Evaluation of an orthogonal pooling strategy for rapid high-throughput screening of proteases.
Evaluation of an orthogonal pooling strategy for rapid high-throughput screening of proteases.
复制标题
用于快速高通量筛选蛋白酶的正交合并策略的评估。
DOI：
10.1089/adt.2007.110
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发表时间：
2008
影响因子：
1.8
通讯作者：
Napper,AndrewD
中科院分区：
文献类型：
--
作者：
Motlekar,Nuzhat;Diamond,ScottL;Napper,AndrewD
Orthogonal pooling was evaluated as a strategy for the rapid screening of multiple cysteine and serine proteases against large compound libraries. To validate the method the human cysteine protease cathepsin B was screened against a library of 64,000 individual compounds and also against the same library mixed 10 compounds per well. The orthogonal pooling method used resulted in each compound being present in two wells, mixed with a different set of nine other compounds in each location. Thus hits were identified based on activity in both locations, avoiding the need for retesting of each component of active mixtures. Hits were tested in dose–response both in the dithiothreitol (DTT)-containing buffer used in the primary HTS and in buffer containing cysteine in place of DTT to rule out artifacts due to oxidative inactivation of the enzyme. Comparison of the confirmed actives from single-compound and mixture screening showed that mixture screening identified all of the actives from single-compound HTS. Based on these results the orthogonal pooling strategy has been used successfully to rapidly screen several cysteine and serine proteases.
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