JP2005509447A - Targeting cells with MAD2 mutations for the treatment and / or prevention of diseases - Google Patents

Abstract

The present invention relates to compositions and methods for identifying one or more second drug targets and their use in identifying a drug or drug candidate, particularly for the treatment of cancer or cell replication disorders. Synthetic lethal screening based on yeast is used to functionally identify and confirm new gene targets for killing cells that are defective in the cell cycle pathway. These newly identified gene targets can be used to develop new therapies to treat cancer, benign cell proliferation, and yeast infections.

Description

【００５７】
そのような治療的利用の他に、そのような示差スクリーニングアッセイ法によって開発された抗真菌剤を、例えば食料品における保存剤、家畜の体重増加を促進するための飼料添加剤、または非生物材料の処理、例えば病院機器および病室の汚染除去のための消毒製剤として用いることができる。
【００５８】
同様にして、哺乳類遺伝子およびショウジョウバエ（Drosophila）のような昆虫遺伝子の阻害を並べて比較すると、本発明の誘導体の中からヒト／哺乳類の酵素および昆虫酵素とを識別する阻害剤を選択することが可能となると考えられる。したがって、本発明は、ショウジョウバエ（Drosophila）のような昆虫の管理のために用いられるように、殺虫剤における本発明の用途および製剤を広く意図する。
【００５９】
さらにもう一つの態様において、本発明の阻害剤化合物のいくつかを、哺乳類の相対物と比較して植物遺伝子に対する阻害特異性に基づいて選択することができる。例えば、植物酵素を阻害するための最大の選択性を有する化合物を選択するために、一つまたは複数のヒト酵素によるディファレンシャルスクリーニングにおいて、植物遺伝子を配置することができる。したがって、本発明は枯葉剤等の形態など農業的応用のための本発明の阻害剤の製剤を特に意図している。
【００６０】
もう一つの局面において、本発明は、一つまたは複数の薬学的に許容されうる担体（添加剤）および／または希釈剤と共に製剤化した、一つまたは複数の治療的有効量の上記の化合物を含む、薬学的に許容されうる組成物を提供する。以下に詳細に説明するように、本発明の薬学的組成物を、以下に関して適合させたものを含む、固体状または液体状での投与のため特に製剤化してもよい：（1）経口投与、例えば飲薬（水溶液もしくは非水溶液、または懸濁液）、錠剤、巨丸剤、粉剤、顆粒、舌に適用するためのペースト；（2）非経口投与としては、例えば滅菌溶液または懸濁液としての皮下注射、筋肉内注射、または静脈内注射；（3）局所適用、例えば、皮膚に適用されるクリーム、軟膏、もしくはスプレーとして；または（4）膣内もしくは直腸内、例えば、ペッサリー、クリーム、もしくはフォームとして。
【００６１】
本明細書において用いられる「治療的有効量」という用語は、第一の遺伝子（MAD1、MAD2、またはその類似体もしくは相同体）に変異を含む動物における細胞の少なくとも一つの亜集団において標的の第二の遺伝子の発現活性を調節し、それによる処理細胞における該タンパク質活性の生物学的帰結を、任意の医学的処置に応用可能な妥当な恩典／リスク比で、妨害することによって、何らかの所望の治療効果を得るために有効である本発明の化合物を含む、化合物、材料、または組成物の量を意味する。
【００６２】
「薬学的に許容される」という用語は、本明細書において、健全な医学的判断の範囲内で、過度の毒性、刺激、アレルギー反応、またはその他の問題もしくは合併症がなく、妥当な利益／リスク比に比例して、ヒトおよび動物の組織と接触して用いることに適している化合物、材料、組成物および／または剤形を意味するために用いられる。
【００６３】
本明細書において用いられる「薬学的に許容される担体」という用語は、一つの臓器、または体内の一部分から体内の他の臓器または他の部分に対象の薬剤を運ぶまたは輸送することに関係する、液体または固体の充填剤、希釈剤、賦形剤、溶媒または封入材料のような、薬学的に許容される材料、組成物、または媒体を意味する。それぞれの担体は、製剤の他の成分と適合性があり、患者に対して有害でないという意味において「許容され」なければならない。薬学的に許容される担体として役立ちうる材料のいくつかの例には：（1）乳糖、ブドウ糖、およびショ糖のような糖；（2）コーンスターチおよびジャガイモデンプンのようなデンプン；（3）セルロースおよび、カルボキシメチルセルロースナトリウム、エチルセルロース、および酢酸セルロースのようなその誘導体；（4）粉末トラガカント；（5）麦芽；（6）ゼラチン；（7）タルク；（8）カカオバターおよび坐剤ロウのような賦形剤；（9）ピーナッツ油、綿実油、ひまわり油、ゴマ油、オリーブ油、トウモロコシ油、および大豆油のような油脂；（10）プロピレングリコールのようなグリコール；（11）グリセリン、ソルビトール、マンニトール、およびポリエチレングリコールのようなポリオール；（12）オレイン酸エチルおよびラウリン酸エチルのようなエステル；（13）寒天；（14）水酸化マグネシウムおよび水酸化アルミニウムのような緩衝剤；（15）アルギン酸；（16）発熱物質不含水；（17）等張生理食塩水；（18）リンゲル液；（19）エチルアルコール；（20）リン酸緩衝液；ならびに（21）薬学的製剤において用いられる他の非毒性適合性物質が含まれる。
【００６４】
上記のように、本発明のチェックポイントタンパク質ならびに上記および下記の他のタンパク質の特定の態様は、アミノ基またはアルキルアミノ基のような塩基性官能基を含んでもよく、したがって、薬学的に許容される酸によって薬学的に許容される塩を形成することができる。この点において、「薬学的に許容される塩」という用語は、本発明の化合物の比較的非毒性の無機酸付加塩および有機酸付加塩を意味する。これらの塩を、本発明の化合物の最終的な単離および精製の際にインサイチューで、または遊離状態の本発明の精製化合物を適切な有機酸または無機酸と個々に反応させ、形成された塩を単離することによって、調製することができる。代表的な塩には、臭化水素塩、塩化水素塩、硫酸塩、亜硫酸塩、リン酸塩、硝酸塩、酢酸塩、吉草酸塩、オレイン酸塩、パルミチン酸塩、ステアリン酸塩、ラウリン酸塩、安息香酸塩、乳酸塩、リン酸塩、トシラート、クエン酸塩、マレイン酸塩、フマル酸塩、コハク酸塩、酒石酸塩、ナフチル酸塩、メシレート、グルコヘプト酸塩、ラクトビオネート、およびラウリルスルホン酸塩等が含まれる。
【００６５】
他の例において、本発明の化合物は、一つまたは複数の酸性官能基を含んでもよく、したがって、薬学的に許容される塩基によって薬学的に許容される塩を形成することができる。これらの例において「薬学的に許容される塩」という用語は、本発明の化合物の比較的非毒性の無機塩基付加塩および有機塩基付加塩を意味する。これらの塩はさらに、化合物の最終的な単離および精製の際にインサイチューで調製することができ、または酸解離状態の精製化合物を、薬学的に許容される金属陽イオンの水酸化物、炭酸塩または炭酸水素塩のような適した塩基と、アンモニアと、または薬学的に許容される有機一級アミン、二級アミン、もしくは三級アミンと個々に反応させることによって、調製することができる。代表的なアルカリ塩類またはアルカリ土類塩には、リチウム塩、ナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩、およびアルミニウム塩等が含まれる。塩基付加塩の形成にとって有用な代表的な有機アミンには、エチルアミン、ジエチルアミン、エチレンジアミン、エタノールアミン、ジエタノールアミン、ピペラジン等が含まれる。
【００６６】
ラウリル硫酸ナトリウムおよびステアリン酸マグネシウムのような湿潤剤、乳化剤、および潤滑剤、ならびに着色剤、放出剤、コート剤、甘味料、香料、保存剤および抗酸化剤も同様に組成物中に存在しうる。
【００６７】
薬学的に許容される抗酸化剤の例にはいかが含まれる：（1）アスコルビン酸、水酸化システイン、重硫酸ナトリウム、メタ重硫酸ナトリウム、亜硫酸ナトリウム等のような水溶性抗酸化剤；（2）アスコルビン酸・パルミテート、ブチルヒドロキシアニソール（BHA）、ブチルヒドロキシトルエン（BHT）、レシチン、没食子酸プロピル、α-トコフェロール等のような脂溶性抗酸化剤；および（3）クエン酸、エチレンジアミン四酢酸（EDTA）、ソルビトール、酒石酸、リン酸等のような金属キレート剤が含まれる。
【００６８】
本発明の製剤には、経口投与、鼻腔内投与、局所投与（頬および舌下を含む）、直腸投与、膣投与、および／または非経口投与に適した製剤が含まれる。製剤は、便宜上単位剤形で存在してもよく、薬学の技術分野において公知の任意の方法によって調製されうる。単一の剤形を生成するために担体材料と組み合わせることができる活性成分の量は、治療される宿主、特定の投与様式に応じて変化すると考えられる。単一の剤形を作製するために担体材料と組み合わせることができる活性成分の量は、一般的に治療効果を生じる化合物の量であると考えられる。一般的に、100％のうち、この量は活性成分の約1％〜約99％、好ましくは約5％〜約70％、最も好ましくは約10％〜約30％の範囲内であろう。
【００６９】
これらの製剤または組成物を調製する方法には、本発明の化合物を担体と、および任意で一つまたは複数の付属成分と結合させる段階が含まれる。一般的に、本発明の化合物を液体担体、または細粒固体担体またはその双方と均一に完全に結合させ、次に必要であれば産物を成形することによって、製剤が調製される。
【００７０】
経口投与に適した本発明の製剤は、活性成分として本発明の化合物の規定量をそれぞれ含む、カプセル剤、カシェ剤、丸剤、錠剤、（香料基剤、通常、ショ糖およびアカシアゴムまたはトラガカントを用いる）ロゼンジ、粉剤、顆粒剤の形状で、または水溶液もしくは非水溶液中での溶液もしくは懸濁液として、または水中油型もしくは油中水型の乳剤として、またはエリキシル剤もしくはシロップ剤として、または（ゼラチンおよびグリセリン、またはショ糖およびアカシアゴムなどの不活性基材を用いる）トローチとしておよび／またはマウスウオッシュ等としての形状であってもよい。本発明の化合物はまた、ボーラス、舐剤、またはペーストとして投与されうる。
【００７１】
経口投与のための本発明の固体剤形（カプセル剤、錠剤、丸剤、糖衣丸、粉剤、顆粒等）において、活性成分を、クエン酸ナトリウムもしくはリン酸二カルシウム、および／または以下のいずれかのような、一つまたは複数の薬学的に許容される担体と混合する：（1）デンプン、乳糖、ショ糖、ブドウ糖、マンニトール、および／またはケイ酸のような充填剤または増量剤；（2）例えば、カルボキシメチルセルロース、アルギネート、ゼラチン、ポリビニルピロリドン、ショ糖および／またはアカシアゴムのような結合剤；（3）グリセロールのような湿潤剤；（4）寒天-寒天、炭酸カルシウム、ジャガイモまたはタピオカデンプン、アルギン酸、特定のケイ酸塩、および炭酸ナトリウムのような崩壊剤；（5）パラフィンのような溶液遅延剤；（6）四級アンモニウム化合物のような吸収加速剤；（7）例えば、セチルアルコールおよびモノステアリン酸グリセロールのような湿潤剤；（8）カオリンおよびベントナイト粘土のような吸収剤；（9）タルク、ステアリン酸カルシウム、ステアリン酸マグネシウム、固体ポリエチレングリコール、ラウリル硫酸ナトリウム、およびその混合物などの潤滑剤；ならびに（10）着色剤。カプセル、錠剤、および丸剤の場合、薬学的組成物はまた緩衝剤を含んでもよい。類似のタイプの固体組成物も同様に、乳糖または牛乳の糖と共に、高分子量ポリエチレングリコール等のような賦形剤を用いて軟および硬充填ゼラチンカプセルにおいて充填剤として用いられうる。
【００７２】
錠剤を、任意に一つまたは複数の補助成分と共に、圧縮または成型によって作製してもよい。圧縮錠剤を、結合剤（例えば、ゼラチンまたはヒドロキシプロピルメチルセルロース）、潤滑剤、不活性希釈剤、保存剤、崩壊剤（例えば、デンプングリコール酸ナトリウムもしくは架橋したカルボキシメチルセルロースナトリウム）、界面活性剤または分散剤を用いて調製してもよい。成型錠剤を、適切な機械において不活性液体希釈剤によって湿らせた粉末化合物の混合物を成型することによって作製してもよい。
【００７３】
本発明の薬学的組成物の錠剤、ならびに糖衣丸、カプセル剤、丸剤および顆粒のようなその他の固体剤形を、任意に、腸溶コーティングおよび製薬技術分野で公知の他のコーティングのようなコーティングおよび外被と共に得ることができる、または調製することができる。それらをまた、例えば、所望の放出プロフィールを提供するための様々な比率のヒドロキシプロピルメチルセルロース、他のポリマーマトリクス、リポソームおよび／またはミクロスフェアを用いることによって、その中の活性成分の遅いまたは制御された放出を提供するように製剤化してもよい。それらを例えば、細菌残留フィルターによる濾過によって、または使用直前に滅菌水または他の滅菌注射可能媒体に溶解することができる滅菌固体組成物の形で滅菌物質を組み入れることによって、滅菌してもよい。これらの組成物はまた、任意に不透明化物質を含んでもよく、そして胃腸管の特定の部位に限って、またはそれらの部位に選択的に、ゆっくりと活性成分を放出する組成物であってもよい。用いることができる包埋組成物の例には、ポリマー物質およびロウが含まれる。活性成分はまた、適当であれば、上記の賦形剤の一つまたは複数を有する、微小封入体の形状でありうる。
【００７４】
本発明の化合物を経口投与するための液体剤形には、薬学的に許容される乳剤、微小乳剤、溶液、懸濁液、シロップ剤、およびエリキシル剤が含まれる。活性成分の他に、液体剤形は、例えば水および他の溶媒のような当技術分野で一般的に用いられる不活性希釈剤、エチルアルコール、イソプロピルアルコール、炭酸エチル、酢酸エチル、ベンジルアルコール、ベンジル安息香酸、プロピレングリコール、1,3-ブチレングリコール、油脂（特に綿実油、落花生油、胚芽油、オリーブ油、ヒマシ油およびゴマ油）、グリセロール、テトラヒドロフリルアルコール、ポリエチレングリコール、およびソルビタンの脂肪酸エステル、ならびにそれらの混合物のような可溶化剤および乳化剤を含んでもよい。
【００７５】
不活性希釈剤の他に、経口組成物はまた、湿潤剤、乳化剤、および懸濁化剤、甘味料、着香料、着色料、香料ならびに保存剤のようなアジュバント（adjuvant）を含みうる。
【００７６】
懸濁剤は、活性化合物の他に、例えば、エトキシル化イソステアリルアルコール、ポリオキシエチレンソルビトール、およびソルビタンエステル、微結晶性（microcrystalline）セルロース、メタ水酸化アルミニウム、ベントナイト、寒天-寒天およびトラガカント、ならびにそれらの混合物などの、懸濁化剤を含んでもよい。
【００７７】
直腸投与または経膣投与のための本発明の薬学的組成物の製剤は、坐剤として示されうり、これは例えば、室温では固体であるが、体温では液体であり、したがって直腸または膣腔内で融解して活性化合物を放出するカカオバター、ポリエチレングリコール、坐剤用ロウ、またはサリチル酸塩を含む一つまたは複数の適切な非刺激性の賦形剤または担体と、本発明の一つまたは複数の化合物を混合することによって調製されうる。
【００７８】
経膣投与に適した本発明の製剤にはまた、当技術分野において適当であることが知られているような担体を含む、ペッサリー、タンポン、クリーム、ゲル、ペースト、フォーム、またはスプレー製剤が含まれる。
【００７９】
本発明の化合物の局所投与または経皮投与のための剤形には、粉末、スプレー、軟膏、ペースト、クリーム、ローション、ゲル、溶液、パッチ、および吸入剤質が含まれる。活性化合物を、滅菌条件下で、薬学的に許容される担体および必要でありうる任意の保存剤、緩衝剤、または噴射剤と共に混合してもよい。
【００８０】
軟膏、ペースト、クリームおよびゲルは、本発明の活性化合物に加えて、動物性脂肪および植物性脂肪、油脂、ロウ、パラフィン、デンプン、トラガカント、セルロース誘導体、ポリエチレングリコール、シリコン、ベントナイト、ケイ酸、タルク、および酸化亜鉛、またはそれらの混合物のような賦形剤を含んでもよい。
【００８１】
粉剤およびスプレーは、本発明の化合物の他に、乳糖、タルク、ケイ酸、水酸化アルミニウム、ケイ酸カルシウム、およびポリアミド粉末、またはそれらの物質の混合物のような賦形剤を含みうる。スプレーは、クロロフルオロヒドロカーボン、ならびにブタンおよびプロパンのような揮発性の非置換炭化水素のような通常の噴射剤をさらに含みうる。
【００８２】
経皮パッチは、本発明の化合物の体内への制御された送達を提供する、さらなる長所を有する。そのような剤形は、適当な媒体に化合物を溶解または分散させることによって作製することができる。化合物の皮膚を介した流入を増加させるために吸収促進剤も同様に用いることができる。そのような流入の速度を、速度調節膜を提供することによって、またはポリマーマトリクスまたはゲル中に化合物を分散させることによって、制御することができる。
【００８３】
眼科用製剤、眼軟膏、点眼液、粉剤、移植片等も同様に、本発明の範囲内であることが意図されている。
【００８４】
非経口投与に適した本発明の薬学的組成物は、抗酸化剤、緩衝剤、静菌剤、意図されたレシピエントの血液と等張な製剤にする溶質、懸濁剤もしくは濃化剤を含んでもよい、一つもしくは複数の薬学的に許容される滅菌等張水溶液もしくは非水溶液、分散液、懸濁液、もしくは乳液、または使用直前に滅菌注射可能溶液または分散剤に戻してもよい滅菌粉末と組み合わせた、本発明の一つもしくは複数の化合物を含む。
【００８５】
本発明の薬学的組成物において用いられうる適切な水溶性担体または非水溶性担体の例には、水、エタノール、（グリセロール、プロピレングリコール、ポリエチレングリコール等のような）ポリオール、およびそれらの適切な混合物、オリーブ油のような植物油、およびオレイン酸エチルのような注射可能な有機エステルが含まれる。適当な流動性を、例えば、レシチンのようなコーティング材料を用いることによって、分散剤の場合には必要な粒子径を維持することによって、および界面活性剤を用いることによって、維持することができる。
