JP2007535962A - 化合物のキャラクタリゼーション法 - Google Patents
化合物のキャラクタリゼーション法 Download PDFInfo
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Abstract
【選択図】 なし
Description
a)複数のウィルスコード化アッセイの第1のセットからのアッセイを、複数の容器を含む第2のセット内に位置する宿主細胞で一過的に発現させ、
b)適宜、被験因子を1以上の容器に添加し、
c)1以上のアッセイの測定を実施し、
d)アッセイの測定値を組合せてデータセットを生成し、
e)データセットを分析して被験因子の機能的特性を求める
ことを含んでなり、上記アッセイの測定を生細胞で非破壊的に実施することを特徴とする方法を提供する。
図1Bは本発明の方法の一つの適用例を示し、ウィルスコード化アッセイの第1のセット又はアレイを使用して第2のセット又はアレイの細胞に形質導入し、第2のセット又はアレイが第1のアレイの4倍であるが、第1のアレイの要素の大きさと間隔を保持しており、例えば96ウェルの第1のアレイから4つの同じ96ウェルの第2のアレイにレプリカ平板培養するものであり、各々の第2のアレイは異なる細胞型を含有している。図1Cは第3の可能な構成を示しており、ウィルスコード化アッセイの第1のセット又はアレイを使用して第2のセット又はアレイの細胞に形質導入するものであり、第2のセット又はアレイが第1のアレイと異なる要素の大きさと間隔で第1のアレイの4倍であり、例えば96ウェルの第1のアレイから4倍レプリカ平板培養して384ウェルのプレートにして各々のアッセイに対して4倍の複製を提供するものである。
本例に記載するアッセイは、MAPKAPキナーゼ2が核から細胞質へトランスロケーションする過程の拮抗作用(アンタゴニスト)と作動作用(アゴニスト)の両方を含む。ヒトの疾病の治療用化合物を同定しようとするときには、アゴニスト又はアンタゴニストの代わりにライブラリーの化合物を使用することができる。培地をデカントして細胞から除去した。幾つかのウェルでは、培地を新しい培地と交換した。他のウェルでは、培地を300ナノモル濃度のアニソマイシン(アゴニスト)を含有する培地と交換した。その他のウェルでは、培地をアゴニスト及びアンタゴニスト(100マイクロモル濃度のSB203580−HCI)を含有する培地と交換した。細胞を90分、37℃でインキュベートした。培地をデカントして細胞から除去し、細胞を2%ホルマリン溶液で15分固定した。細胞をPBSで洗浄し、Hoechst 33342の2.5マイクロモル濃度溶液を加えて核を染色した。このアッセイの結果を図5に示す。
第2のアッセイでは、グルココルチコイド受容体(GR)が細胞質から核へトランスロケーションする過程の作動作用を例示する。GFPに連結されたGRを含有する100MOIの組換えアデノウィルスベクターを用い、上述の通りHeLa細胞を調製した。アッセイの1時間前に、組織培養培地を、活性炭で処理した血清を含有する培地で1時間37℃で交換した。この培地をデカントし、同じ血清と様々な濃度のデキサメタゾン(800、400、200、100、50、25ナノモル濃度)を含有する培地で20分37℃で交換した。GRの細胞質から核へのトランスロケーションに影響し得るライブラリーからの化合物を含ませることができる。次に、培地をデカントして細胞から除去し、細胞を固定し、上述の通り処理した。このアッセイの結果を図6に示す。
Claims (35)
- 宿主細胞内での情報伝達経路及び/又は細胞プロセスに対する被験因子の活性及び/又は機能のインビトロキャラクタリゼーション方法であって、
a)複数のウィルスコード化アッセイの第1のセットからのアッセイを、複数の容器を含む第2のセット内に位置する宿主細胞で一過的に発現させ、
b)適宜、被験因子を1以上の容器に添加し、
c)1以上のアッセイの測定を実施し、
d)アッセイの測定値を組合せてデータセットを生成し、
e)データセットを分析して被験因子の機能的特性を求める
ことを含んでなり、上記アッセイの測定を生細胞で非破壊的に実施することを特徴とする方法。 - 1種以上の異なるウィルスコード化アッセイを各容器内の宿主細胞で発現させる、請求項1記載の方法。
- 各容器が異なる宿主細胞を含む、請求項1又は請求項2記載の方法。
- 各容器が同一の宿主細胞又は同一の宿主細胞の遺伝的変異体を含む、請求項1又は請求項2記載の方法。
- アデノウィルスベクターを用いてウィルスコード化アッセイを宿主細胞で発現させる、請求項1乃至請求項4のいずれか1項記載の方法。
- 組換えヒト昆虫ウィルスベクターを用いてウィルスコード化アッセイを宿主細胞で発現させる、請求項1乃至請求項4のいずれか1項記載の方法。
