Mannheim). For the Tat-TAR gel retardation reaction, the method of Huq et al. (Nucleic Acids Research, 1999, 27: 1084-1093) was used to obtain 0.5 mM MgCl_TwoAnd a TK buffer containing a 12-mer Tat peptide (YGRKKRRQRRRP (SEQ ID NO: 31); one-letter amino acid code). For the 16S rRNA-gentamicin reaction, TKM buffer was used, utilizing the method of Huq et al. Within a 20 μl reaction volume,³²50 pmol of P-cytidine labeled oligonucleotide and gentamicin sulfate (Sigma) or short Tat peptide (Tat_47-58) Was heated in TK or TKM buffer at 90 ° C. for 2 minutes and allowed to cool at room temperature (approximately 24 ° C.) for 2 hours. Then, 10 μl of 30% glycerol was added to each reaction tube and all samples were loaded on a TBE non-denaturing polyacrylamide gel and electrophoresed at 1200-1600 volt-hour at 4 ° C. The gel was exposed to an intensifying screen and radioactivity was quantified using a Typhoon phosphor imager (Molecular Dynamics).
[0183]
7.2.background
One method used to demonstrate the interaction of small molecules with natural RNA structures such as ribosomes is a method called chemical footprint or toe printing (Moazed & Noller, 1987, Nature 327). Woodcock et al., 1991, EMBO J. 10: 3099-3103; Yoshizawa et al., 1998, EMBO J. 17: 6437-6448). Described herein is the use of a gel mobility shift assay to monitor RNA-small molecule interactions. This approach allows rapid visualization of small molecule-RNA interactions based on differences in mobility between RNA alone and small molecule-RNA complexes. To confirm this approach, RNA oligonucleotides corresponding to the gentamicin binding site on the well-characterized 16S rRNA (Moazed & Noller, 1987, Nature 327: 389-394) and similarly well-characterized HIV The HIV-1 TAT protein binding site on the -1 TAR element (Huq et al., 1999, Nucleic Acids Res. 27: 1084-1093) was selected. The purpose of these experiments is to lay the foundation for using high-throughput chromatography techniques such as microcapillary electrophoresis for drug discovery.
[0184]
7.3.result
Gel shift assay, Tat_47-58Performed using peptides and TAR RNA oligonucleotides. As shown in FIG. 1, when the products are separated on a 12% non-denaturing polyacrylamide gel, a clear shift is seen in the presence of the Tat peptide. In reactions lacking the peptide, only free RNA is seen. These observations confirm previous reports made with other Tat peptides (Hamy et al., 1997, Proc. Natl. Acad. Sci. USA 94: 3548-3553; Huq et al., 1999). , Nucleic Acids Res. 27: 1084-1093).
[0185]
Based on the results in FIG. 1, it was hypothesized that RNA interactions with small molecules could also be visualized using this method. As shown in FIG. 2, the addition of various concentrations of gentamicin to the RNA oligonucleotide corresponding to the 16S rRNA A site results in a mobility shift. These results demonstrate that binding of the small molecule gentamicin to RNA oligonucleotides with defined structures in solution can be monitored using this approach. Further, as shown in FIG. 2, gentamicin concentrations as low as 10 ng / ml cause mobility shifts.
[0186]
To determine if lower concentrations of gentamicin were sufficient to cause a gel shift, the same as shown in FIG. 2 except that the concentration of gentamicin was in the range of 100 ng / ml to 10 pg / ml. An experiment was performed. As shown in FIG. 3, gel mobility shift occurs even when the gentamicin concentration is as low as 10 pg / ml. In addition, the results shown in FIG. 3 demonstrate that the shift is specific to the 16S rRNA oligonucleotide, and that gel migration occurs when incubated with 10 pg / ml gentamicin using an irrelevant oligonucleotide corresponding to the HIV TAR RNA element. No degree shift occurs. In addition, if low concentrations of 10 pg / ml gentamicin cause a gel mobility shift, screening small amounts of compounds from a diverse library of compounds in this manner can detect changes to RNA structural motifs. Must be.