【００８６】
これらの組成物はまた、保存剤、湿潤剤、乳化剤および分散剤のようなアジュバントを含んでもよい。微生物の作用の阻害は、例えばパラベン、クロロブタノール、フェノールソルビン酸等のような様々な抗菌剤および抗真菌剤を含めることによって、確保されうる。同様に、糖、塩化ナトリウム等のような等張剤が組成物に望ましく含まれうる。さらに、注射可能な薬学的形態の持続的な吸収は、モノステアリン酸アルミニウムおよびゼラチンのような吸収を遅らせる物質を含めることによってもたらされうる。
【００８７】
場合によっては、薬物の効果を持続させるために、皮下注射または筋肉内注射からの薬物の吸収を遅らせることが望ましい。これは、水溶性が低い結晶またはアモルファス材料の液体懸濁液を用いることによって達成されうる。薬物の吸収速度はその溶解速度に依存し、その結果、結晶の大きさおよび結晶形に依存しうる。または、非経口投与剤形の吸収の遅延は、油性媒体に薬物を溶解または懸濁することによって行われる。
【００８８】
注射可能デポー剤形は、本発明の化合物の微小封入マトリクスを、ポリラクチド-ポリグリコリドのような生体分解性のポリマーにおいて形成することによって作製される。ポリマーに対する薬物の比、および用いる特定のポリマーの特性に応じて、薬物の放出速度を制御することができる。その他の生体分解性ポリマーの例には、ポリ（オルトエステル）およびポリ（無水物）が含まれる。デポー注射可能製剤はまた、生体組織と適合性であるリポソームまたは微小乳液中に薬物を捕獲することによって調製される。
【００８９】
本発明の化合物を医薬品としてヒトおよび動物に投与する場合、それ自体を投与するか、または例えば0.1％〜99.5％（より好ましくは0.5％〜90％）の活性成分を薬学的に許容される担体と組み合わせて含む、薬学的組成物として投与することができる。
【００９０】
本発明の調製物を、経口投与、非経口投与、局所投与、または直腸内投与してもよい。それらは当然、それぞれの投与経路に適した形状で投与される。例えば、それらは、錠剤、またはカプセル剤、注射剤、吸入剤、眼科用ローション、軟膏、坐剤等、注射、注入、または吸入投与；ローションまたは軟膏による局所投与；および坐剤による直腸内投与によって投与される。経口投与が好ましい。
【００９１】
本明細書において用いられる「非経口投与」および「非経口的に投与する」という用語は、腸内および局所投与以外の投与様式、通常注射による投与を意味し、静脈内、筋肉内、動脈内、髄腔内、嚢内、眼窩内、心臓内、皮内、腹腔内、経気管、皮下、表皮下、関節内、嚢下、くも膜下、脊髄内、および槽内の注射および注入が含まれるがこれらに限定されない。
【００９２】
本明細書において用いられる「全身投与」、「全身に投与する」、「末梢投与」、および「末梢に投与する」という用語は、本明細書において、これが患者の神経系に入って、したがって代謝および他の類似の過程に供されるように、中枢神経系に直接投与する以外の化合物、薬物、または他の材料を投与すること、例えば皮下投与を意味する。
【００９３】
これらのペプチドおよび化合物を、例えばスプレーによる経口投与、鼻腔内投与、直腸内投与、膣内投与、非経口投与、槽内投与、および粉末、軟膏、または点眼液のような、経頬投与および舌下投与を含む局所的投与を含む、任意の適切な経路によって治療するために、ヒトおよびその他の動物に投与してもよい。
【００９４】
選択した投与経路にかかわらず、適切な水和型で用いられうる本発明の化合物および／または本発明の薬学的組成物は、当業者に公知である従来の方法によって薬学的に許容される投与剤形に製剤化される。
【００９５】
本発明の薬学的組成物における活性成分の実際の投与レベルを、患者に対して毒性を示さずに、特定の患者、組成物および投与様式に対して所望の治療反応を得るために有効な活性成分の量を得るために変更してもよい。
【００９６】
選択した用量レベルは、用いる本発明の特定の化合物、もしくはそのエステル、塩、もしくはアミドの活性、投与経路、投与時期、用いられる特定の化合物の排泄速度、治療期間、他の薬物、用いられる特定の化合物と組み合わせて用いられる化合物および／もしくは材料、治療すべき患者の年齢、性別、体重、状態、全身健康、および既往、ならびに医学の技術分野において公知の類似の要因を含む、様々な要因に依存すると考えられる。
【００９７】
当技術分野において通常の技術を有する医師または獣医師は、必要とされる薬学的組成物の有効量を容易に決定して処方することができる。例えば、医師または獣医師は薬学的組成物において用いられる本発明の化合物の用量を、所望の効果を得るために必要な用量より低いレベルで開始して、所望の効果が得られるまで用量を徐々に増加させることができる。
【００９８】
一般的に、本発明の化合物の適切な一日量は、治療効果を生じるために有効な最低用量である化合物の量であると考えられる。そのような有効量は、一般的に上記の要因に依存すると考えられる。一般的に、示された鎮痛効果のために用いる場合、本発明の化合物の患者への静脈内投与量、脳室内投与量および皮下投与量は、1日あたり体重1キログラムあたり約0.0001mg〜約100mgの範囲であると考えられる。
【００９９】
望ましいならば、活性化合物の有効な一日量を、1日を通じて適当な間隔で個別に2回、3回、4回、5回、6回またはそれ以上に分けて、任意に単位剤形で投与してもよい。
【０１００】
本発明の化合物を単独で投与することができるが、薬学的製剤（組成物）として化合物を投与することが好ましい。
【０１０１】
有糸分裂チェックポイントに欠損が起こると、染色体の喪失、細胞周期の乱れ、および多倍数体をもたらす。これらの欠損は腫瘍発生において役割を果たしうる。Mad1およびMAD2は、有糸分裂チェックポイントの制御を補助する遺伝子である。したがって、MAD1またはMAD2に変異を有する細胞をターゲティングすることは、癌の予防において実行可能な戦略である。
【０１０２】
MAD1および／またはMAD2における変異と不適合な変異を有する遺伝子は、有糸分裂チェックポイントの欠損を有する細胞を特異的に根絶して、機能的有糸分裂チェックポイントを有する細胞を残すことができる化合物に対する潜在的な薬物標的を提供する。そのような遺伝子における変異は、MAD1および／またはMAD2によって合成的に致死性である。
【０１０３】
本発明を全般的に記述したが、これは本発明の特定の局面および態様を説明する目的のために単に含まれるのであって本発明を制限することを意図していない、以下の実施例を参考にしてより容易に理解されるであろう。
【０１０４】
実施例1．MAD1変異によって合成的に致死性である遺伝子
以下の株を試験した：

【０１０５】
したがって、4つの遺伝子における変異（TUB1、CIN8、STU1、およびSFI1）は、MAD1変異によって合成的に致死である。すなわち、これらの株は、TUB1、CIN8、SFI1、またはSTU1プラスミドによってそれぞれ形質転換されない限り、MAD1プラスミドがないと増殖できない。TUB1はαチューブリンをコードする。CIN8は、キネシン関連微小管運動タンパク質をコードする。STU1は、微小管関連タンパク質をコードする。SFI1は、新規タンパク質をコードする。
【０１０６】
実施例2．MAD2によって合成的に致死性である遺伝子のスクリーニング
プラスミドをMAD2遺伝子に関する酵母細胞変異体に導入する。このプラスミドは、Ty1と呼ばれる著しいレトロトランスポジションエレメントの発現および転移（transposition）を可能にする。ゲノムへの転移後、細胞の大部分が、ゲノムにランダムに挿入されたトランスポゾンの一つまたはいくつかのコピーを有する。細胞において、トランスポゾンのコピーが遺伝子に挿入されれば、その不活化がNAD2によって合成的に致死となり、MAD2および第二の遺伝子の双方がこの細胞内で不活化されるために、そのような細胞は分裂を停止すると考えられる。本発明者らは、数世代にわたって細胞を増殖させることによってそのような転移を見出す。そのような転移を有し、かつ分裂しない細胞は、活発に分裂する細胞の集団から急速に失われる。増殖選択機構の後、本発明者らは、選択した遺伝子におけるそのような転移の有無に関してゲノム全体を調査する。転移が無作為な過程であるため、特定の遺伝子にそのような転移が存在しなければ、この遺伝子は合成致死の候補となる。
【０１０７】
本実験は、機能的MAD2を有する細胞における平行選択によって制御される。そのような細胞では、機能的MAD2を有する細胞が増殖選択を生き残るために、（第二の）合成致死遺伝子候補において転移が起こるはずである。
【０１０８】
次に、MAD2による新規合成致死遺伝子候補を遺伝的に確認する。
【０１０９】
実施例3．微小管重合化、紡錘極体、微小管運動、および動原体の欠損は全て、紡錘体チェックポイントを活性化することによって有糸分裂中に細胞を停止させる
材料および方法：
材料および方法に関する詳細な説明に関しては、その全体が参照として本明細書に組み入れられる、ハードウィック（Hardwick）, K.G.、リ（Li）, R.、ミストロット（Mistrot）, C.、チェン（Chen）, R. H.、ダン（Dann）, P.、ラドナー（Rudner）, A.、およびミュレイ（Murray）, A. W.、「多くの異なる紡錘体成分における障害は、出芽酵母サッカロミセス・セレビジエ（Saccharomyces serevisiae）における紡錘体チェックポイントを活性化する（Lesions in many different spindle components activate the spindle checkpoint in the budding yeast Saccharomyces serevisiae）」、Genetics 152、509〜518（1999）を参照されたい。S288Cと同系であるmps2-1およびtub2-403株ならびにそれらの誘導体を除き、用いた株は全てW303（MATa ade2-1 can1-100 ura3-1、leu2-3、112his3-11、15trp1-1）と同系である。
【０１１０】
MAD依存的チェックポイントを活性化する有糸分裂欠損の種類を調べるために、mad変異と、有糸分裂の特定の段階の実行において欠損を引き起こす変異との相互作用を調べる。これらには微小管脱重合を阻害する変異、紡錘極体複製を防止する変異、中心体結合タンパク質を除去する変異、および37℃で細胞を有糸分裂中に停止させるcdc変異が含まれる。有糸分裂変異を、2つの種類、すなわち条件的致死変異および非必須遺伝子におけるヌル変異において試験する。mad変異と、C2または有糸分裂中に停止を引き起こすcdc変異との相互作用を決定するために、mad cdc二重変異体の特性をcdc変異体単独の特性と比較する。cdc変異体の細胞周期停止点が紡錘体チェックポイントに依存していなければ、cdc mad二重変異体は、cdc変異体単独と全く同じ挙動を示す；細胞周期が非許容温度で停止し、許容温度でインキュベートすると、単一変異体および二重変異体が同じ速度で生存力を喪失する。これに対して、cdc変異体の停止が紡錘体チェックポイントに依存していれば、チェックポイントの不活性化により、たとえ紡錘体が欠損していても、mad cdc二重変異体は制限温度において有糸分裂すると考えられる。したがって、そのようなcdc変異をmad変異と組み合わせると、株を非許容温度でインキュベートした場合に、有糸分裂中に停止できず、cdc変異体単独より速く生存力を喪失する二重変異体が産生されると考えられる。
【０１１１】
実施例4．分析した遺伝子の誘導型発現に基づく合成致死スクリーニング
微小管運動が欠損している半数体をmad株と交配させることにより、実験を実施する。好ましい態様において、mad変異に関して既にヘテロ接合である株において微小管運動遺伝子を喪失させるために、遺伝子破壊を用いて、ヘテロ接合性を作製する実験を行う。この後者の方法は、kar3のような増殖の遅い株が抑制性変異を獲得する可能性を消失させるが、これはより面倒な方法である。
【０１１２】
劣性の合成致死変異を含むハイブリッドを野生型株と交配させて、胞子を形成させ、解剖し、胞子4連菌を、合成致死表現型の分離に関する適当な単一変異パターンに関してチェックする。そのような分離パターンを示す変異体を相補性によってクローニングする。酵母をスクリーニングするこの方法および他の方法の変法は公知であり（例えば、米国特許第5,912,154号；第5,908,752号；第5,876,951号；第5,869,287号；第5,866,338号；第5,789,184号；第5,674,996号；第5,578,477号；第5,527,896号；第5,352,581号；第5,175,091号；および第5,139,936号を参照のこと）、特定の目的に関してどれを選択するかを当業者は容易に理解している。
【０１１３】
実施例5．ヒトcDNAライブラリーによる酵母株の相補
本実施例において、異常なMAD1またはMAD2を有する酵母における上記の合成致死の代理になるタンパク質をコードするヒトcDNAが、それによって同定される原理を提供する。異常なMAD1またはMAD2を含む酵母株を構築する。ヒト胎児線維芽細胞から調製したmRNAを用いるcDNAライブラリーを、ヒトcDNAの発現のためのADHのような構成性酵母プロモーターを含む酵母菌（S. serevisiae）ベクターにおいて構築する（詳細に関しては、例えば米国特許第5,783,661号；第5,952,195号を参照のこと）。ライブラリーを宿主MAD1酵母株またはMAD2酵母株にトランスフェクトして、独立した形質転換体10⁵個をレプリカ平板法によってグルコースまたはガラクトース上での増殖能に関してスクリーニングする。その結果、増殖パターンがヒトcDNAの発現によって救出されるいくつかの形質転換体が単離される。それらのいくつかは既に公知の遺伝子、例えばCDC14のヒト相同体、アベルソンマウス白血病ウイルス腫瘍遺伝子のヒト相同体ABL1、線維芽細胞増殖因子受容体bfr-2であるが、いくつかはあまり知られておらず、タンパク質キナーゼのeph/elkファミリーにおける上皮細胞受容体、カルシウムチャネルタンパク質、またはヒトWee1キナーゼのような遺伝子に関連するか、または「相同」であるように思われる。
【０１１４】
実施例6．薬学的アンタゴニストおよびアゴニストのスクリーニング
本発明において同定された細胞周期またはDNA複製障害を治療するために有用な薬物を、現在では上記の確立された酵母モデルを用いてスクリーニングすることができる。化学化合物、有機化合物および無機化合物、ペプチド、またはペプチド模倣物、アンチセンス分子、抗体等のようないくつかの種類の薬物をスクリーニングすることができる。化学ライブラリーのハイスループットスクリーニングを作製および実施する方法は十分に確立されており、当業者に公知である。細胞周期調節において得られた見通しを用いて、酵母に基づくアッセイ法において選択された候補物質をスクリーニングするため、かつ哺乳類細胞または細胞株をさらに調べることによってその治療的可能性を評価するための薬物発見プログラムを確立する。本質的に、アッセイ法は、致死性を観察する対照系および薬物が添加される試験系からなり、その効果は対照系と試験系間の差に基づいて評価される。薬物が致死性を増強するか、またはこれを逆転させれば、この薬物は第一の遺伝子および第二の遺伝子によって制御される生化学経路において特異的である可能性がある、候補薬物と見なされる。特異性は多くの特異性決定試験、例えば、標的遺伝子またはその産物との結合能によって、当初認められた効果を逆転させる二重変異体の導入によって、遺伝子産物の酵素活性に及ぼす影響に関するインビトロ試験等によって、さらに試験される。これらの薬学的薬剤は、予測可能的に細胞周期の局面を理想的に阻害して、それによって癌またはその他の細胞周期関連臨床状態における有望性の決定を可能にすると考えられた。試験した化合物のいくつかは、薬学的に許容される濃度で活性であることが判明する。これらには、トリブチレート（4-フェニル酪酸ナトリウム塩、またはナトリウムフェニルブチレート）、当技術分野で公知の手段によって調製される様々なベンゾピラン誘導体が含まれるが、これらに限定されない。例えば、米国特許第5,359,115号；第5,362,899号；第5,288,514号；第5,733,920号または国際公開公報第94/08051号；国際公開公報第92/10092号；国際公開公報第93/09668号；国際公開公報第91/07087号；国際公開公報第93/20242号を参照のこと。したがって、本発明の化合物の1000個〜100,000個以上のオーダーのディバーソマー（diversomer）の多様なライブラリーを合成することができ、PCT国際公開公報第94/09135号に記載のように、阻害剤を検出するためのハイスループットアッセイ法を用いることによって、生物活性を迅速にスクリーニングすることができる。組み合わせ（combinatorial）合成方法の概論、ならびにライブラリースクリーニングおよび脱重畳積分解析方法に関しては、例えばE. M. ゴードン（Gordon)ら、（1994）J. Med. Chem. 37：1385〜1401、およびそれらに引用された参考文献を参照されたい。
【０１１５】
実施例7．良性細胞増殖または悪性細胞増殖による臨床病態の治療
