Quantitative cell-based protein degradation assays to identify and classify drugs that target the ubiquitin-proteasome system.
Quantitative cell-based protein degradation assays to identify and classify drugs that target the ubiquitin-proteasome system.
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DOI：
10.1074/jbc.m110.215319
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发表时间：
2011-05-13
期刊：
影响因子：
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通讯作者：
Deshaies RJ
中科院分区：
文献类型：
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作者：
Chou TF;Deshaies RJ
我们已经产生了一组双报告人细胞系，并设计了一个追逐协议，以量化蛋白酶体降解的泛素融合降解（UFD）底物，泛素连接酶CRL 2 VHL底物，和泛素非依赖性底物。靶向泛素-蛋白酶体系统的不同方面的充分表征的抑制剂可以通过其独特的底物稳定模式来区分，从而能够将测试化合物指定为蛋白酶体、泛素链形成或感知、CRL活性或UFD-p97途径的抑制剂。我们证实，降解的UFD，而不是CRL 2 VHL或泛素非依赖性底物依赖于p97的活性。我们优化了我们的一套检测方法，以建立适合高通量筛选的条件，然后通过筛选160种细胞渗透性蛋白激酶抑制剂来验证其性能。该筛选将Syk抑制剂III鉴定为不可逆的p97/vasolin蛋白抑制剂（IC 50 = 1.7 μm），其通过D2 ATP酶结构域内的Cys-522起作用。我们的工作建立了一个高通量筛选兼容的管道，用于识别和分类小分子，cDNA或siRNA，靶向泛素-蛋白酶体系统的组成部分。
We have generated a set of dual-reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of a ubiquitin fusion degradation (UFD) substrate, a ubiquitin ligase CRL2VHL substrate, and a ubiquitin-independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin-proteasome system can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of the proteasome, ubiquitin chain formation or perception, CRL activity, or the UFD-p97 pathway. We confirmed that degradation of the UFD but not the CRL2VHL or ubiquitin-independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk inhibitor III as an irreversible p97/vasolin containing protein inhibitor (IC50 = 1.7 μm) that acts through Cys-522 within the D2 ATPase domain. Our work establishes a high-throughput screening-compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the ubiquitin-proteasome system.