[0187]
Further analysis of the gentamicin-RNA interaction indicated that the interaction was Mg and temperature dependent. As shown in FIG._TwoIn the absence of (TK buffer), 1 mg / ml gentamicin must be added to the reaction to cause a gel shift.
[0188]
Similarly, the reaction temperature when adding gentamicin is important. When gentamicin is present in the reaction during the entire denaturation / regeneration cycle, ie when gentamicin is added to 90 ° C or 85 ° C, a gel shift is seen (data not shown). In contrast, when gentamicin is added after the regeneration step has proceeded to 75 ° C., no mobility shift occurs. These results are consistent with the idea that gentamicin can recognize and interact with RNA structures formed early in the regeneration process.
[0189]
8.Example: Dye-labeled target bound to a small molecule compound by capillary electrophoresis RNA Identification
The results reported in this example show that the interaction between a Tat peptide and a peptide, such as TAR RNA, and its target RNA can be monitored by a gel shift assay in an automated capillary electrophoresis system.
[0190]
8.1.Materials and methods
8.1.1.buffer
Tris-potassium chloride (TK) buffer contains 50 mM Tris-HCl pH 7.4, 20 mM KC1, 0.1% Triton X-100, and 0.5 mM MgCl_TwoConsists of Tris-borate-EDTA (TBE) buffer consists of 45 mM Tris-borate pH 8.0, and 1 mM EDTA. Tris-potassium magnesium chloride (TKM) buffer is 50mM Tris-HCl pH 7.4, 20mM KC1, 0.1% Triton X-100 and 5mM MgCl_TwoConsists of
[0191]
8.1.1.Gel shift analysis using capillary electrophoresis
RNA oligonucleotides were purchased from Dharmacon Inc. (Lafayette, CO). 5'-fluorescein-labeled HIV-1 TAR element TAR RNA (5'-GGCGUCACACCUUCGGGUGAAGUCGCC-3 '(SEQ ID NO: 30); Huq et al., 1999, Nucleic Acids Research. 27: 1084-1093; Hwang et al., 1999, Proc. Natl. Acad. Sci. USA 96: 12997-13002) was used. For the Tat-TAR gel shift reaction, the method of Huq et al. (Nucleic Acids Research, 1999, 27: 1084-1093) was used to obtain 0.5 mM MgCl 2._TwoAnd a TK buffer containing a 12-mer Tat peptide (YGRKKRRQRRRP (SEQ ID NO: 31); one-letter amino acid code). Within a 20 μl reaction volume, 50 pmol of labeled oligonucleotide and a short Tat peptide (Tat_47-58) Was heated in TK or TKM buffer at 90 ° C. for 2 minutes and allowed to cool to room temperature (approximately 24 ° C.) for 2 hours. The reaction solution was supplied on an SCE9610 automatic capillary electrophoresis apparatus (SpectruMedix; State College, Pennsylvania).
[0192]
8.2.result
As reported in the example in Section 7 above, the interaction between the peptide and RNA can be monitored by a gel shift assay. It was hypothesized that the interaction between the peptide and RNA could be monitored by an automated capillary electrophoresis system by a gel shift assay. To test this hypothesis, a gel shift assay using an automated capillary electrophoresis system was performed by Tat._47-58Performed using peptides and TAR RNA oligonucleotides. As shown in FIG. 5, a clear shift is seen when the concentration of the added Tat peptide is increased using the capillary electrophoresis system in the presence of the Tat peptide. In reactions lacking peptide, only peaks corresponding to free RNA are observed. These observations confirmed previous reports made with the Tat peptide (Hamy et al., 1997, Proc. Natl. Acad. Sci. USA 94: 3548-3553; Huq et al., 1999, Nucleic Acids Res. 27: 1084-1093).
[0193]
The invention is not to be limited in scope by the specific examples described herein. Indeed, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and accompanying drawings in addition to these descriptions. Such modifications are intended to fall within the scope of the appended claims.