次に、酵母に基づくアッセイ法において陽性とスクリーニングされた薬物を、過度の細胞増殖によって引き起こされる疾患の治療について試験する。そのような疾患のほとんどが悪性疾患、すなわち多様な種類の癌であり、以下のような固形腫瘍および白血病であるが、これらに限定されない：アプドーマ、分離腫、鰓腫、悪性カルチノイド症候群、カルチノイド心疾患、癌（例えば、ウォーカー癌、基底細胞癌、基底有棘細胞癌、ブラウン-ピアース（Brown-Pearce）癌、腺管癌、エールリッヒ腫瘍、クレブス2腫瘍、メルケル細胞腫、粘液性癌腫、非小細胞肺癌、燕麦細胞癌、乳頭状癌、硬性癌、細気管支、細気管支癌、気管支原生癌、扁平上皮細胞癌、および移行上皮癌）、組織球障害、白血病（例えば、B細胞白血病、混合型白血病、ヌル細胞白血病、T細胞白血病、慢性T細胞白血病、HTLV-II関連白血病、急性リンパ性白血病、慢性リンパ性白血病、肥満細胞性白血病、および骨髄性白血病）、悪性組織球増殖症、ホジキン病、免疫増殖性小（immunoproliferative small）、非ホジキンリンパ腫、形質細胞腫、細網組織増殖症、黒色腫、軟骨芽細胞腫、軟骨腫、軟骨肉腫、線維腫、線維肉腫、巨細胞腫、組織球腫、脂肪腫、脂肪肉腫、中皮腫、粘液腫、粘液肉腫、骨腫、骨肉腫、ユーイング肉腫、滑膜腫、腺線維腫、腺リンパ腫、癌肉腫、脊索腫、頭蓋咽頭腫、未分化胚細胞腫、過誤腫、間葉腫、中腎腫、筋肉腫、エナメル上皮腫、セメント質腫、歯牙腫、奇形種、胸腺腫、栄養膜腫（trophoblastic tumor）、腺癌、腺腫、胆管腫、コレステリン腫、円柱腫、嚢胞腺癌、嚢胞腺腫、顆粒膜細胞腫、男性胚細胞腫、肝癌、汗腺腫、島細胞腫（islet cell tumor）、ライディヒ細胞腫（Leydig cell tumor）、乳頭腫、セルトリ細胞腫、卵胞膜細胞腫、平滑筋腫、平滑筋肉腫、筋原細胞腫、筋腫、筋肉腫、横紋筋腫、横紋筋肉腫、脳室上皮腫、神経節細胞腫、神経膠腫、髄芽細胞腫、髄膜腫、神経鞘腫（neurilemmoma）、神経芽腫、神経上皮腫、神経線維腫、神経腫、傍神経節腫、非クロム親和性傍神経節腫（paraganglioma nonchromaffin）、被角血管腫、好酸球増多随伴性血管類リンパ組織増殖症、硬化性血管腫（angioma sclerosing）、血管腫症、グロムス血管腫、血管内皮腫、血管腫、血管外皮細胞腫、血管肉腫、リンパ管腫、リンパ管筋腫（lymphangiomyoma）、リンパ管肉腫、松果体腫、癌肉腫、軟骨肉腫、葉状嚢肉腫、線維肉腫、血管肉腫、平滑筋肉腫、白血肉腫、脂肪肉腫、リンパ管肉腫、筋肉腫、粘液肉腫、卵巣癌、横紋筋肉腫、肉腫、新生物（例えば、骨新生物、胸部新生物、消化器系新生物、結腸直腸新生物、肝臓新生物、膵臓新生物、下垂体新生物、精巣新生物、眼窩新生物、頭頸部新生物、中枢神経新生物、耳道（acoustic）新生物、骨盤新生物、気道新生物、および尿性器新生物）、神経線維腫症、子宮頸部異形成。
【０１１６】
いくつかの疾患は、過度だが良性の細胞増殖（すなわち、非悪性）によって起こる。そのような疾患の例は、以下である：線維症、良性前立腺過形成、アテローム性動脈硬化症、再狭窄、糸球体硬化症、ケロイド、乾せん、黒子、角化症、線維性軟ゆう、伝染性軟属腫、性病いぼ、皮脂腺過形成（sebaceous hyperplasia）、尖圭コンジローム、血管腫、静脈湖、軟骨皮膚炎（chondrodermatitis）、化膿性肉芽腫、化膿性汗腺炎、ケロイド、角化棘細胞腫、白斑症、多発性皮脂嚢腫、睫毛乱生症、表層上皮母斑（superficial epithelial nevus）、ポリープ、接合部母斑（junctional nevus）、化膿性肉芽腫、結節性痒疹、皮膚線維腫、脂腺腫、およびその他の皮膚疾患、ならびに例えばカポジ肉腫、乳頭腫のような非悪性新生物疾患。
【０１１７】
次に、ナトリウムフェニルブチレートまたはベンゾピラン誘導体の効果を、インビボにおけるヒト神経膠芽細胞腫細胞の腫瘍発生表現型について決定する。フィッシャー344系ラットの右脳半球の深部白質に同系9L神経膠肉腫細胞（4×10⁴個）を接種した。その後、皮下に移植した浸透圧ミニポンプを用いて、動物をナトリウムフェニルブチレート（550 mg/kg/日、皮下投与）による2週間連続治療に供した。対照のラットにおいてはミニポンプを生理食塩水で満たした。データの統計分析には、フィッシャーの正確検定（Fisher's Exact Test）を用いた。薬物による神経膠芽腫細胞の処理によって、時間および用量依存的な増殖の停止が起こり、これに付随して同様にDNA合成も減少した。4mMフェニルブチレートによる連続的な処理を4日〜6日行うと、増殖が約50％阻害される。フェニルアセテートの中間代謝物であるフェニルブチレートは腫瘍細胞複製を阻害したが、最終代謝物であるフェニルアセチルグルタミンは不活性である。選択的な腫瘍の細胞分裂の停止に加えて、フェニルアセテートとフェニルブチレートはいずれも、無胸腺マウスにおける細胞骨格中間体フィラメントのパターンの変化、接着独立性の喪失、および腫瘍形成性の減少によって明らかになるように、細胞の成熟および非悪性表現型への変換を促進した。腫瘍の挙動におけるこれらの顕著な変化は、増殖制御、血管新生、および免疫抑制に関係するサイクリン遺伝子の発現の変化を伴う。
【０１１８】
上記に引用したインターネットの典拠、特許、出版物、およびその中に含まれる引用文献は全て、それぞれの全体を参照するために特に参照として本明細書に組み入れられる。
【０１１９】
以上のように本発明を説明したが、これが様々に変化させられうることは明らかであろう。そのような変更は、本発明の趣旨および範囲から逸脱しないと見なされ、そのような改変は全て、添付の特許請求の範囲に含まれると解釈される。[0001]
1. Field of Invention
The present invention relates to compositions and methods of identifying eukaryotic genes that are important in the cell life cycle. More specifically, the invention relates to yeast genes and mammalian counterparts such as human genes that are essential in cell growth and death. Methods and compositions are provided for identifying and treating cell cycle-related gene dysfunctions in which diseases and clinical conditions such as malignancies and other related diseases occur.
[0002]
2. Background of the Invention
Genetic manipulation of the yeast genome provides a convenient model for identifying essential genes required for eukaryotic cell replication, proliferation and death. The complete DNA sequence of Saccharomyces serevisiae strain S288C was determined in April 1996 by international cooperation by more than 100 laboratories. A database containing general information and a yeast genome of about 6000 genes has been published, for example: http://www.ncbi.nlm.nih.gov/Yeast; http://genome-www.stanford.edu/Saccharomyces / Can be found on public websites. Other databases exist as well, and links to these databases and other websites are equally suitable for the purposes of the present invention. By way of example, Yeast GenBank (a collection of all genbank sequences from Saccharomyces serevisiae); Swiss plot from Saccharomyces serevisiae, which is incorporated herein by reference; Yeast Swiss-Prot (Saccharomyces serevisiae) A collection of protein sequences); including, but not limited to, YPD (yeast protein database maintained by Proteome, Inc.), and periodic updates thereof. Methods for manipulating yeast are well established and known to those skilled in the art and can be found on public websites by using appropriate keywords, such as the keywords yeast and protocol among others. .
[0003]
Most of the sequences of all yeast genes are known and are located on their respective chromosomes, but many of these genes, particularly those that have no counterpart in other species, or known genes It is still difficult to predict the biological function of genes that do not show sufficient sequence homology. In other words, determining the sequence of a gene is easier than assigning a meaningful function to the gene. Only half of the 6000 yeast genes are known. Furthermore, it is not clear how even a gene with a known function is modified when the function or regulation of such a gene is combined with the regulatory mechanism or product of another gene. Thus, with respect to the problem of identifying a known or unknown function of a gene when combined with other genes, the technology is still very unpredictable.
[0004]
To date, this work has been accomplished one by one, whereby researchers can use known genetic engineering and screening methods to transfer such methods or techniques to each specific gene of interest or each specific gene. Applied to the combination. Yeast is often selected as a model because of its simplicity of operation and the ability to screen a large number of candidates in a relatively short period of time. Yeast has the highest recombination and gene conversion rates among the organisms examined, several orders of magnitude higher than mammals. Later in the genome, it can be demonstrated by a continuous gene knockout project in yeast (tetrad analysis, eg http://bioinformatics.weizmann.ac.il/pub/software/mac/mactetrad69.readme ) Confirms that approximately 1 out of 6 gene products is essential for the life of the cell under the conditions examined (deletion of those gene products is lethal). A reasonable fraction of the gene product examined is an enzyme, but other genes either have an unknown function or the function does not fit a priori assumptions.
[0005]
There are several US patents that provide insights into the means of screening and identifying yeast genes and their respective functions. For example, US Pat. Nos. 5,916,752 and 5,698,686, incorporated herein by reference, disclose telomerase compositions and screening methods; US Pat. Nos. 5,821,076 and 5,756,305 disclose essential survival genes. And US Pat. Nos. 5,585,245 and 5,503,977 relate to ubiquitin split protein sensors. A commonly used approach is screening by known gene knockout and complementarity. The term “complementarity” is used herein to refer to homology with a mutated gene element such that the gene element rescues the cell from the effects of the mutation when introduced into the cell (see, eg, US Pat. No. 5,527,896). Is a genetic term intended to mean (at least 40% are homologous). . For example, MEC-1 DNA rescues mec-1 deficiency in mec-1, cdc9-8 cells through complementation process, or RAD9 rescues rad9 deficiency in rad9, cdc9-8 cells through complementation process, and MEC-1 (or RAD9) DNA with such ability is called “complementary cDNA”. Similarly, human CDC34 is homologous to yeast CDC34 and can complement mutations in single (or double mutant) cells, eg, single mutant cells of yeast strain cdc34. For further details and examples, see Hartwell et al. US Pat. No. 5,866,338, incorporated herein by reference. Means for alternating reference of yeast and human genes are currently achievable, such as the “XREF2” program found on http://www.ncbi.nlm.nih.gov/XREFdb/, which is incorporated herein by reference. Can be used. By these methods, for example, DNA replication accessory factors from yeast Saccharomyces serevisiae and their human counterpart genes, such as DNA polymerase α, were identified.
[0006]
In the context of identifying anti-cancer drug targets, the first defect would be a mutation in a gene conserved from yeast to human that is often deregulated in the tumor (eg, overexpression of cyclin). A gene product with a mutation that specifically kills cells with the first deficiency may produce a significant therapeutic benefit due to inactivation in the tumor (i.e., the first drug target) (i.e., the first Constitutes the next of one deficit). In principle, if there are two mutations that have an additional negative effect on a single essential biological pathway, or the mutation inactivates two different but functionally overlapping pathways In some cases, synthetic lethality can occur.
