文章基本信息

标题：Establishment and Application of a High Throughput Model for Rho Kinase Inhibitors Screening Based on Fluorescence Polarization
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作者：WeiGang Duan ; LiXin Sun ; Jun Liu 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2006
卷号：29
期号：6
页码：1138-1142
DOI：10.1248/bpb.29.1138
出版社：The Pharmaceutical Society of Japan
摘要：Rho kinase (ROCK) inhibitors are effective candidates for neural or cardiovascular disorders. High throughput model for screening ROCK inhibitors is a basic foundation to pick up ROCK inhibitors from thousands of compounds for drug developing. The high throughput model was established based on purified recombinant rat ROCK catalytic domain (rROCK-CD) from Escherichia coli ( E. coli ). There are two steps of reaction in the model: incubation of 5.0 μl recombinant rROCK-CD (2.0 μg/ml), 5.0 μl different compounds, 5.0 μl fluorescent S6-peptide (200 n M ), and 5.0 μl ATP (10 μ M ) at 37 °C for 60 min was made the first reaction, and the second reaction was made by incubating them with additional 60 μl binding reagent at ambient temperature for 30 min. The phosphorylated S6 peptide can bind to a binding reagent, and the fluorescence varies from low polarization to high according to the amount of the phosphorylated peptide. IC₅₀ was calculated based on polarization variation. Compound, which IC₅₀ was less than 10 μ M , was recognized as a lead compound which taken bioactivity evaluation in PC12 by observing neurite outgrowth. The Z ′-factor of the model is 0.81 (above 0.5). The model screened five lead compounds from 3294, which promoted neurite outgrowth to different extent. The results suggested that the model is suitable for high throughput screening (HTS), and the five lead compounds are worth of further investigation.
关键词：Rho kinase inhibitor;high throughput screening;fluorescence polarization