[0194]
Various publications are cited herein, the disclosures of which are incorporated by reference in their entirety.
The present invention can be described by the following embodiments, which are listed in the following numbered paragraphs.
[0195]
1. A method for identifying a test compound that binds to a target RNA molecule, comprising: (a) combining a library of detectably labeled target RNA molecules and a test compound with a library of labeled target RNA and a test compound; Contacting under conditions that allow direct binding to the member to form a detectably labeled target RNA: test compound complex; (b) the detectably labeled target RNA formed in step (a) Separating the target RNA: test compound complex from the uncomplexed target RNA molecule and the test compound; and (c) determining the structure of the test compound bound to the RNA in the RNA: test compound complex The method comprising:
[0196]
2. The method of paragraph 1, wherein the target RNA molecule contains an HIV TAR element, an internal ribosome entry site, a "slippery site", an unstable element, or an adenylate uridylate-rich element.
[0197]
3. RNA molecules are tumor necrosis factor α (“TNF-α”), granulocyte-macrophage colony stimulating factor (“GM-CSF”), interleukin 2 (“IL-2”), interleukin 6 (“IL -6 "), vascular endothelial growth factor (" VEGF "), human immunodeficiency virus I (" HIV-1 "), hepatitis C virus (" HCV "-genotypes 1a and 1b), ribonuclease P RNA (" RN Parasite 1), which is an element derived from mRNA for apoptosis protein X-linked inhibitors ("XIAP"), or Survivin.
[0198]
4. Detectably labeled RNA is labeled with a fluorescent dye, phosphorescent dye, ultraviolet dye, infrared dye, visible dye, radioactive label, enzyme, spectral colorimetric label, affinity tag, or nanoparticle , Paragraph 1.
[0199]
5. The test compound is a peptoid; a random bio-oligomer; a diversomer such as hydantoin, benzodiazepine and dipeptide; a vinylog polypeptide; a non-peptide peptidomimetic; an oligocarbamate; a peptidyl phosphonate; a peptide nucleic acid library; Or a carbohydrate library; and a combinatorial library comprising a library of small organic molecules including, but not limited to, a library of benzodiazepines, isoprenoids, thiazolidinones, metathiazanones, pyrrolidines, morpholino compounds, or diazepindiones. The method of paragraph 1 selected from:
[0200]
6. Screening a library of test compounds comprises contacting the test compound with a target nucleic acid, preferably in the presence of an aqueous solution comprising a combination of buffers and salts that approximate or mimic physiological conditions. , Paragraph 1.
[0201]
7. The method of paragraph 6, wherein said aqueous solution optionally further comprises non-specific nucleic acids, including DNA, yeast tRNA, salmon sperm DNA, homoribopolymer, and non-specific RNA.
[0202]
8. The method of paragraph 6, wherein said aqueous solution further comprises a buffer, a combination of salts, and optionally, a detergent or surfactant. In other embodiments, the aqueous solution further comprises about 0 mM to about 100 mM KCl, about 0 mM to about 1 M NaCl, and about 0 mM to about 200 mM MgCl._TwoOr a combination of salts of In a preferred embodiment, the salt combination is about 100 mM KCl, about 500 mM NaCl, and about 10 mM MgCl_TwoIt is. In another embodiment, the solution optionally comprises about 0.01% to about 0.5% (w / v) of a detergent or surfactant.
[0203]
9. Any method of detecting alteration of the physical properties of the target nucleic acid complexed with the test compound from the unbound target nucleic acid may be used to form a complex with the complexed target nucleic acid in the method of paragraph 1. Untargeted nucleic acids may be separated. In a preferred embodiment, electrophoresis is used to separate a complexed target nucleic acid and a non-complexed target nucleic acid. In a preferred embodiment, the electrophoresis is capillary electrophoresis. In other embodiments, utilizing fluorescence spectroscopy, surface plasmon resonance, mass spectrometry, scintillation proximity assays, structure-activity relationships by NMR spectroscopy ("SAR"), size exclusion chromatography, affinity chromatography, and nanoparticle aggregation The complexed target nucleic acid and the uncomplexed target nucleic acid are separated.