[0007]
MAD2 is known to interact with Bet1p, Cdc20p, and Mad1p at the protein-protein level. MAD2 is present in two pools in vivo: one in the low molecular weight fraction and the second in the larger Mad1p-containing fraction. The association with Mad1p is very robust and unaffected by the cell cycle stage (Chen, RH, Brady, DM, Smith, D., Murray, AW, and Hardwick, KG, “Sbudding yeast spindle checkpoint. Depends on a robust complex of Mad1 and Mad2 proteins (The Spindle Checkpoint of Budding Yeast Depends on the Tight Complex between the Mad1 and Mad2 Proteins), Mol Biol Cell 10, 2607-2618 (1999)). Furthermore, Mad2p and Mad3p remain bound to Cdc20p throughout the cell cycle and binding of Mad2p to Cdc20p requires Mad1p (Hwang, LH, Lau, LF, Smith, DL, Mistrot, CA, Hardwick, KG , Hwang, ES, Amon, A. and Murray, AW, “Budding Yeast Cdc20: A Target of the Spindle Checkpoint”, Science 279, 1041-4 (1998)) . However, cdc20 mutants resistant to spindle checkpoints do not form bonds with Mad1p, Mad2p, and Mad3p (Hwang, LH, Lau, LF, Smith, DL, Mistrot, CA, Hardwick, KG , Hwang, ES, Amon, A. and Murray, AW, “Budding Yeast Cdc20: A Target of the Spindle Checkpoint”, Science 279, 1041-4 (1998)) , Binds to different phosphorylated isoforms of Mad1p (Chen, RH, Brady, DM, Smith, D., Murray, AW, and Hardwick, KG, “The budding yeast spindle checkpoint is a tight anchor for the Mad1 and Mad2 proteins. The Spindle Checkpoint of Budding Yeast Depends on a Tight Complex between the Mad1 and Mad2 Proteins ”, Mol Biol Cell 10, 2607-2618 (1999)). MAD2 is involved in gene interactions with CIN8, CTF13, and KAR3. Although little information is available on the exact function of MAD2, this gene is thought to be required for cell cycle delay and viability during mitosis in cells treated with low concentrations of nocodazole or benomyl (Wang, Y. and Burke, DJ, “The checkpoint gene required to delay cell division in response to nocodazole is responsive to damaged centromere function in the yeast Saccharomyces serevisiae ( Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae), Mol Cell Biol. 15, 6838-44 (1995); Li, R. and Murray, AW, “Presence in budding yeast. Feedback control of mitosis in budding yeast. ”, Cell 66, 519-31 (1991)). Mad1p, Mad2p, Mad3p, and Cdc20p form a complex responsible for the spindle checkpoint required for exit from mitosis (Hwang, LH, Lau, LF, Smith, DL, Mistrot, CA, Hardwick , KG, Hwang, ES, Amon, A. and Murray, AW, “Budding Yeast Cdc20: A Target of the Spindle Checkpoint”, Science 279, 1041-4 (1998). )) And also required for cell cycle delay in ctf13 mutants (Wang, Y. and Burke, DJ, “The checkpoint gene required to delay cell division in response to nocodazole is the yeast Saccharomyces cerevisiae. (Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae) " Mol Cell Biol. 15, 6838-44 (1995)). Mad1p-Mad2p complex may be involved in the regulation of late prophase complex (APC) and binding to Mad1p is required for its spindle checkpoint function and hyperphosphorylation of Mad1p (Chen, RH, Brady, DM, Smith, D., Murray, AW, and Hardwick, KG, “The Spindle Checkpoint of Saccharomyces cerevisiae depends on a robust complex of Mad1 and Mad2 proteins (The Spindle Checkpoint of Budding Yeast Depends on a Tight Complex between the Mad1 and Mad2 Proteins) ”, Mol Biol Cell 10, 2607-2618 (1999)). Overproduction of Bud1-5p from the GAL promoter causes mitotic delay and the phenotype depends on other checkpoint genes including BUB2, BUB3, MAD1, MAD2, and MAD3 (Farr, KA and Hoyt, MA, "Bub1p kinase activates the Saccharomyces cerevisiae spindle assembly checkpoint.", Mol. Cell Biol. 18, 2738-2747 (1998) )), These checkpoint genes are essential in preventing Pds1p degradation and in preventing Mcd1p dissociation from the chromosome after disruption of the mitotic spindle by nocodazole, but they are also responsible for Clb2p degradation or Clb2p-Cdc28p Is not essential for the inactivation of S. (Alexandru, G., Zachariae, W., Schleiffer, A., and Nasmyth, K, “Separation of sister chromatids and re-replication of chromosomes. Is regulated by different mechanisms depending on spindle damage (Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage.) ", Embo Journal 18, 2707-2721 (1999)). Response to disruption of mitotic spindles by nocodazole may occur through a Mad2p-dependent pathway that prevents Pds1p degradation and sister chromatid segregation, and a Bub2p-dependent pathway that prevents Clb2p degradation and DNA re-replication (Alexandru, G., Zachariae, W., Schleiffer, A., and Nasmyth, K, “Separation of sister chromatids and chromosomal re-replication are regulated by different mechanisms depending on spindle damage. (Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage.) ", Embo Journal 18, 2707-2721 (1999)). Analysis of gene interactions suggests that Mad1p, Mad2p, Mad3p and Bub1p function in the same regulatory pathway (Alexandru, G., Zachariae, W., Schleiffer, A., and Nasmyth, K, “sister staining. Separation of chromosomes and re-replication of chromosomes are regulated by different mechanisms depending on spindle damage (Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage.), Embo Journal 18 2707-2721 (1999)). MAD2 is also mentioned to be necessary for G2 delay of sgt1-3 mutants at limited temperatures (Kitagawa, K., Skowyra, D., Elledge, SJ, Harper, JW, and Hieter, P. ), “SGT1 encodes an essential component of the kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex ) ", Mol Cell 4, 21-33 (1999)).
[0008]
The yeast MAD2 gene or its gene product includes human mitotic feedback control protein, T cell receptor α, human plasma membrane calcium pump, rat GTPase Rab8b, human Mad2-like protein, spindle assembly checkpoint component, mouse presence Like mitotic checkpoint component Mad2, spindle assembly checkpoint protein Mad2p, cell cycle checkpoint protein, DNA gyrase A subunit, SCS2, huntingtin, and RNA polymerase II (DNA directed) 220 kD protein Mammalian homologues and analogs. For example, MAD2 has 41% identity to the Xenopus homolog and 40% homology to the human homolog hsMAD2. Xenopus and human homologues bind to nonadherent centromeres in metaphase and nocodazole-treated cells and disappear from centromere in metaphases in untreated cells (Chen, RH, Waters, JC, Salmon, ED, and Murray, AW), “Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores”, Science 274, 242-6 (1996). Li, Y., and Benezra, R., “Identification of human mitotic checkpoint gene (hsMAD2)”, Science 274, 246-8 (1996)).
[0009]
Mutants are known, for example, the MAD2-1 mutation is changed to Zervos Mxi1, which is a protein that specifically interacts with Max and binds to the Myc-Max recognition site by changing the codon of Trp94 to a stop codon. (Cell 79, 389 (1994)). The null allele has the same phenotype as MAD2-1 and the toxic effects of benomyl are partially inhibited by S phase elongation in MAD2 mutants treated with low concentrations of hydroxyurea ( Li, R. and Murray, AW, “Feedback control of mitosis in budding yeast.” Cell 66, 519-31 (1991)). In addition, disruption of mitotic spindles by nocodazole or benomyl usually results in high levels of Clb2p and Clb3p arrest, and high levels of Cdc28p-Clb kinase activity, but MAD2 mutants do not stabilize the Clb protein. (Minshull, J., Straight, A., Rudner, AD, Dernburg, AF, Belmont, A., and Murray AW, “Protein phosphatase 2A regulates MPF activity and sister chromatid binding in budding yeast ( Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast.), Curr. Biol. 6, 1609-20 (1996); Li, R. and Murray, AW, “Feedback control of mitosis in budding yeast ( Feedback control of mitosis in budding yeast.), Cell 66, 519-31 (1991)). Null mutants have been shown not to prevent separation of sister chromatids after disruption of the mitotic spindle by nocodazole, but to prevent cytokinesis and DNA re-replication (Alexandru, G., Zachariae , W., Schleiffer, A., and Nasmyth, K, “Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms depending on spindle damage. are regulated by different mechanisms in response to spindle damage. ”, Embo Journal 18, 2707-2721 (1999)). When null mutant cells are treated with the mitotic spindle disruptor nocodazole, they show normal degradation of Pds1p and normal dissociation of Mcd1p from the chromosome (Alexandru, G., Zachariae, W., Schleiffer, A. , And Nasmyth, K, “Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage.) ", Embo Journal 18, 2707-2721 (1999)). In general, null mutants maintain high levels of Clb2p-Cdc28p activity and are unable to replicate their DNA in the presence of nocodazole (Alexandru, G., Zachariae, W., Schleiffer, A., and Nasmyth, K, “Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage. .) ", Embo Journal 18, 2707-2721 (1999)). If the spindle formation is incomplete, the mutant cells cannot stop the cell cycle before mitosis, causing cell death (Chen, RH, Waters, JC, Salmon, ED, and Murray, AW, “Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores”, Science 274, 242-26 (1996); Li, R. and Murray, AW, “Feedback control of mitosis in budding yeast.”, Cell 66, 519-31 (1991)), mutants overload Mad1p in the presence of microtubule disrupting drugs. Inability to phosphorylate (Hardwick, KG, and Murray, AW, “A phosphoprotein component of the spindle assembly checkpoint in budding yeast.”, J Cell Biol 131, 709 20 (1995)). Deletion of MAD2 causes disruption of mitotic checkpoint arrest as induced by nocodazole (Fraschini, R., Formenti, E., Lucchini, G., and Piatti, S., “Bub2 in Budding yeast "Budding yeast Bub2 is localized at spindle pole bodies and activates the mitotic checkpoint via a different pathway from Mad2", J Cell Biol 145 , 979-991 (1999)), as well as mutants treated with benomyl, lethality follows division (Li, R. and Murray, AW, “Feedback control of mitosis in Saccharomyces cerevisiae). in budding yeast.), Cell 66, 519-31 (1991)). Overproduction of the human homologue hsMAD2 inhibits thiabedazole sensitivity of the cpf1 null mutant (Li, Y., and Benezra, R., “Identification of human mitotic checkpoint gene: hsMAD2) ", Science 274, 246-8 (1996)), overproduction of Xenopus homologue Mad2 cannot inhibit the benomyl sensitivity of MAD2 mutants (Chen, RH, Waters, JC, Salmon, ED, and Murray, AW), “Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores”, Science 274, 242-6 (1996)).
[0010]
MAD2 mutants are involved in gene interactions with other mutated second genes (double mutants). For example, double mutants with kar3, cln8, or cbf1 have significantly reduced viability compared to single mutants (Hardwick, KG, Li, R., Mistrot, C., Chen, RH Dann, P., Rudner, A., and Murray, AW, “Damages in many different spindle components activate spindle checkpoints in the budding yeast Saccharomyces serevisiae (Lesions in many different spindle components activate the spindle checkpoint in the budding yeast Saccharomyces serevisiae) ”, Genetics 152, 509-518 (1999)). Double mutants with cdc23, cdc15, or cdc20 are less defective than any single mutant (Hardwick, KG, Li, R., Mistrot, C., Chen, RH, Dann, P., Rudner , A., and Murray, AW, “Damages in many different spindle components activate the spindle checkpoint in the budding yeast Saccharomyces serevisiae. the budding yeast Saccharomyces serevisiae) ", Genetics 152, 509-518 (1999)). However, the MAD2 bub2 double mutant has a more severe checkpoint deficiency than both single mutants (Fraschini, R., Formenti, E., Lucchini, G., and Piatti, S., “Bub2 in Budding yeast "Budding yeast Bub2 is localized at spindle pole bodies and activates the mitotic checkpoint via a different pathway from Mad2", J Cell Biol 145, 979-991 (1999)). Other double mutants, such as the MAD2-1 mutation, inhibit the mitotic delay of the ctf7-203 mutant (Skibbens, RV, Corson, LB, Koshland, D., and Hieter, P., “Ctf7p Is essential for sister chromatid binding and links the mitotic chromosome structure to the DNA replication machinery (Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinary), Genes And Development 13 307-319 (1999)). The ipl1-321 MAD2 double mutant, like the MAD single mutant, cannot stop in the cell cycle due to the absence of a chromosome segregator in either (Biggins, S., Severin, FF, Bhalla, N., Sassoon, I., Hyman, AA, and Muray, AW, “The conserved protein kinase Ipl1 regulates microtubule binding in budding yeast. to kinetochores in budding yeast.), Genes And Development 13, 532-544 (1999)). Mad2 and Bub2 double null mutants can undergo Clb2p and Clb3p protein degradation, DNA re-replication, and cytokinesis after disruption of the mitotic spindle by nocodazole (Alexandru, G., Zachariae, W., Schleiffer, A., and Nasmyth, K, “Sister chromatid separation and chromosome re-duplication are regulated by different. mechanisms in response to spindle damage.), Embo Journal 18, 2707-2721 (1999)), and DNA replication in MAD2 bub2 double null mutants treated with nocodazole appears to depend on Cdc14p (Alexandru , G., Zachariae, W., Schleiffer, A., and Nasmyth, K, “Sister chromatid segregation and chromosome re-replication are regulated by different mechanisms depending on spindle damage (Sister chromati d separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage.), Embo Journal 18, 2707-2721 (1999)). Some mutants, such as the glc7-10 MAD2 double mutant, phenotypically display wild-type DNA content and wild-type spindle elongation and DNA segregation (Sassoon, I., Severin, FF , Andrews, PD, Taba, MR, Kaplan, KB, Ashford, AJ, Stark, MJR, Sorger, PK, and Hyman, AA, “Regulation of Saccharomyces serevisiae centromere by type 1 phosphatase Glc7p (Regulation of Saccharomyces serevisiae kinetochores by the type 1 phosphatase Glc7p) ", Genes And Development 13, 545-555 (1999)). Other mutants, such as the MAD2 chl1 double mutant, have only a slow growth phenotype (Li, R. and Murray, AW, “Feedback control of mitosis in budding yeast. budding yeast.) ", Cell 66, 519-31 (1991)). Bud2p and Mad2p are not in the same supergroup, because Bub2p-dependent DNA re-replication in nocodazole-treated MAD2 null mutant cells is avoided by overproduction of Tem1p.
[0011]
Finally, double mutants that cause cell death, such as mutants that overproduce Mps1p, tub2-403 MAD2 mutants that lose viability at 14 ° C (showing that tubulin activates the spindle checkpoint ), And mps2-1 MAD2 double mutants that lose viability at 37 ° C. (Hardwick, KG, Li, R., Mistrot, C., Chen, RH, Dann, P., Rudner, A.) , And Murray, AW, “Damages in many different spindle components activate the spindle checkpoint in the budding yeast, Saccharomyces serevisiae. Saccharomyces serevisiae) ", Genetics 152, 509-518 (1999)).
[0012]
Given the role of mad1 and MAD2 in the cell life cycle, further information on these genes is desirable to identify new pathways for cancer prevention and treatment.
[0013]
3. Summary of invention
Accordingly, an object of the present invention is to provide yeast mutants as models of tumor cells or other deregulated cells corresponding to various diseases.
[0014]
Massive screening and genetic analysis identifies compounds or drug targets that specifically kill tumor cells. In this context, examples of compounds that stimulate the activity of the found lethal mutants are provided. Therefore, therapeutic agents developed from identification of essential genes in eukaryotes are expected. Such identified genes (hereinafter referred to as “second genes”) or their products serve as new targets for therapeutic procedures based on mechanisms that are different or similar to those of existing drugs. Such compounds inhibit the function of the gene product identified by the methods disclosed herein, for example, by exhibiting a phenotype or form similar to that found in the original mutant strain.
[0015]
In accordance with one aspect of the invention, a collection of variants is systematically screened to identify genes and / or gene products that are targets for the drug. For example, a drug may act as an antagonist by reversibly or preferably irreversibly binding to an identified gene or gene product target and thereby impairing function. Loss of the target function (or synthesis or complete processing) of the gene product will inhibit tumor cell growth and preferably will lead to death of the tumor cell. This aspect includes exposing a gene product corresponding to the wild type sequence of the second gene identified by the methods disclosed herein to a test agent; and the function of the gene product (preferably selecting In general, a method of identifying an anti-cancer agent comprising the step of selecting a disordered agent.
[0016]
In certain embodiments of the invention, the first genetic defect is a defect that is preferably found in or associated with tumor cells or cells that have been deregulated in the cell cycle. Alternatively, the first genetic defect provided by the methods of the invention is similar or homologous to a defect found in or associated with mammalian or human tumor cells, or chromosomally abnormal cells. The term “homologous” means a direct relationship in a gene “family” in which a particular sequence or region is tightly conserved among members of the family. For example, MAD2 has 41% identity with the Xenopus homolog and 40% identity with the human homolog hsMAD2. On the other hand, “similar” genes may have similar or “similar” functions, but they are not directly related (ie, no sequence is conserved between similar genes). Within this context, such “similar” genes are referred to as “functionally homologous” and thereby refer to gene products that have a function similar to that in mammalian cells in yeast cells. For example, Xenopus and human homologues bind non-adherent centromere before metaphase in nocodazole-treated cells and disappear from centromere in metaphase in untreated cells (Chen, RH , Waters, JC, Salmon, ED, and Murray, AW), “Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores”, Science 274, 242 ~ 6 (1996); Li, Y. and Benezra, R., "Identification of human mitotic checkpoint gene (hsMAD2)", Science 274, 246-8 (1996) ). In certain embodiments of the invention, analogs and homologues of MAD2 include human mitotic feedback control protein, T cell receptor α, human plasma membrane calcium pump, rat GTPase Rab8b, human Mad2-like protein, spindle Body assembly checkpoint component, mouse mitosis checkpoint component Mad2, spindle assembly checkpoint protein Mad2p, cell cycle checkpoint protein, DNA gyrase A subunit, SCS2, huntingtin, and RNA polymerase II (DNA-directed) Including but not limited to 220 kD protein.
[0017]
In the methods of the invention, the first gene deficiency can result in a change, loss, or inhibition of function, eg, cell function. Thus, the methods of the present invention relate to defects in MAD1 and / or MAD2.
[0018]
In general, the affected functions can vary over a wide range. Affected functions include tumor growth inhibition, DNA damage checkpoint, DNA mismatch repair, nucleotide excision repair, o6-methylguanine reversal, double-strand break repair, DNA helicase function, signal transduction, cell cycle control Or includes, but is not limited to, apoptosis. In certain embodiments of the invention, the life cycle of a cell includes, but is not limited to, a cell having a defective mitotic checkpoint. Such a defect can be effectively modeled by a first genetic defect in other organisms such as Drosophila.
[0019]
It has been found that the method of the invention can result in a specific second site mutation, which can be lethal to cells having the first gene defect. Such a second site mutation can be achieved, for example, in a gene selected from the group consisting of TUB1, CIN8, STU1, SFI1, TUB2, and MPS2.
[0020]
Double variants comprising MAD1 or MAD2 and a second gene and / or ORF (open reading frame) are also within the scope of the present invention.