- 前記アッセイが検出可能なタンパク質を含む、請求項1乃至請求項6のいずれか1項記載の方法。
- 検出可能なタンパク質が蛍光タンパク質又は改変蛍光タンパク質である、請求項7記載の方法。
- 蛍光タンパク質が緑色蛍光タンパク質(GFP)又は変性GFPである、請求項7又は請求項8記載の方法。
- 改変緑色蛍光タンパク質(GFP)が、F64L、Y66H、Y66W、Y66F、S65T、S65A、V68L、Q69K、Q69M、S72A、T203I、E222G、V163A、I167T、S175G、F99S、M153T、V163A、F64L、Y145F、N149K、T203Y、T203Y、T203H、S202F及びL236Rからなる群から選択される1以上の突然変異を含む、請求項9記載の方法。
- 前記検出可能なタンパク質が酵素である、請求項7記載の方法。
- 前記酵素が、β−ガラクトシダーゼ、ニトロ還元酵素、アルカリホスファターゼ及びβ−ラクタマーゼからなる群から選択される、請求項11記載の方法。
- 前記アッセイが、MAPKAPキナーゼ2、グルココルチコイド受容体(GCCR)、上皮成長因子(EGF)、ERK1、アンドロゲン受容体(AR)、STAT3、NFAT1、SMAD2、AKT1−PH、FYVE−PH、PLC−PH及びRac1からなる群から選択される、請求項1乃至請求項12のいずれか1項記載の方法。
- 前記宿主細胞が真核細胞であり、該細胞が遺伝子改変されたものでも或いは遺伝子改変されていないものでもよい、請求項1乃至請求項13のいずれか1項記載の方法。
- 前記真核細胞が哺乳動物細胞である、請求項14記載の方法。
- 前記宿主細胞がヒト細胞である、請求項14又は請求項15記載の方法。
- 前記被験因子が化学作用因子又は物理作用因子である、請求項1乃至請求項16のいずれか1項記載の方法。
- 前記化学作用因子が有機又は無機化合物である、請求項17記載の方法。
- 前記有機化合物が、ペプチド、ポリペプチド、タンパク質、炭水化物、脂質、核酸、ポリヌクレオチド及びタンパク質核酸からなる群から選択される、請求項18記載の方法。
- 前記物理作用因子が電磁放射線である、請求項17記載の方法。
- ステップb)を行わずに、前記被験因子の非存在下で当該方法を実施する、請求項1乃至請求項20のいずれか1項記載の方法。
- 分析ステップe)が、被験因子の存在下で得られた各アッセイ測定値を、被験因子の非存在下での同じアッセイの測定値と比較する、請求項1乃至請求項20のいずれか1項記載の方法。
- 被験因子の非存在下でのアッセイ測定値が既知であってデータベースに記憶されている、請求項22記載の方法。
- 前記アッセイをイメージング装置を用いて測定する、請求項1乃至請求項23のいずれか1項記載の方法。
- 前記イメージング装置がIN Cell Analyzer 3000又はIN Cell Analyzer 1000である、請求項24記載の方法。
- 細胞集団に対する被験因子の活性及び/又は機能の自動化キャラクタリゼーション装置であって、請求項1乃至請求項25のいずれか1項記載の方法をイメージング装置及びコンピューター化されたデータ処理装置と共に用いることを含む、装置。
- 宿主細胞内での情報伝達経路及び/又は細胞プロセスに対する被験因子の活性及び/又は機能のキャラクタリゼーションのための複数のウィルスコード化アッセイを備える部品のキット。
- 前記ウィルスコード化アッセイがアデノウィルスベクター又は組換えヒト昆虫ウィルスベクターを含む、請求項27記載の部品のキット。
- 前記アッセイが、MAPKAPキナーゼ2、グルココルチコイド受容体(GCCR)、上皮成長因子(EGF)、ERK1、アンドロゲン受容体(AR)、STAT3、NFAT1、SMAD2、AKT1−PH、FYVE−PH、PLC−PH及びRac1からなる群から選択される、請求項27又は請求項28記載の部品のキット。
- 前記アッセイが検出可能なタンパク質を含む、請求項27乃至請求項29のいずれか1項記載の部品のキット。
- 前記検出可能なタンパク質が蛍光タンパク質又は改変蛍光タンパク質である、請求項30記載の部品のキット。
- 前記検出可能なタンパク質が酵素である、請求項30記載の部品のキット。
- 前記酵素が、β−ガラクトシダーゼ、ニトロ還元酵素、アルカリホスファターゼ及びβ−ラクタマーゼからなる群から選択される、請求項32記載の部品のキット。
- 前記キットがさらに酵素に対する基質を備える、請求項32又は請求項33記載の部品のキット。
- 前記酵素がニトロ還元酵素であり、前記基質がその基質である、請求項34記載の部品のキット。
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