A High-Throughput Screening Method for Identification of Inhibitors of the Deubiquitinating Enzyme USP14.
A High-Throughput Screening Method for Identification of Inhibitors of the Deubiquitinating Enzyme USP14.
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DOI：
10.1002/9780470559277.ch120078
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发表时间：
2012-12-01
影响因子：
--
通讯作者：
King, Randall W
中科院分区：
文献类型：
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作者：
Lee, Byung-Hoon;Finley, Daniel;King, Randall W
去泛素化酶（DUBs）逆转了泛素化的过程，在人类中有近100种。原则上，DUB代表有前途的药物靶点，因为几种酶与人类疾病有关。DUBs的异肽酶活性可以通过用药物样化合物靶向催化位点来选择性地抑制。值得注意的是，哺乳动物26 S蛋白酶体与三种主要DUB相关：RPN 11，UCH 37和USP 14。由于USP 14的泛素“链修剪”活性可抑制蛋白酶体功能，因此USP 14的抑制剂可刺激蛋白酶体降解。我们最近建立了一种高通量筛选（HTS）方法来鉴定USP 14特异性的小分子抑制剂。本文中的方案涵盖了制备检测试剂、对USP 14抑制剂进行HTS以及进行HTS后分析的必要程序。Curr.方案4：311-330 © 2012，John Wiley & Sons，Inc.
Deubiquitinating enzymes (DUBs) reverse the process of ubiquitination, and number nearly 100 in humans. In principle, DUBs represent promising drug targets, as several of the enzymes have been implicated in human diseases. The isopeptidase activity of DUBs can be selectively inhibited by targeting the catalytic site with drug-like compounds. Notably, the mammalian 26S proteasome is associated with three major DUBs: RPN11, UCH37, and USP14. Because the ubiquitin 'chain-trimming' activity of USP14 can inhibit proteasome function, inhibitors of USP14 can stimulate proteasomal degradation. We recently established a high-throughput screening (HTS) method to identify small-molecule inhibitors specific for USP14. The protocols in this article cover the necessary procedures for preparing assay reagents, performing HTS for USP14 inhibitors, and carrying out post-HTS analysis. Curr. Protoc. Chem. Biol. 4:311-330 © 2012 by John Wiley & Sons, Inc.