[0021]
The invention also includes contacting a cell having a mutated MAD1 gene, a mutated MAD2 gene or an analog or homolog thereof with a drug; A mutant MAD1 gene, a mutant MAD2 gene, or analogs or homologues thereof, depending on whether the drug regulates the activity of a synthetically lethal wild-type second gene Methods of identifying drugs that inhibit cell proliferation or replication are provided. Without limiting to the above genes, KAR3, CIN8, KIP1, CIK1, CTF19, CBF1, yeast, human cytoplasmic linker protein, human restin, mouse synaptonemal complex protein, mouse testis protein, human desmoplakin, mouse Rho Related to rat SCP1 gene product, human desmoplakin I, rat ROK-α, bovine Rho related kinase, human AH antigen, human non-muscle myosin heavy chain, human NuMA protein, human SP-H antigen, human KIAA0378, human myosin Other preferred comprising at least one second gene or product thereof selected from the group consisting of weak similarity, human KIAA0336, human mouse CC1 analog, rat heart myosin heavy chain 5, human endosomal protein and combinations thereof The second gene is equally suitable.
[0022]
The object of the present invention further includes the identification of a drug or drug candidate. Thus, after the second drug target is elucidated, the second drug target can be used to interact with the second drug target, for example a drug that can impair its physiological activity or Drug candidates can be screened. Thus, the present invention may provide drugs or drug candidates that interact, bind to or inhibit specific gene products. Such gene products can include, but are not limited to, the above examples. The drug or drug candidate desirably exhibits the ability to inhibit or stop the growth of human tumors or benign neoplastic tumors. Preferably, the assay consists of a control system that observes lethality and a test system that adds the drug and evaluates the effect based on the difference between the control system and the test system. If the drug promotes or cancels lethality, the drug is considered a candidate drug that may be specific in the biochemical pathway controlled by the first and second genes . Specificity is further tested by a number of specificity determination tests, such as in vitro tests on the effect of the first gene and the second gene on the enzymatic activity of the gene product, such as the ability to target the gene or its product. Are tested by introducing double mutants that cancel the perceived effects.
[0023]
Thus, an object of the present invention is to provide a means for controlling unwanted proliferation or differentiation of eukaryotic cells. The term `` undesirable growth '' refers to unwanted cell growth, such as neoplastic or hyperplastic growth, e.g. keloids intended for wound healing, treatment of restenosis, true, whether by cell transformation. Applies to infection by nuclear pathogens, and other undesirable smooth muscle proliferation, cosmetic applications, retinopathy due to diabetes, etc. Similarly, the term “undesirable differentiation” refers to an undesirable change in cell differentiation such that the differentiated cell returns to an earlier state and restores or retains proliferative capacity in an unnatural or “undesirable” manner. Applies to
[0024]
The present invention provides prostate cancer, melanoma, adult cancer, and hyperplastic lesions, such as benign prostate hyperplasia and papilloma, by administering a therapeutically effective amount of the drug or a pharmaceutically acceptable derivative thereof. Methods of treating malignant conditions such as childhood cancer, eg, brain tumors of glial origin, astrocytoma, Kaposi's sarcoma, adenocarcinoma of the lung, and leukemia are provided.
[0025]
Most preferably, administration of the drug or drug candidate results in tumor cell death, reduction of neoplastic tissue and cancer treatment. Without being limited to cancer, other clinical diseases are expected to be treatable by the compounds of the present invention. These diseases include, for example, cardiovascular diseases such as restenosis, in which smooth muscle cells proliferate excessively in blood vessels after angioplasty and can lead to blood flow restriction and death.
[0026]
Without being limited to the above-mentioned diseases, the present invention also provides Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida crusi (Candida krusei), Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flusigalus, sperus lus flavus (Aspergillus niger), Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absisi Provide a means of treating various fungal or yeast pathogens selected from the group consisting of Absidia corymbifera, Absidia ramosa, and Mucor pusillus, or combinations thereof.
[0027]
The present invention also embodies diagnostic and prognostic assays that assess abnormal phenotypes and aggressiveness by detecting the expression of MAD1 or MAD2 protein or the expression of regulatory genes or gene products thereof.
[0028]
It is an object of the present invention to selectively interact with the production of at least one gene product in a cell population in which the drug contains at least one first gene defect, and when the cell population is exposed to the drug, cell division in the cell population Further included is a pharmaceutical composition comprising a drug in a pharmaceutically acceptable carrier or diluent that selectively halts. Such gene products are encoded or regulated by human genes that are similar or homologous to yeast genes.
[0029]
The object of the present invention further includes means of gene therapy with a gene product in a cell population comprising at least one first gene defect that selectively stops cell division in the cell population when the cell population is exposed to a drug. Such gene products are encoded or regulated by human genes that are similar or homologous to yeast genes.
[0030]
A synthetic lethal screening method is a mutation in a first gene of interest, and a yeast mutation with a second target gene that, when present in combination with the first gene mutation in the variant, determines the regulation of cell growth or death Disclosed on the basis of body lethality, the screening assays of the invention utilize single and double mutants that can complement or inhibit the function of the first gene of interest. An experimental example for realizing the present invention for identifying a novel gene involved in human cell cycle control is provided. These genes include, but are not limited to, yeast TUB1, CIN8, STU1, SFI1, TUB2, MPS2, and others, and analogs and homologues of these genes. These genes encode proteins that are involved in aspects including but not limited to aspects of microtubule and spindle formation. The screening assays of the invention utilize single and double mutants that can complement or inhibit the function of the first gene of interest (eg, mad1 or mad2). An experimental example for realizing the present invention for identifying a novel gene related to cell proliferation is provided.
[0031]
Objects of the present invention further include means of using gene therapy to induce synthetic lethality in cells containing at least one deletion in the first gene, such as MAD1, MAD2, or homologues or analogs thereof It is. In the context of the present invention, methods of inducing synthetic lethality include MAD1, MAD2, or homologues thereof, which result in disruption of the second gene resulting in the synthetically lethal cell as a result of the action or expression of the polynucleotide. Alternatively, the method includes introducing the polynucleotide into a cell containing at least one deletion in the first gene, such as an analog. Disturbances of the second gene include, but are not limited to, upregulation, downregulation, removal or destruction of the second gene. Polynucleotides suitable for use in the present invention include triplex forming oligonucleotides, antisense polynucleotides, RNA-I (for review, Nature 402: 128-129, incorporated herein by reference in its entirety. 1999), and ribozymes that specifically target a second gene. Second genes useful in this regard include the previously mentioned genes, TUB1, CIN8, STU1, SFI1, TUB2, and MPS2, and their respective analogs and homologues.
[0032]
The use of antisense polynucleotides and their applications are described, for example, in the editions of Mol and Van der Krul, “Basics of Antisense Nucleic Acids and Proteins,” which are incorporated herein by reference in their entirety. And Antisense Nucleic Acids and Proteins Fundamentals and Applications, New York, NY, (1992). Suitable antisense oligonucleotides are at least 15 nucleotides in length, and up to 15 nucleotides, and include an untranslated coding sequence and associated coding sequence upstream of the second gene. As will be apparent to those skilled in the art, the optimal length of an antisense oligonucleotide is determined by the strength of interaction between the antisense oligonucleotide and its complementary sequence on the mRNA, the temperature and ionic environment at which translation occurs, the antisense It depends on the nucleotide sequence of the oligonucleotide, the presence of secondary and tertiary structures in the mRNA and / or antisense oligonucleotide, and the preferred mode of delivery. For example, soluble antisense oligonucleotides have been used to inhibit transcription / translation of target genes (Ching et al., Proc. Natl. Acad. Sci., Each incorporated herein by reference. USA 86: 10006-10010, 1989; Broder et al., Ann. Int. Med. 113: 604-618 (1990); Loreau et al., FEBS Letters 274: 53-56, 1990; Holcenberg et al., International Publication. Publication No. 91/11535; International Publication No. 91/09865; International Publication No. 91/04753; International Publication No. 90/13641; and European Patent No. 386563). Suitable target sequences for antisense polynucleotides include intron-exon junctions (to prevent proper splicing), regions where DNA / RNA hybrids prevent mRNA transport from the nucleus to the cytoplasm, initiation factor binding sites, Included are ribosome binding sites and sites that interfere with ribosome progression. A particularly preferred target region for antisense polynucleotides is the 5 ′ untranslated region of the second gene selected.
[0033]
Antisense polynucleotides that target the second gene of choice also include the target polynucleotide sequence in a suitable expression vector in which the DNA molecule is inserted downstream of the promoter in the opposite direction compared to the gene itself. Can be prepared by inserting a DNA molecule comprising The expression vector can then be transduced, transformed, or transfected into suitable cells to allow expression of the antisense polynucleotide. Alternatively, antisense oligonucleotides may be synthesized using standard manual or automated synthesis techniques.
[0034]
Synthesized oligonucleotides were electroporated (eg, Yang et al., Nucl. Acids. Res. 23: 2803-2810, 1995), calcium phosphate precipitation, microinjection, poly-L-ornithine / DMSO (Dong et al., Nucle. Acids. Res. 21: 771-772, 1993) may be introduced into suitable cells. The selection of a suitable antisense oligonucleotide administration method will be apparent to those skilled in the art. With respect to synthesized oligonucleotides, the stability of antisense oligonucleotide-mRNA hybrids can be increased by adding stabilizers to the oligonucleotide. Stabilizers include intercalete agents that are covalently attached to one or both ends of the oligonucleotide. Oligonucleotides can be made resistant to nucleases by modification of the phosphodiester backbone, for example by introducing phosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates, phosphoramidates, or phosphorodithioates . Oligonucleotides can also be rendered nuclease resistant by synthesizing oligonucleotides with deoxyribonucleotide alpha anomers, as generally described in Mol and Van der Krul (ibid).
[0035]
In another embodiment, the polynucleotide-based inhibitors of the present invention include triplex forming oligonucleotides, sequence specific DNA binding agents that interfere with transcription of the target gene. Triplex forming oligonucleotides are generally generally incorporated herein by reference in their entirety, Maher, Bioessays 14: 807-815, 1992; Gee et al., Gene 149: 109-114. Noonberg et al., Gene 149: 123-126, 1994); Song et al., Ann. NY Acad. Sci. 761: 97-108, 1995; Westin et al., Nuc. Acids. Res. 23: 2184-1219, 1995); and Wand and Glazer, J. Biol. Chem. 207: 22595-22901, 1995). These oligonucleotides select gene transcription by physically forming a triple helix complex on double-stranded DNA at physiological conditions and physically preventing contact of RNA polymerase or transcription factors to the DNA template. To inhibit. Also, for example, International Publication No. 95/25818; International Publication No. 95/20404; International Publication No. 94/15616; International Publication No. 94/04550, each of which is incorporated herein by reference. And International Publication No. 93/09788. Triplex forming oligonucleotides can include either nucleotide or non-nucleotide tails to facilitate inhibition of transcription factor binding. As one example, triplex-forming oligonucleotides target the following second genes: TUB1, CIN8, STU1, SFI1, TUB2, and MPS2 and their respective analogs and homologues.
[0036]
For polynucleotide-based inhibitors, the selection of a suitable sequence will depend, for example, on the type of inhibitor (ie, triplex-forming oligonucleotide or antisense polynucleotide) and the species being treated. . It may be preferable to select sequences that are conserved among species so that they can be used in readily available animal models.
[0037]
The present invention also provides compositions and methods relating to the use of ribozyme inhibitors to inhibit second gene expression. Ribozymes can be administered in a variety of ways, including by gene therapy targeting the desired cells. The ribozymes of the present invention are sequence-specific targets for RNA transcripts of the gene of interest. For example, a ribozyme can be designed to specifically inhibit transcription of a second gene. Each ribozyme molecule is designed to include a catalytically active moiety capable of cleaving a second gene RNA, and further includes a flanking sequence having a nucleotide sequence complementary to a portion of the targeted RNA. Including. The flanking sequence anneals the ribozyme to RNA in a site-specific manner. However, the sequence adjacent to the target sequence is double-stranded with the target RNA and only requires sufficient complementarity for the catalytically active portion of the ribozyme to cleave at the target site. It need not be absolutely complementary. Thus, only sufficient complementarity is necessary to hybridize the ribozyme to the target RNA under physiological conditions. As used herein, the term “ribozyme” refers to an RNA molecule having an enzymatic activity capable of cleaving or splicing other discrete RNA molecules in a nucleotide base sequence specific manner. . The term catalytic RNA molecule or enzyme RNA molecule is complementary in the substrate binding region to a specific RNA target (eg, TUB1, CIN8, STU1, SFI1, TUB2, or MPS2 RNA) and directs RNA at that target. It means an RNA molecule that has an enzymatic activity that is active to cleave and / or combine, thereby changing the target molecule. In a preferred embodiment of the invention, the enzyme RNA molecule is formed in a hammer head motif, but the ribozyme is also a hairpin motif, hepatitis delta virus, group I intron or RNase P RNA (RNA guide sequence). In relation to). Examples of hammerhead motifs are described in Rossi et al., AIDS Res. Hum. Retrovir. 8: 183, (1992), and hairpin motifs are described in Hampel et al., Biochem. 28: 4929, (1989), and Hampel et al., Nucl. Acids. Res. 18: 299, (1990), the hepatitis delta virus motif is exemplified in Perrotta and Been, Biochem. 31:16, (1992), and the RNase P motif is Guerrier -Takada et al., Cell 35: 849, (1983), and examples of group I intron motifs are described in Cech et al., US Pat. No. 4,987,071, the disclosures of each of which are hereby incorporated by reference. Incorporated into the book. These specific motifs are not limiting in the present invention and those skilled in the art will have specific substrate binding sites in which the enzyme RNA molecules of the present invention are complementary to one or more target RNA regions, which are molecules It will be appreciated that it has a nucleotide sequence in or around the substrate binding site that confers RNA cleavage activity.
[0038]
The flanking sequences upstream and downstream of the ribozyme catalytic site may include portions of any length that effectively confer the desired degree of target specificity for the ribozyme. Preferably, the flanking sequence comprises from about 4 to about 24 nucleotides, more preferably from about 6 to about 15 nucleotides, generally from about 9 to 12 nucleotides, resulting in an RNA sequence comprising a cleavage site Base pairing with the substrate sequence occurs immediately upstream and downstream. Polynucleotide inhibitors targeting different parts of the target DNA or corresponding RNA, such as triplex forming oligonucleotide inhibitors, antisense oligonucleotide inhibitors, ribozyme inhibitors, etc., or combinations of such inhibitors Can be delivered to the targeted cells in a wide variety of ways to facilitate inhibition of the gene of interest. Oligonucleotides can be administered as synthetic oligonucleotides or expressed from expression vectors. Oligonucleotides can be administered ex vivo, ie, contacted with target cells taken from an individual or other cell source, treated back, or oligonucleotides can be administered in vivo. When administered ex vivo, depending on the particular cell population, the target cell is generally mitogenic, eg serum mitogen (SCF, IL-3, EPO, TPO, etc.) or the like To be exposed to.
[0039]
These and other objects of the invention will be apparent from the disclosure provided herein.
[0040]
4. Detailed Description of the Invention
The present invention provides yeast mutants as models for cancer and other diseases associated with deregulation of cell proliferation and replication. Yeast is a very useful model for studying human diseases. For example, as of January 20, 1996, the following human diseases and clinically important pathologies have been identified as a match between the human gene and the yeast S. serevisiae gene / protein: : Hereditary nonpolyposis colon cancer; cystic fibrosis; Wilson disease; glycerol kinase deficiency; adrenoleukodystrophy; telangiectasia ataxia; amyotrophic lateral sclerosis; myotonic dystrophy; Congenital choroid deficiency; structural dysplasia; gyrus defect; Tomzen's disease; Wilms tumor; chondrogenic dysplasia; Menkes syndrome; multiple endocrine neoplasia 2A; Wiscott-Aldrich syndrome; Duchenne Muscular dystrophy; aniridia; gonadal dysplasia; early-onset breast and ovarian cancer; chronic granulomatosis; epidermolytic palmokeratoderma; Denburg syndrome; multiple adenomatous colon polyps; type 2 neurofibromatosis; Kalman syndrome; tuberous sclerosis; renal cysts; Aarskog-Scott syndrome; Marfan syndrome; Huntington's disease; spinocerebellar ataxia; Syndrome; Fragile X syndrome; Emery-Dreis muscular dystrophy; Retinoblastoma; MacLeod syndrome; Norie disease; von Hippel-Lindau disease; Alzheimer's disease; hyperreflexia; (See http://www.ncbi.nlm.nih.gov/Bassett/Yeast/ and updated versions thereof incorporated herein by reference). Means for alternating reference of yeast and human genes are currently available, e.g., the `` XREF2 '' program found at http://www.ncbi.nlm.nih.gov/XREFdb, incorporated herein by reference. Can be used. Abnormalities in the components of the cell cycle monitoring system have been identified in human cancer and other diseases as described above. These abnormalities include cyclin changes (80% to 90% of tumors), p53 changes (50% to 60% of tumors), and DNA mismatch repair (of several types of tumors such as the colon and pancreas) 10% to 20%). The first genetic change is often loss of function, so drug discovery programs that focus on these deficiencies require recovery of lost function. Another approach is to identify which other protein (s) selectively kill cells with the first defect when inhibited.
