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Sample translations submitted: 1
Japanese to English: (WO2012039414) SUBSTITUTED POLYCYCLIC CARBAMOYL PYRIDONE DERIVATIVE PRODRUG General field: Science Detailed field: Medical: Pharmaceuticals
Source text - Japanese [1525] 試験例１：キャップ依存的エンドヌクレアーゼ（ＣＥＮ）阻害活性の測定
１）基質の調製
5’末端のGを2リン酸化修飾、且つ2’位の水酸基をメトキシル化修飾し、5’末端から6番目のUをCy3標識、3’末端をBHQ2標識した30merRNA（5’-pp-[m2’-O]GAA UAU（-Cy3） GCA UCA CUA GUA AGC UUU GCU CUA-BHQ2-3’：日本バイオサービス社製）を購入し、EPICENTRE社製のスクリプトキャップ(ScriptCap)システムを使ってcap構造を付加した（産物はm7G [5’]-ppp-[5’] [m2’-O]GAA UAU（-Cy3） GCA UCA CUA GUA AGC UUU GCU CUA（-BHQ2）-3’）。これを変性ポリアクリルアミドゲル電気泳動法にて分離・精製し、基質として使用した。
２）酵素の調製
RNPは定法に従いウイルス粒子から調製した（参考文献：VIROLOGY(1976) 73, p327-338 OLGA M. ROCHOVANSKY）。具体的にはA/WSN/33ウイルス1x103 PFU/mL、200μLを10日齢発育鶏卵に接種し、37℃で2日間培養後、鶏卵のしょう尿液を回収した。20%スクロースを用いた超遠心分離によりウイルス粒子を精製し、TritonX-100とリソレシチンを用いてウイルス粒子を可溶化後、30-70%グリセロール密度勾配を用いた超遠心分離によりRNP画分(50～70％グリセロール画分)を採取し、酵素液（約1nMのPB1・PB2・PA複合体を含む）として使用した。
３）酵素反応
ポリプロピレン製の384穴プレートに酵素反応液（組成： 53 mM Tris-塩酸塩 (pH7.8)、1mM MgCl2、1.25 mM ジチオスレイトール、80mM NaCl、12.5％グリセロール、酵素液0.15μＬ）を2.5μＬ分注した。次にジメチルスルホキシド(DMSO)で段階的に希釈した被検化合物溶液0.5μＬ、ポジティブコントロール(PC)及びネガティブコントロール(NC)には、DMSO 0.5μＬを加え、よく混合した。次に基質溶液（1.4nM基質RNA、0.05%Tween20）2μＬを加えて反応を開始し、室温で60分間インキュベートした後、反応液1μＬを10μＬ のHi-Di Formamide溶液（サイジングマーカーとしてGeneScan 120 Liz Size Standardを含む：アプライドバイオシステム（ABI）社製。）に加え、反応を停止した。NCは反応開始前にEDTA（4.5mM）を加えることで予め反応を停止させた（表記濃度は全て終濃度である）。
３）阻害率（IC50値）の測定
反応停止させた溶液を85 ℃で5分間加熱し、氷上で2分間急冷後、ABI PRIZM 3730ジェネティックアナライザで分析した。解析ソフトABI Genemapperによりキャップ依存的エンドヌクレアーゼ産物のピークを定量し、PC、NCの蛍光強度をそれぞれ0％阻害、100％阻害として被検化合物のCEN反応阻害率(％)を求めた後、カーブフィッティング ソフトウェア （XLfit2.0：Model 205（IDBS社製）など）を使ってIC50値を求めた。親化合物である被検物質のIC50値を表２２～３４に示す。
Translation - English WO2012/039414
[1525] Experimental example 1: Cap-dependent endonuclease (CEN) inhibitory activity measurement
1) Preparation of substrate
The 5’-G being modified by diphosphorylation as well as having a 2’-methoxy modification; in addition, the 6th position U from the 5’ end being labeled and the 3’end being labeled with BHQ2 making a 30mer RNA of the following sequence: 5’-pp-[m-2’ –O]GAA UAU (-Cy3) GCA UCA CUA GUA AGC UUU GCU CUA-BH2-3’. This oligo was purchased from Japan Bioscience Inc., using the Epicenter manufacturer of Scrip tCap System appended to the cap construction (product of m7G[5’]-ppp-[5’] [m2-O]GAA UAU(-Cy3) GCA UCA CUA GUA AGC UUU GCU CUA (-BHQ2) -3’.
This [oligo] was separated and purified on a denaturing Polyacrylamide gel by electrophoresis and then used as a substrate.
2) Preparation of enzyme
RNP was produced from virus particles according to a protocol (Reference: Olga M. Rochovansky, Virology (1976) vol 73, pp 327-338). Explicitly, 200 µL of A/WSN/33 virus 1 x 103 PFU/ml was use to inoculate 10 day old chicken eggs. After 2 days of incubation at 37 °C, the fluid from chorioallantoic membrane in chicken eggs was collected. The virus particles were purified by ultracentrifugation separation using 20% sucrose; After the virus particles were solubilized using Triton X-100 and lysolecithin, the RNP fraction was collected (from the 50-70% glycerol fraction) by ultracentrifugation separation using a 30-70% glycerol gradient.
3) Enzyme reaction
In a polypropylene 384-well plate, was added 2.5 µL of the enzymatic reaction liquid (composition: 53 mM Tris-HCl (pH 7.8), 1 mM MgCl2, 1.25 mM dithiothreitol, 80 mL NaCl, 12.5% glycerol, enzyme solution 0.15 µL). Next, 0.5 µl of serial dilutions of the compound of interest in DMSO, positive control, or negative control in DMSO 0.5 µL was added followed by thorough mixing. Next he substrate solution (1.4 nM substrate RNA and 0.05% Tween20) 2 µL was added to start the reaction. After incubation at room temperature for 60 minutes, the reaction was stopped by addition to the reaction solution 10 µL of Hi-DI Formamide solution (GeneScan 120 Liz Size Standard (ABI Inc.) used as a sizing marker). For the negative control, before starting the reaction, EDTA (4.5 mM) was added in advance of stopping the reaction. (The noted concentrations are all final concentrations.)
3) Measurement of inhibitory ratios (IC50 levels)
The solutions of stopped reactions were heated to 85 °C for 5 minutes and then cooled on ice for 2 minutes followed by analysis on an ABI PRIZM 3730 genetic analyzer. Using the AMI Genemapper analysis software, the product peak of cap dependent endonuclease was quantitated. After the positive and negative control fluorescence strengths were fixed at 0% and 100% respectively, the inhibitory percentage (%) was determined for the test compounds using curve fitting software (XLfit 2.0 Model 205 IDBS Inc.) for determination of the IC50 levels. The IC50 values are shown for test compounds which are parent compounds is shown in Figures 22 to 34.
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Years of experience: 16. Registered at ProZ.com: Jan 2013.
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I have extensive experience in chemistry, medicinal chemistry, pharmacology. I have experience in translating patents in these fields into English. I have written nearly 100 patents and scientific manuscripts in English. Thus, I have a good understanding of the accepted formats of patents and scientific manuscripts in English.
Keywords: translations of patents and scientific manuscripts for chemistry, biochemistry, and pharmacology
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