[0204]
10. The structure of the test compound in the RNA: test compound complex of paragraph 1 is determined, in part, by the type of library of test compounds. In a preferred embodiment where the combinatorial library is a small organic molecule library, the structure of the test compound is determined using mass spectroscopy, NMR, or vibrational spectroscopy.
[Brief description of the drawings]
[0205]
FIG. 1 shows a gel shift analysis to detect peptide-RNA interactions. Increasing concentrations (0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM) of Tat in TK buffer_47-5850 pmol TAR RNA oligonucleotide was added to the 20 μl reaction containing the peptide. The reaction mixture was then heated at 90.degree. C. for 2 minutes and gradually cooled to 24.degree. 10 ml of 30% glycerol was added to each sample and applied to a 12% native polyacrylamide gel. The gel was electrophoresed in TBE buffer at 4 ° C. and 1200 volt-hour. After electrophoresis, the gel was dried and the radioactivity was quantified using a phosphor imager. The concentration of added peptide is shown above each lane.
FIG. 2 shows that gentamicin interacts with an oligonucleotide corresponding to 16S rRNA. In a TKM buffer, a 20 μl reaction solution containing increasing concentrations of gentamicin (1 ng / ml, 10 ng / ml, 100 ng / ml, 1 μg / ml, 10 μg / ml, 50 μg / ml, 500 μg / ml) was added to an RNA oligonucleotide. In addition to 50 pmol, the mixture was heated at 90 ° C for 2 minutes and gradually cooled to 24 ° C. Then 10 ml of 30% glycerol was added to each sample and applied to a 13.5% non-denaturing polyacrylamide gel. The gel was electrophoresed in TBE buffer at 4 ° C. and 1200 volt-hour. After electrophoresis, the gel was dried and the radioactivity was quantified using a phosphor imager. The concentration of gentamicin added is indicated above each lane.
FIG. 3 shows that the presence of gentamicin 10 pg / ml results in a gel mobility shift in the presence of 16S rRNA oligonucleotide. A 20 μl reaction containing increasing concentrations of gentamicin (100 ng / ml, 10 ng / ml, 1 ng / ml, 100 pg / ml, and 10 pg / ml) was added to 50 pmol of RNA oligonucleotide in TKM buffer and described in FIG. Treated as follows.
FIG. 4 shows MgCl_TwoShows weak binding of gentamicin to the 16S rRNA oligonucleotide in the absence of. The reaction mixture containing gentamicin (1 mg / ml, 100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml, and 10 ng / ml) was added to a TKM buffer containing MgCl 2._TwoWas processed as described in FIG. 2 except that it did not contain
FIG. 5 is a gel shift analysis to detect peptide-RNA interactions. Increasing concentrations (0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM) of Tat in TK buffer_47-5850 pmol of TAR RNA oligonucleotide was added to the reaction solution containing the peptide. The reaction mixture was then heated at 90 ° C. for 2 minutes and gradually cooled to 24 ° C. The reaction solution was supplied to an SCE9610 automatic capillary electrophoresis apparatus (SpectruMedix; State College, Pennsylvania). The peak corresponds to the amount of free TAR RNA ("TAR") or Tat-TAR complex ("Tat-TAR"). The concentration of added peptide is shown below each lane.

Claims (1)

A method for identifying a test compound that binds to a target RNA molecule, comprising:
(a) Detectable by contacting a library of test compound with a detectably labeled target RNA molecule under conditions that allow direct binding of the labeled target RNA to a member of the library of test compound libraries Forming a target RNA: test compound complex labeled at:
(b) separating the detectably labeled target RNA: test compound complex formed in step (a) from the uncomplexed target RNA molecule and the test compound by capillary gel electrophoresis;
(c) The method comprising determining the structure of the test compound bound to the RNA in the RNA: test compound complex by mass spectroscopy.

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