[0041]
General information and databases containing yeast genomes are officially available and can be found, for example, on official websites whose contents and links are incorporated herein by reference: http: // bioinformayics.weizmann.ac.il;http://ourworld.compuserve.com/homepages/C Velten / yeast.htm;h ttp: //www.ncbi.nlm.nih.gov/YeastHttp://genome-www.stanford.edu/Saccharomyces/;genome-ftp.stanford.edu (directory / yeast / genome_seq);http://vectordb.atcg.com/vectordb/;http://www.mpimg-berlin-dahlem.mpg.de/~andy/GN/S.cerevisiae/Or http://www.mips.biochem.mpg.de/proj/yeast. Other databases exist as well, and links to these databases and other websites are equally suitable for the purposes of the present invention. By way of example, Yeast GenBank (collection of all GenBank sequences derived from Saccharomyces serevisiae), the contents of which are incorporated herein by reference; Yeast Swiss-Prot (derived from Saccharomyces serevisiae) Swiss-Prot protein sequence collection); YPD (yeast protein database maintained by Proteome, Inc.), and regular updates thereof. Methods for manipulating yeast are well established and known to those skilled in the art, for example,www.goshen.edu/bio/yeast,www.fhcrc.org/~gottschling,andwww.sacs.ucsf.edu/home/HerskowitzLabFound on official websites such as
[0042]
The invention features a method for identifying mutant organisms having condition-sensitive lethal mutations and subsequent gene products. The disclosed methods are useful for high-throughput screening of genomic or mutant libraries to rapidly identify genes that are essential for survival and corresponding gene products. A lethal mutation results in a gene or protein that is not functional under limiting conditions (ie, in tumor cells). Non-functional genes can have a defect in the promoter, which can result in decreased or abnormal gene expression. Non-functional proteins can have structural defects that cause inappropriate protein folding or abnormal proteolysis. As a result of improper protein folding, folding, recognition of the original substrate, and / or substrate binding and release partially or completely fail.
[0043]
The present invention relates to novel compounds capable of inhibiting a specific gene such that the inhibited gene is incompatible with mutation MAD1 or MAD2. Accordingly, use for inhibition of cell proliferation is preferred for the compounds of the present invention. In particular, the compounds of the invention can be useful for treating subjects with excessive or abnormal cell proliferation.
[0044]
There are a variety of pathological cell growth conditions in which the compounds of the present invention can provide therapeutic benefits, and a common strategy is inhibition of abnormal cell growth. To illustrate, cell types that exhibit pathological or abnormal growth include various cancers and leukemias, psoriasis, bone disease, fibroproliferative disorders involving connective tissue, atherosclerosis, and other smooth muscle proliferative disorders. As well as chronic inflammation.
[0045]
There are a variety of pathological cell growth conditions in which the compounds of the present invention can provide therapeutic benefits, and a common strategy is inhibition of abnormal cell growth. To illustrate, cell types showing pathological or abnormal growth include various cancers, leukemias, psoriasis, bone diseases, fibroproliferative disorders involving connective tissue, atherosclerosis, and other smooth muscle proliferative disorders. As well as chronic inflammation. In this sense, agents that mimic synthetic lethality found with a second mutation that becomes lethal in combination with a mutation in MAD2, MAD1, analogs or homologues thereof may be useful in the treatment of human T-cell leukemia ( Jin et al., Cell 93: 81-91, 1998).
[0046]
In addition to proliferative abnormalities, for example, treatment of differentiation abnormalities caused by tissue dedifferentiation may (optionally) involve a reentrant reentry into mitosis. Such degenerative disorders include chronic neurodegenerative diseases of the nervous system including Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis and the like, and spinocerebellar degeneration. Other differentiation abnormalities include, for example, abnormalities associated with connective tissue, such as those that can occur by dedifferentiation of chondrocytes or bone cells, and vascular abnormalities associated with dedifferentiation of endothelial tissue and smooth muscle cells, degenerative changes in glandular cells. Includes gastric ulcers that are characterized, and kidney diseases that are characterized by poor differentiation, such as Wilms tumor.
[0047]
The present invention is also directed to the treatment of a disease selected from the group consisting of: Apdoma, sarcoma, atheroma, malignant carcinoid syndrome, carcinoid heart disease, cancer (eg, Walker cancer, basal cell carcinoma, basal cancer) Spine cell cancer, Brown-Pearce cancer, Ductal cancer, Ehrlich tumor, Krebs 2 tumor, Merkel cell tumor, Mucinous carcinoma, Non-small cell lung cancer, Oat cell carcinoma, Papillary carcinoma, Hard cancer, Fine cancer Bronchial, bronchiolar cancer, primary bronchial carcinoma, squamous cell carcinoma, and transitional cell carcinoma), histiocytic disorder, leukemia (eg, B cell leukemia, mixed leukemia, null cell leukemia, T cell leukemia, chronic T cell leukemia, HTLV-II-related leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mast cell leukemia, and myeloid leukemia), malignant histiocytosis, Hodgkin's disease, immunoproliferative small ), Non-Hodgkin lymphoma, plasmacytoma, reticuloproliferative disorder, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumor, histiocytoma, lipoma, liposarcoma, Mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovial tumor, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, craniopharyngioma, anaplastic germ cell tumor, hamartoma, between Lobar, medium nephroma, myoma, enamel epithelioma, cementoma, odontoma, teratomas, thymoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, columnar tumor, Cystadenocarcinoma, cystadenoma, granulosa cell tumor, male germ cell tumor, liver cancer, sweat adenoma, islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor, follicular membrane cell Tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myoma, rhabdomyosarcoma, rhabdomyosarcoma, ventricle Epithelioma, gangliocytoma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, non-chromium Paraganglioma nonchromaffin, horned hemangioma, eosinophilia-associated angiolymphoid lymphoproliferative disorder, angioma sclerosing, hemangioma, glomus hemangioma, hemangioendothelioma , Hemangioma, hemangiocarcinoma, angiosarcoma, lymphangioma, lymphphangiomyoma, lymphangiosarcoma, pineal tumor, carcinosarcoma, chondrosarcoma, phyllosarcoma, fibrosarcoma, hemangiosarcoma, smooth muscle Tumor, leukosarcoma, liposarcoma, lymphangiosarcoma, myoma, myxosarcoma, ovarian cancer, rhabdomyosarcoma, sarcoma, neurofibromatosis, cervical dysplasia, fibrosis, benign prostatic hyperplasia, atherosclerotic artery Sclerosis, restenosis, glomerulosclerosis, keloid, psoriasis, mole, keratosis Fibromyalgia, contagious molluscum, sexually transmitted warts, sebaceous hyperplasia, scabaceous hyperplasia, hemangioma, venous lake, chondrodermatitis, suppurative granuloma, purulent scabyenitis, keloid , Keratinous pheochromocytoma, leukoplakia, multiple sebaceous cysts, lashes, superficial epithelial nevus, polyps, junctional nevus, purulent granuloma, nodular eruption, Dermatofibroma, sebinomas, papillomas, and combinations thereof.
[0048]
In addition to therapeutic applications (eg, for both human and veterinary use), the compounds of the present invention control cell proliferation and / or differentiation status in vitro, for example by controlling mitotic checkpoints. It will be apparent that it can be used as a cell culture additive to do so.
[0049]
Similarly, differential screening assays can be used to select compounds of the invention having specificity for non-human yeast enzymes. Accordingly, compounds that act specifically against eukaryotic pathogens, such as antifungal agents or antiparasitic agents, can be selected from the inhibitors of the present invention. To illustrate, the inhibitors of the present invention can be used to treat candidiasis, an opportunistic infection that commonly occurs in debilitated immunosuppressed patients. Use these same inhibitors to treat these infections in patients with leukemia and lymphoma, those receiving immunosuppressive treatment, and patients with a predisposition such as diabetes mellitus or AIDS, where fungal infections are particularly problematic can do.
[0050]
Once a synthetically lethal gene is identified in combination with a mutation in the first gene MAD1, MAD2, or a homologue or analogue thereof, a screening method for drugs that can mimic the second mutation can be developed. . Such agents are useful therapeutics within the meaning of the present invention for treating diseases associated with abnormal growth. The second gene of the present invention treats proliferative diseases such as cancer, where the disease is characterized by a mutation in the first gene, such as a mutation in MAD1, MAD2, or an analog or homologue thereof. Used in assays to identify therapeutic agents to do. In these aspects of the invention, the second gene, wherein such regulation results in inhibition or reduction of the activity of the second gene product, up-regulation, down-regulation, loss or destruction of said second gene Alternatively, screening assays may be performed to identify agents that include candidate drugs that modulate the activity of the second gene product or analog or homologue thereof. In this sense, the assay may use whole cells expressing the target gene, cell lysate containing the target gene product, or a purified target gene product. In certain assays of the invention, under appropriate conditions, the test agent is incubated with a reaction mixture containing target gene-expressing cells for a time sufficient for the test agent to modulate the activity of the target gene product, and a control sample is prepared. Incubate under the same conditions in the absence of the test agent. As used herein, modulation of the activity of a target gene product includes, for example, up-regulating or increasing the activity of the target gene product by direct binding to the gene product and transcription or translation of the target gene product. This includes, but is not limited to, increasing or decreasing the activity of the target gene product by down-regulating. Aspects of the invention include high-throughput screening assays that are designed to identify modulators of target gene products.
[0051]
In one assay of the invention, a candidate drug is identified by the following method: a) Exposing a test cell line containing a cell having a deletion or mutation in the first gene, such as MAD1, MAD2, to the candidate drug B) a first gene, such as MAD1, MAD2, or an analog or homologue thereof, wherein the candidate drug that causes a lower survival rate in the test cell line than the control cell line is a therapeutic drug candidate drug, and A control cell line comprising cells having a deletion or mutation in a second gene whose variant is synthetically lethal when combined with the first gene mutation, such as MAD1, MAD2 or analogs or homologues thereof Comparing the viability of the cell to the viability of the test cell line. In the context of the present invention, viability is measured as the ability of a cell to regenerate itself, a non-viable cell does not divide or cannot divide, a cell that does not stop at any stage of the cell cycle, undergoes apoptosis, or an apoptotic pathway Cells that progress according to, and dead cells. Means for determining the presence or absence of viable and non-viable cells will be apparent to those skilled in the art and include vital dye staining, chromosome enrichment measurements, and the like.
[0052]
Another embodiment is a method for identifying compounds useful in the treatment of proliferative diseases such as cancer characterized by mutations in the MAD1 gene, MAD2 gene or analogs or homologues thereof, comprising the following steps: 1 ) In the mutant form, the test compound affects the gene product of the wild-type gene product of the second gene that is synthetically lethal when combined with the mutant MAD1 gene, mutant MAD2 gene, or an analog or homologue thereof. Contacting the test compound under conditions and for a period of time sufficient; 2) comparing the activity of the gene product in the presence of the test compound to the activity of the gene product in the absence of the test compound. In this exemplary assay, the compound capable of reducing the activity of the second gene product or analog or homologue thereof is a cancer characterized by a mutation in the MAD1 gene, MAD2 gene, or an analog or homologue thereof. Or identified as useful for the treatment of other proliferative diseases. In another embodiment of the present invention, a cell having a wild-type second gene or analog or homologue thereof is contacted with a test compound, and the expression and / or activity of the tol gene product or analog or homologue thereof is determined. A whole cell assay is performed that is evaluated and compared to the expression and / or activity of the wild-type second gene product in cells not exposed to the test compound. In this aspect, compounds that reduce the expression or activity of the gene product are useful for the treatment of cancer or other proliferative diseases characterized by mutations in the MAD1 gene, MAD2 gene, or analogs or homologues thereof.
[0053]
For the purposes of the assays described herein, assays for modulation of the gene product of the second gene are known to those skilled in the art. As will be apparent to those skilled in the art, the second gene target is suitable for measuring the modulation of protein activity so that one skilled in the art will determine the assay used to characterize the gene and its respective mutation. Will determine the appropriate assay. For example, deoxyribonucleotides of ribonucleotides using the methods of Thelander et al. (J. Biol. Chem. 255: 7426-7432, 1980) and Engstrom et al. (Biochemistry 18: 2941-2948, 1979). Increase or decrease in enzymatic activity of ribonucleotide reductase (RNR1 and RNR2 gene products), direct measurement to thymidylate kinase (CDC8 gene product) activity, by direct reduction to nuclease and polymerase activity, for example, Yon ( Yong) and Campbell (J. Biol. Chem. 259: 14394-14398, 1984), phosphorylated or deoxythymidine monophosphate, and endo and 5 'exonuclease activity of RAD27 protein, and POL1 Determine using such an assay by measuring the polymerase activity associated with Door can be. Protein activity can also be determined by measuring the increase or decrease in binding of the gene product to its ligand or substrate, eg, by visualization using antibody staining or immunoprecipitation. On the other hand, structural proteins such as tubulin (TUB1 and TUB2 gene products) can be measured by measuring the quality and quantity of tubulin produced by the cells.
[0054]
For purposes of illustration, the assay methods described herein are used to determine candidiasis, aspergillosis, mucormycosis, spore buds, geotrichumosis, cryptococcosis, chromocytosis, coccidiosis, and conidiosis. (Conidiosporosis), histoplasmosis, mazuramosis, linosporiasis, nocardiosis, paraactinomycosis, penicillosis, moniriosis, or ultimately to inhibit at least one fungus related to mycosis such as sporotrichosis Can be screened for potentially useful drugs.
[0055]
Table 1 shows a synthetic lethal screening strategy to identify a second target. The table below shows some of the second site mutations that were found to be lethal in combination with the indicated first genetic defects (also showing human homologues). Once the second target is identified in the model system, it is reasonable to match some conditions before initiating a high-throughput screen for inhibitors of mammalian homologues of these gene products. It must first be confirmed that synthetic lethality occurs in mammalian cells (both matched cell line pairs and tumor cell lines) where the first and second targets are inactivated as well. This requires the use of mammalian inducible gene disruption techniques such as ribozymes, antisense molecules, or dominant-negative strategies. The second gene target that is most susceptible to small molecule inhibition (eg, an enzyme with a well-defined substrate) is an obvious first choice for detailed analysis, so the pharmacology of each putative drug target Real feasibility must be determined at the same time. Only after these tests are completed can a standard high-throughput screen for inhibitors of identified second gene targets of these mammals be initiated.
[0056]
[Table 1] Yeast strain: MAD2

[0057]
In addition to such therapeutic uses, antifungal agents developed by such differential screening assays, such as preservatives in foodstuffs, feed additives to promote livestock weight gain, or non-biological materials Can be used as a disinfectant formulation for the treatment of, for example, decontamination of hospital equipment and hospital rooms.
[0058]
Similarly, comparing side-by-side inhibition of mammalian genes and insect genes such as Drosophila, it is possible to select inhibitors that distinguish human / mammalian and insect enzymes from the derivatives of the present invention. It is thought that it becomes. Accordingly, the present invention broadly contemplates the uses and formulations of the present invention in insecticides, as used for the management of insects such as Drosophila.
[0059]
In yet another embodiment, some of the inhibitor compounds of the invention can be selected based on their inhibitory specificity for plant genes relative to their mammalian counterparts. For example, a plant gene can be placed in a differential screen with one or more human enzymes to select the compound with the greatest selectivity for inhibiting the plant enzyme. Accordingly, the present invention specifically contemplates the formulation of the inhibitors of the present invention for agricultural applications such as defoliant forms.
[0060]
In another aspect, the present invention provides one or more therapeutically effective amounts of the above compounds formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. Pharmaceutically acceptable compositions comprising are provided. As explained in detail below, the pharmaceutical compositions of the invention may be specifically formulated for administration in solid or liquid form, including those adapted for: (1) oral administration; For example, a drug (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, paste for application to the tongue; (2) For parenteral administration, eg as a sterile solution or suspension (3) topical application, eg as a cream, ointment, or spray applied to the skin; or (4) vaginal or rectal, eg pessary, cream, Or as a form.
