Application of micro arrayed compound screening (μARCS) to identify inhibitors of caspase-3
Application of micro arrayed compound screening (μARCS) to identify inhibitors of caspase-3
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DOI：
10.1089/108705702320351169
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发表时间：
2002-08-01
影响因子：
--
通讯作者：
Warrior, U
中科院分区：
文献类型：
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作者：
Gopalakrishnan, SM;Karvinen, J;Warrior, U
微阵列化合物筛选（muARCS）是雅培公司开发的一种小型化超高通量筛选平台。在这种格式中，8640个离散化合物被发现并干燥到聚苯乙烯片上，聚苯乙烯片具有与96孔板相同的足迹。使用荧光铕螯合物和达布西利猝灭剂标记的肽底物，采用均匀的时间分辨荧光分析格式（LANCE）来鉴定caspase-3的抑制剂。将caspase-3酶注入薄琼脂糖凝胶中，将其放置在含有测试化合物的薄片上。然后将含有半胱天冬酶底物的第二种凝胶置于酶凝胶之上以启动反应。Caspase-3切割底物并从猝灭剂中分离铕，产生时间分辨荧光信号，使用ViewLux电荷耦合器件成像系统检测。caspase-3的潜在抑制剂在明亮的荧光背景上显示为黑点。将muARCS检测格式的结果与常规96孔板筛选格式的结果进行比较。
Micro Arrayed Compound Screening (muARCS) is a miniaturized ultra-high-throughput screening platform developed at Abbott Laboratories. In this format, 8640 discrete compounds are spotted and dried onto a polystyrene sheet, which has the same footprint as a 96-well plate. A homogeneous time-resolved fluorescence assay format (LANCE) was applied to identify the inhibitors of caspase-3 using a peptide substrate labeled with a fluorescent europium chelate and a dabcyl quencher. The caspase-3 enzyme was cast into a thin agarose gel, which was placed on a sheet containing test compounds. A second gel containing caspase substrate was then laid above the enzyme gel to initiate the reaction. Caspase-3 cleaves the substrate and separates the europium from the quencher, giving rise to a time-resolved fluorescent signal, which was detected using a ViewLux charge-coupled device imaging system. Potential inhibitors of caspase-3 appeared as dark spots on a bright fluorescent background. Results from the muARCS assay format were compared to those from a conventional 96-well plate-screening format.