[0061]
As used herein, the term “therapeutically effective amount” refers to the target number in at least one subpopulation of cells in an animal that contains a mutation in the first gene (MAD1, MAD2, or an analog or homolog thereof). By modulating the expression activity of the two genes and thereby interfering with the biological consequences of the protein activity in the treated cells with a reasonable benefit / risk ratio applicable to any medical treatment. By an amount of a compound, material or composition comprising a compound of the invention that is effective to obtain a therapeutic effect is meant.
[0062]
The term “pharmaceutically acceptable” is used herein within reasonable medical judgment, without undue toxicity, irritation, allergic reactions, or other problems or complications. Used in proportion to the risk ratio to mean compounds, materials, compositions and / or dosage forms suitable for use in contact with human and animal tissues.
[0063]
The term “pharmaceutically acceptable carrier” as used herein relates to carrying or transporting a subject drug from one organ, or part of the body, to another organ or other part of the body. Means a pharmaceutically acceptable material, composition or medium, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose And carboxymethylcellulose sodium, ethylcellulose, and derivatives thereof such as cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository waxes Excipients; (9) fats and oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) glycerin, sorbitol, mannitol, and Polyols such as polyethylene glycol; (12) Ethyl oleate and Lauri (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic compatible materials used in pharmaceutical formulations.
[0064]
As noted above, certain aspects of the checkpoint proteins of the present invention and other proteins described above and below may contain basic functional groups such as amino groups or alkylamino groups and are therefore pharmaceutically acceptable. Pharmaceutically acceptable salts can be formed with certain acids. In this regard, the term “pharmaceutically acceptable salts” refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts were formed either in situ during the final isolation and purification of the compounds of the invention, or by reacting the purified compounds of the invention in the free state individually with the appropriate organic or inorganic acid. It can be prepared by isolating the salt. Typical salts include hydrobromide, hydrogen chloride, sulfate, sulfite, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate , Benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptate, lactobionate, and lauryl sulfone Acid salts and the like are included.
[0065]
In other examples, the compounds of the present invention may contain one or more acidic functional groups and can thus form pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these examples, the term “pharmaceutically acceptable salts” refers to the relatively non-toxic inorganic and organic base addition salts of the compounds of the present invention. These salts can further be prepared in situ during the final isolation and purification of the compound, or the acid dissociated purified compound can be converted to a pharmaceutically acceptable metal cation hydroxide, It can be prepared by reacting individually with a suitable base, such as carbonate or bicarbonate, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Typical alkali salts or alkaline earth salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
[0066]
Wetting agents such as sodium lauryl sulfate and magnesium stearate, emulsifiers and lubricants, as well as colorants, release agents, coating agents, sweeteners, flavorings, preservatives and antioxidants may be present in the composition as well. .
[0067]
Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants such as ascorbic acid, cysteine hydroxide, sodium bisulfate, sodium metabisulfate, sodium sulfite and the like; (2 ) Fat-soluble antioxidants such as ascorbic acid palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc .; and (3) citric acid, ethylenediaminetetraacetic acid ( EDTA), metal chelators such as sorbitol, tartaric acid, phosphoric acid and the like are included.
[0068]
The formulations of the present invention include formulations suitable for oral, intranasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations may conveniently be present in unit dosage form and may be prepared by any method known in the pharmaceutical arts. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of 100%, this amount will be in the range of about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.
[0069]
Methods for preparing these formulations or compositions include the step of bringing a compound of the invention into association with a carrier and optionally with one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the invention with liquid carriers, or finely divided solid carriers or both, and then, if necessary, shaping the product.
[0070]
Formulations of the present invention suitable for oral administration include capsules, cachets, pills, tablets, (fragrance bases, usually sucrose and acacia gum or tragacanth, each containing a defined amount of the compound of the present invention as an active ingredient In the form of lozenges, powders, granules, or as solutions or suspensions in aqueous or non-aqueous solutions, or as oil-in-water or water-in-oil emulsions, or as elixirs or syrups, or It may be in the form of a troche (using an inert base such as gelatin and glycerin or sucrose and acacia gum) and / or as a mouthwash. The compounds of the present invention can also be administered as boluses, electuaries or pastes.
[0071]
In a solid dosage form (capsule, tablet, pill, dragee, powder, granule, etc.) of the present invention for oral administration, the active ingredient is sodium citrate or dicalcium phosphate, and / or any of the following: (1) a filler or bulking agent such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; ) Binders such as, for example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or acacia gum; (3) wetting agents such as glycerol; (4) agar-agar, calcium carbonate, potato or tapioca starch Disintegrants such as alginic acid, certain silicates, and sodium carbonate; (5) solution retarders such as paraffin (6) Absorption accelerators such as quaternary ammonium compounds; (7) Wetting agents such as cetyl alcohol and glycerol monostearate; (8) Absorbents such as kaolin and bentonite clay; (9) Talc. Lubricants such as calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) colorants. In the case of capsules, tablets, and pills, the pharmaceutical composition may also include a buffer. Similar types of solid compositions can be used as fillers in soft and hard-filled gelatin capsules with excipients such as high molecular weight polyethylene glycols as well as lactose or milk sugar.
[0072]
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be combined with binders (eg gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (eg sodium starch glycolate or crosslinked sodium carboxymethylcellulose), surfactants or dispersants. You may prepare using. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
[0073]
Tablets of the pharmaceutical composition of the present invention, and other solid dosage forms such as dragees, capsules, pills and granules, optionally such as enteric coatings and other coatings known in the pharmaceutical arts Can be obtained or prepared with coatings and jackets. They can also be slow or controlled of the active ingredients therein, for example by using various ratios of hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres to provide the desired release profile. It may be formulated to provide release. They may be sterilized, for example, by filtration through a bacterial residue filter, or by incorporating sterile substances in the form of a sterile solid composition that can be dissolved in sterile water or other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying substances, and may be compositions that slowly release the active ingredient only at or selectively to specific areas of the gastrointestinal tract. Good. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in the form of micro-inclusions, if appropriate, with one or more of the above-described excipients.
[0074]
Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms include inert diluents commonly used in the art, such as water and other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl Benzoic acid, propylene glycol, 1,3-butylene glycol, fats and oils (especially cottonseed oil, peanut oil, germ oil, olive oil, castor oil and sesame oil), fatty acid esters of glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and sorbitan, and their Solubilizing agents such as mixtures and emulsifiers may be included.
[0075]
In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring and preserving agents.
[0076]
Suspensions include, in addition to the active compounds, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and Suspending agents may be included, such as mixtures thereof.
[0077]
Formulations of the pharmaceutical composition of the present invention for rectal or vaginal administration may be shown as suppositories, for example, it is solid at room temperature but liquid at body temperature and is therefore intrarectal or vaginal. One or more suitable non-irritating excipients or carriers including cocoa butter, polyethylene glycol, suppository wax, or salicylate that melts at room temperature to release the active compound, and one or more of the present invention Can be prepared by mixing the compounds.
[0078]
Formulations of the present invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing carriers as known to be suitable in the art. It is.
[0079]
Dosage forms for topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalant formulations. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.
[0080]
Ointments, pastes, creams and gels contain animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicon, bentonite, silicic acid, talc in addition to the active compounds of the present invention. And excipients such as zinc oxide, or mixtures thereof.
[0081]
Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. The spray may further comprise conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.
[0082]
Transdermal patches have the added advantage of providing controlled delivery of the compounds of the invention into the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such inflow can be controlled by providing a rate regulating membrane or by dispersing the compound in a polymer matrix or gel.
[0083]
Ophthalmic formulations, eye ointments, eye drops, powders, implants and the like are also intended to be within the scope of this invention.
[0084]
Pharmaceutical compositions of the present invention suitable for parenteral administration include antioxidants, buffers, bacteriostats, solutes, suspensions or thickeners that make the isotonic formulation with the blood of the intended recipient. One or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions, or emulsions that may be included, or sterile that may be returned to a sterile injectable solution or dispersion immediately before use. Contains one or more compounds of the invention in combination with a powder.
[0085]
Examples of suitable water-soluble or water-insoluble carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and their suitable Mixtures, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate are included. The proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
[0086]
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Inhibition of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. Similarly, isotonic agents such as sugars, sodium chloride and the like may be desirably included in the composition. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
[0087]
In some cases, it may be desirable to delay the absorption of the drug from subcutaneous or intramuscular injection in order to sustain the effect of the drug. This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility. The absorption rate of a drug depends on its dissolution rate, and as a result can depend on the crystal size and crystal form. Alternatively, delayed absorption of a parenteral dosage form is accomplished by dissolving or suspending the drug in an oil medium.
[0088]
Injectable depot forms are made by forming microencapsule matrices of the compounds of the invention in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the characteristics of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with living tissues.
[0089]
When the compound of the present invention is administered as a pharmaceutical to humans and animals, it is administered per se or, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) of the active ingredient is a pharmaceutically acceptable carrier And can be administered as a pharmaceutical composition comprising.
[0090]
The preparations of the present invention may be administered orally, parenterally, topically, or rectally. They will of course be administered in a form suitable for the respective route of administration. For example, they can be administered by tablet, or capsule, injection, inhalation, ophthalmic lotion, ointment, suppository, etc., injection, infusion, or inhalation administration; topical administration by lotion or ointment; and rectal administration by suppository Be administered. Oral administration is preferred.
[0091]
As used herein, the terms “parenteral administration” and “administer parenterally” refer to modes of administration other than enteral and topical administration, usually by injection, intravenous, intramuscular, intraarterial. Including intrathecal, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and intracisternal injections and infusions It is not limited to these.
[0092]
As used herein, the terms “systemic administration”, “systemic administration”, “peripheral administration”, and “peripheral administration” refer herein to the entry into the nervous system of a patient and thus metabolism. As used in and other similar processes, it means administration of a compound, drug, or other material other than direct administration to the central nervous system, eg, subcutaneous administration.
[0093]
These peptides and compounds can be administered, for example, by oral administration by spray, intranasal administration, rectal administration, vaginal administration, parenteral administration, buccal administration, and buccal administration and tongue, such as powders, ointments, or eye drops. Administration to humans and other animals may be made for treatment by any suitable route, including topical administration, including sub-administration.
[0094]
Regardless of the chosen route of administration, the compounds of the invention and / or the pharmaceutical compositions of the invention that can be used in an appropriate hydrated form are administered pharmaceutically acceptable by conventional methods known to those skilled in the art. Formulated into a dosage form.
[0095]
The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is effective to obtain the desired therapeutic response for a particular patient, composition and mode of administration without showing toxicity to the patient. Variations may be made to obtain component amounts.
[0096]
The selected dose level will depend on the activity of the particular compound of the invention used, or its ester, salt, or amide, route of administration, timing of administration, excretion rate of the particular compound used, duration of treatment, other drugs, particular used A variety of factors, including compounds and / or materials used in combination with other compounds, the age, sex, weight, condition, general health and history of the patient to be treated, and similar factors known in the medical arts It is thought that it depends.
[0097]
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start a dose of a compound of the invention used in a pharmaceutical composition at a level lower than that required to achieve the desired effect, and gradually increase the dose until the desired effect is achieved. Can be increased.
[0098]
In general, a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective amount will generally depend on the above factors. In general, when used for the indicated analgesic effect, intravenous, intraventricular and subcutaneous doses of a compound of the invention to a patient will be about 0.0001 mg / kg to about 0.0001 mg / kg body weight per day. It is considered to be in the 100 mg range.
[0099]
If desired, the effective daily dose of the active compound may be divided into 2, 3, 4, 5, 6 or more times at appropriate intervals throughout the day, optionally in unit dosage forms. It may be administered.
[0100]
While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
[0101]
Defects in the mitotic checkpoint result in chromosome loss, cell cycle disruption, and polyploidy. These defects can play a role in tumor development. Mad1 and MAD2 are genes that help control mitotic checkpoints. Therefore, targeting cells with mutations in MAD1 or MAD2 is a viable strategy in preventing cancer.
[0102]
A gene having a mutation incompatible with the mutation in MAD1 and / or MAD2 can specifically eradicate cells having a defect in mitotic checkpoint and leave a cell having a functional mitotic checkpoint Provides potential drug targets for Mutations in such genes are synthetically lethal by MAD1 and / or MAD2.
[0103]
Although the invention has been generally described, the following examples are included solely for the purpose of illustrating particular aspects and embodiments of the invention and are not intended to limit the invention. It will be more easily understood with reference.
[0104]
Example 1. Genes that are synthetically lethal due to MAD1 mutations
The following strains were tested:

[0105]
Thus, mutations in the four genes (TUB1, CIN8, STU1, and SFI1) are synthetically lethal due to the MAD1 mutation. That is, these strains cannot grow without the MAD1 plasmid unless transformed with the TUB1, CIN8, SFI1, or STU1 plasmid, respectively. TUB1 encodes α-tubulin. CIN8 encodes a kinesin-related microtubule motility protein. STU1 encodes a microtubule-associated protein. SFI1 encodes a novel protein.
[0106]
Example 2 Screening for genes that are synthetically lethal by MAD2
The plasmid is introduced into a yeast cell mutant for the MAD2 gene. This plasmid allows for the expression and transposition of a significant retrotransposition element called Ty1. After transfer to the genome, the majority of cells have one or several copies of the transposon randomly inserted into the genome. In a cell, if a copy of the transposon is inserted into a gene, the inactivation is synthetically lethal by NAD2, and both MAD2 and the second gene are inactivated in this cell, so that cell Is thought to stop dividing. We find such metastases by growing cells over several generations. Cells that have such metastases and do not divide rapidly are lost from a population of actively dividing cells. After the growth selection mechanism, we examine the entire genome for the presence or absence of such transfer in selected genes. Since metastasis is a random process, if no such metastasis exists for a particular gene, this gene is a candidate for synthetic lethality.
[0107]
This experiment is controlled by parallel selection in cells with functional MAD2. In such cells, transfer should occur in the (second) synthetic lethal gene candidate in order for cells with functional MAD2 to survive growth selection.
[0108]
Next, a new synthetic lethal gene candidate by MAD2 is genetically confirmed.
[0109]
Example 3 Microtubule polymerization, spindle pole body, microtubule motility, and centromere deficiency all stop cells during mitosis by activating spindle checkpoints
Materials and methods:
For a detailed description of materials and methods, see Hardwick, KG, Li, R., Mistrot, C., Chen, which is incorporated herein by reference in its entirety. ), RH, Dann, P., Rudner, A., and Murray, AW, “The obstacles in many different spindle components are spindles in the budding yeast Saccharomyces serevisiae. See Genetics 152, 509-518 (1999), “Activating spindle checkpoints in the budding yeast Saccharomyces serevisiae”. Except for the mps2-1 and tub2-403 strains that are syngeneic to S288C and their derivatives, all strains used were W303 (MATa ade2-1 can1-100 ura3-1, leu2-3, 112his3-11, 15trp1-1) And related.
[0110]
To investigate the types of mitotic defects that activate MAD-dependent checkpoints, the interaction between mad mutations and mutations that cause defects in the execution of specific stages of mitosis is investigated. These include mutations that inhibit microtubule depolymerization, mutations that prevent spindle pole body replication, mutations that eliminate centrosome-bound proteins, and cdc mutations that cause cells to arrest during mitosis at 37 ° C. Mitotic mutations are tested in two types: conditional lethal mutations and null mutations in non-essential genes. To determine the interaction of mad mutations with C2 or cdc mutations that cause arrest during mitosis, the characteristics of the mad cdc double mutant are compared with those of the cdc mutant alone. If the cell cycle stop point of the cdc mutant is not dependent on the spindle checkpoint, the cdc mad double mutant behaves exactly like the cdc mutant alone; the cell cycle stops at non-permissive temperatures and is permissive When incubated at temperature, single and double mutants lose viability at the same rate. In contrast, if the cdc mutant stoppage is dependent on the spindle checkpoint, the mad cdc double mutant will be at the limiting temperature, even if the spindle is missing, due to checkpoint inactivation. It is thought to be mitotic. Therefore, when such a cdc mutation is combined with a mad mutation, double mutants that fail to stop during mitosis and lose viability faster than the cdc mutant alone when the strain is incubated at non-permissive temperatures. It is thought to be produced.
[0111]
Example 4 Synthetic lethal screening based on inducible expression of analyzed genes
Experiments are carried out by crossing a haploid that lacks microtubule movement with a mad strain. In a preferred embodiment, experiments are performed to create heterozygosity using gene disruption to lose microtubule motility genes in strains that are already heterozygous for the mad mutation. This latter method eliminates the possibility that a slow-growing strain such as kar3 will acquire a suppressive mutation, but this is a more cumbersome method.
[0112]
Hybrids containing recessive synthetic lethal mutations are mated with wild type strains to form spores, dissected, and spore quadruples are checked for the appropriate single mutation pattern for separation of the synthetic lethal phenotype. Mutants that exhibit such separation patterns are cloned by complementation. Variations on this and other methods of screening yeast are known (eg, US Pat. Nos. 5,912,154; 5,908,752; 5,876,951; 5,869,287; 5,866,338; 5,789,184; 5,674,996; No. 5,578,477; No. 5,527,896; No. 5,352,581; No. 5,175,091; and No. 5,139,936), one of ordinary skill in the art readily understands which one to choose for a particular purpose.
[0113]
Example 5. Complementation of yeast strains with a human cDNA library
In this example, the human cDNA encoding a protein that is a surrogate for the above synthetic lethality in yeast with aberrant MAD1 or MAD2 provides the principle by which it is identified. A yeast strain containing an abnormal MAD1 or MAD2 is constructed. A cDNA library using mRNA prepared from human fetal fibroblasts is constructed in a S. serevisiae vector containing a constitutive yeast promoter such as ADH for expression of human cDNA (for details see eg U.S. Pat. Nos. 5,783,661; 5,952,195). The library was transfected into the host MAD1 or MAD2 yeast strain and transformed into independent transformants 10^FiveIndividuals are screened for growth potential on glucose or galactose by replica plating. As a result, several transformants are isolated whose growth pattern is rescued by expression of human cDNA. Some of them are already known genes, such as the human homologue of CDC14, the human homologue ABL1 of the Abelson murine leukemia virus oncogene, and the fibroblast growth factor receptor bfr-2. It appears to be related or “homologous” to genes such as epithelial cell receptors, calcium channel proteins, or human Weel kinase in the eph / elk family of protein kinases.
[0114]
Example 6 Screening for pharmaceutical antagonists and agonists
Drugs useful for treating cell cycle or DNA replication disorders identified in the present invention can now be screened using the established yeast models described above. Several types of drugs can be screened, such as chemical compounds, organic and inorganic compounds, peptides or peptidomimetics, antisense molecules, antibodies and the like. Methods for generating and performing high-throughput screening of chemical libraries are well established and known to those skilled in the art. Drugs for screening candidate substances selected in yeast-based assays using prospects obtained in cell cycle regulation and for assessing their therapeutic potential by further examining mammalian cells or cell lines Establish a discovery program. In essence, the assay consists of a control system that observes lethality and a test system to which a drug is added, the effect of which is evaluated based on the difference between the control system and the test system. If the drug enhances or reverses lethality, the drug is considered a candidate drug that may be specific in the biochemical pathway controlled by the first and second genes It is. Specificity is a number of specificity-determining tests, for example, in vitro tests on the effect of gene products on enzyme activity by introducing double mutants that reverse the originally observed effect by the ability to bind to the target gene or its product Further testing by, etc. These pharmaceutical agents were thought to predictably ideally inhibit cell cycle aspects, thereby enabling promising determinations in cancer or other cell cycle related clinical conditions. Some of the compounds tested are found to be active at pharmaceutically acceptable concentrations. These include, but are not limited to, tributyrate (4-phenylbutyric acid sodium salt, or sodium phenylbutyrate), various benzopyran derivatives prepared by means known in the art. For example, U.S. Patent Nos. 5,359,115; 5,362,899; 5,288,514; 5,733,920 or International Publication No. 94/08051; International Publication No. 92/10092; International Publication No. 93/09668; No. 91/07087; see WO 93/20242. Accordingly, a diverse library of diversomers of the order of 1000 to 100,000 or more of the compounds of the present invention can be synthesized, and inhibitors described in PCT International Publication No. 94/09135 can be synthesized. By using a high-throughput assay for detection, biological activity can be rapidly screened. For an overview of combinatorial synthesis methods, as well as library screening and deconvolution integral analysis methods, see, for example, EM Gordon et al. (1994) J. Med. Chem. 37: 1385-1401, and See the referenced references.
[0115]
Example 7. Treatment of clinical conditions by benign or malignant cell proliferation
Next, drugs screened positive in a yeast-based assay are tested for the treatment of diseases caused by excessive cell proliferation. Most of these diseases are malignant diseases, ie various types of cancer, including but not limited to solid tumors and leukemias such as: Apdoma, sebaceous tumor, atheroma, malignant carcinoid syndrome, carcinoid heart Disease, cancer (eg, Walker cancer, basal cell carcinoma, basal squamous cell carcinoma, Brown-Pearce cancer, ductal carcinoma, Ehrlich tumor, Krebs 2 tumor, Merkel cell carcinoma, mucinous carcinoma, non-small Cell lung cancer, oat cell carcinoma, papillary carcinoma, rigid carcinoma, bronchiole, bronchiole carcinoma, primary bronchial carcinoma, squamous cell carcinoma, and transitional cell carcinoma), histiocytic disorder, leukemia (eg, B cell leukemia, mixed type) Leukemia, null cell leukemia, T cell leukemia, chronic T cell leukemia, HTLV-II related leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, mast cell leukemia, and myeloid leukemia) Malignant histiocytosis, Hodgkin's disease, immunoproliferative small, non-Hodgkin lymphoma, plasmacytoma, reticulo-tissue hyperplasia, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fiber Sarcoma, giant cell tumor, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovial tumor, adenofibroma, adenolymphoma, carcinosarcoma, notochord Tumor, craniopharyngioma, undifferentiated germinoma, hamartoma, mesenchyme, midnephroma, sarcoma, ameloblastoma, cementoma, odontoma, teratocarcinoma, thymoma, trophoblastic tumor , Adenoma, adenoma, cholangioma, cholesteatoma, columnar tumor, cystadenocarcinoma, cystadenoma, granulosa cell tumor, male germ cell tumor, liver cancer, sweat adenoma, islet cell tumor, Leydig cell tumor (Leydig cell tumor), papilloma, Sertoli cell tumor, follicular cell tumor, leiomyoma, flat Leiomyosarcoma, myoblastoma, myoma, myoma, rhabdomyosarcoma, rhabdomyosarcoma, ventricular epithelioma, ganglion celloma, glioma, medulloblastoma, meningioma, schwannoma ( neurilemmoma), neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, horned hemangioma, eosinophilia associated blood vessels Lymphoid tissue hyperplasia, angioma sclerosing, hemangiomatosis, glomus hemangioma, hemangioendothelioma, hemangioma, vascular dermatocytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphatic vessel Sarcoma, pineal tumor, carcinosarcoma, chondrosarcoma, phyllosarcoma, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myoma, myxosarcoma, ovarian cancer, rhabdomyosarcoma Sarcomas, neoplasms (eg, bone neoplasms, breast neoplasms, digestive neoplasms, colorectal neoplasms, Visceral neoplasms, pancreatic neoplasms, pituitary neoplasms, testicular neoplasms, orbital neoplasms, head and neck neoplasms, central nerve neoplasms, acoustic neoplasms, pelvic neoplasms, airway neoplasms, and urogenital organs Neoplasm), neurofibromatosis, cervical dysplasia.
[0116]
Some diseases are caused by excessive but benign cell proliferation (ie non-malignant). Examples of such diseases are: fibrosis, benign prostatic hyperplasia, atherosclerosis, restenosis, glomerulosclerosis, keloid, psoriasis, mole, keratosis, fibromyalgia, infection Molluscum pneumonia, sexually transmitted warts, sebaceous hyperplasia, warts, hemangioma, venous lake, chondrodermatitis, purulent granuloma, purulent spondylodenitis, keloid, keratinous squamous cell tumor , Leukoplakia, multiple sebaceous cysts, eyelashes, superficial epithelial nevus, polyps, junctional nevus, purulent granuloma, nodular urticaria, dermal fibroma, sebaceous adenoma , And other skin diseases, and non-malignant neoplastic diseases such as Kaposi's sarcoma, papilloma.
[0117]
Next, the effect of sodium phenylbutyrate or a benzopyran derivative is determined on the oncogenic phenotype of human glioblastoma cells in vivo. In the deep white matter of the right hemisphere of Fisher 344 rats, syngeneic 9L gliosarcoma cells (4 × 10^FourInoculated). Thereafter, the animals were subjected to continuous treatment with sodium phenylbutyrate (550 mg / kg / day, subcutaneous administration) for 2 weeks using an osmotic minipump implanted subcutaneously. In control rats, the minipump was filled with saline. Fisher's Exact Test was used for statistical analysis of the data. Treatment of glioblastoma cells with drugs resulted in a time- and dose-dependent growth arrest that was accompanied by a similar decrease in DNA synthesis. When treated with 4 mM phenylbutyrate for 4-6 days, growth is inhibited by about 50%. Phenylbutyrate, an intermediate metabolite of phenylacetate, inhibited tumor cell replication, but the final metabolite, phenylacetylglutamine, is inactive. In addition to selective tumor cell arrest, both phenylacetate and phenylbutyrate are associated with altered cytoskeletal intermediate filament patterns, loss of adhesion independence, and decreased tumorigenicity in athymic mice. As can be seen, it promoted cell maturation and conversion to a non-malignant phenotype. These significant changes in tumor behavior are accompanied by changes in the expression of cyclin genes involved in growth control, angiogenesis, and immunosuppression.
[0118]
All of the Internet authorities, patents, publications, and references cited therein, cited above, are hereby specifically incorporated by reference in their entirety for each reference.
[0119]
Although the invention has been described above, it will be apparent that this can be varied in many ways. Such modifications are considered to be within the spirit and scope of the present invention, and all such modifications are intended to be included within the scope of the appended claims.

Claims (22)

A method of identifying a drug that inhibits the growth or replication of cells having a mutated MAD2 gene or analog or homologue thereof, comprising the following steps:
(A) contacting a cell having a mutated MAD2 gene or an analog or homologue thereof with a drug; and (b) synthetically if mutated and present in combination with a mutated MAD2 gene or an analog or homologue thereof. Determining whether a drug modulates the activity of a wild-type second gene that is lethally lethal. 2. The method of claim 1, wherein the cell is a tumor cell. The method of claim 1, further comprising the step of comparing the result of step (b) with control cells grown without drug. 4. The method according to claim 3, wherein the control cell is a normal cell. 4. The method of claim 3, wherein the control cell is a tumor cell. 4. The method of claim 3, wherein the control cell has a mutated MAD2 gene or an analog or homologue thereof. 2. The method of claim 1, wherein the second gene is selected from the group consisting of analogs and homologues TUB1, CIN8, SFI1, STU1, and combinations thereof. A method for identifying a compound useful in the treatment of tumor cells having a mutated MAD2 gene or a homologue or analogue thereof, comprising the following steps:
(A) contacting, in its mutant form, a second gene product that is synthetically lethal when combined with a mutant MAD2 gene or a homologue or analog thereof, with a test compound; and (b) Determining the activity of the second gene product. 9. The method of claim 8, further comprising comparing the activity of the second gene product in the presence of the test compound with the activity of the gene product in the absence of the second gene product. 9. The method of claim 8, wherein the second gene is selected from the group consisting of analogs and homologues TUB1, CIN8, SFI1, STU1, and combinations thereof. A screening assay system for identifying drugs, including the following:
(A) a control yeast cell with a deletion in the MAD2 gene, analogs or homologues thereof, and a wild-type second gene whose mutant form is synthetically lethal when combined with a deletion in MAD2;
(B) a test yeast cell line comprising a drug and yeast with a MAD2 deletion; and (c) in a test system compared to a control system in which positive drug candidates are identified by a decrease in the survival rate of the test cell line. Detector for comparing MAD2 survival rate. A method for screening for the presence of benign or malignant cell proliferation in a tissue sample, comprising the following steps:
a) providing a tissue sample from an individual suspected of having cancer;
b) assessing the expression of MAD2 in cells of the tissue sample; and
c) comparing the MAD2 expression of the tissue sample with the MAD2 expression of the control sample, wherein the presence of abnormal expression of MAD2 in the test sample compared to the expression in the control sample indicates the presence of cancer. A drug can selectively interact with at least one second gene or gene product in a target cell containing a mutated or deleted first MAD2 gene or gene product, thereby exposing the target cell to the drug A pharmaceutical composition comprising an effective amount of a drug, and then a pharmaceutically acceptable carrier or diluent, wherein cell division is stopped or cells are selectively killed. 14. The pharmaceutical composition of claim 13, wherein the drug comprises an oligonucleotide, a gene product, a homologue or analog of the oligonucleotide or gene product, a small molecule, or a peptide mimetic. A method of treating a human or animal hosting or potentially having a disease associated with a MAD2 mutation, comprising providing a pharmaceutical composition according to claim 13, and human or Treating the animal with an effective amount of a pharmaceutical composition. 14. The method of claim 13, wherein the disease is selected from the group consisting of: Apdoma, sarcoma, atheroma, malignant carcinoid syndrome, carcinoid heart disease, cancer (eg, Walker cancer, basal cell carcinoma, basal spine cell) Cancer, Brown-Pearce cancer, ductal carcinoma, Ehrlich tumor, Krebs 2 tumor, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, rigid carcinoma, bronchiole, Bronchiolocarcinoma, primary bronchial carcinoma, squamous cell carcinoma, and transitional cell carcinoma), histiocytic disorder, leukemia (eg, B cell leukemia, mixed leukemia, null cell leukemia, T cell leukemia, chronic T cell leukemia, HTLV- II-related leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mast cell leukemia, and myeloid leukemia), malignant histiocytosis, Hodgkin's disease, immunoproliferative small, non-Hodge Lymphoma, plasmacytoma, reticulocytosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumor, histiocytoma, lipoma, liposarcoma, mesothelioma , Myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovial, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, craniopharynoma, anaplastic germoma, hamartoma, mesenchyme, Medium nephroma, myoma, enamel epithelioma, cementoma, odontoma, teratocarcinoma, thymoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, columnar tumor, cystadenocarcinoma Cystadenoma, granulosa cell tumor, male germinoma, liver cancer, sweat adenoma, islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor, follicular cell tumor, smooth Myoma, leiomyosarcoma, myoblastoma, myoma, myoma, rhabdomyosarcoma, rhabdomyosarcoma, ventricular epithelioma, god Gangliocytoma, glioma, medulloblastoma, meningioma, neurolemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, non-chromophilic paranervous Paraganglioma nonchromaffin, horned hemangioma, eosinophilia-associated angiolymphoid lymphoproliferative disorder, angioma sclerosing, hemangioma, glomus hemangioma, hemangioendothelioma, hemangioma, Hemangiosarcoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pineal gland, carcinosarcoma, chondrosarcoma, phyllosarcoma, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma , Liposarcoma, lymphangiosarcoma, myoma, myxosarcoma, ovarian cancer, rhabdomyosarcoma, sarcoma, neurofibromatosis, cervical dysplasia, fibrosis, benign prostatic hyperplasia, atherosclerosis, re Stenosis, glomerulosclerosis, keloid, psoriasis, mole, keratosis, fibrous softness , Molluscum contagiosum, sexually transmitted warts, sebaceous hyperplasia, warts, hemangioma, venous lake, chondrodermatitis, suppurative granuloma, suppurative scab, keloid, keratinization Squamous cell tumor, leukoplakia, multiple sebaceous cysts, lash infestation, superficial epithelial nevus, polyp, junctional nevus, purulent granuloma, nodular urticaria, dermal fibroma , Sebinomas, papillomas, and combinations thereof. 16. The method of claim 15, wherein the disease comprises a yeast infection. 16. The method of claim 15, wherein the disease comprises breast cancer. An agent is capable of selectively interacting with at least one second gene or gene product found in cells having at least one first gene defect, and the gene product is a human isozyme Effective amount of drug and pharmaceutically acceptable selected from TUB1, CIN8, SFI1, STU1, and combinations thereof (or lethal gene encodes the human isozyme TUB1, CIN8, SFI1, STU1) A method of treating cancer cells having an abnormal accumulation of MAD2 gene or an analog or homologue thereof, comprising administering a pharmaceutical composition comprising a carrier or diluent. At least one second gene and at least one first gene MAD2, wherein the first gene MAD2 is mutated so that synthetic lethality results in up-regulation, down-regulation, removal or destruction of the second gene, Or a recombinant eukaryotic cell comprising an analog or homologue thereof. 21. The recombinant eukaryotic cell of claim 20, wherein the at least one second gene or product thereof is selected from the group consisting of analogs and homologues TUB1, CIN8, SFI1, STU1, and combinations thereof. 21. The recombinant eukaryotic cell of claim 20, wherein the first gene has been removed or destroyed.