Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes.
Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes.
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通过基于荧光活性探针的高通量筛选，通过高通量筛选来鉴定未表征化酶的选择性抑制剂。
DOI：
10.1038/nbt.1531
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发表时间：
2009-04
影响因子：
46.9
通讯作者：
Cravatt, Benjamin F.
中科院分区：
文献类型：
--
作者：
Bachovchin, Daniel A.;Brown, Steven J.;Rosen, Hugh;Cravatt, Benjamin F.
基于靶点的高通量筛选(HTS)是发现蛋白质小分子调节剂的关键。典型的酶筛选方法依赖于广泛定制的底物分析，这不适用于特征不佳的生化活性的目标。在这里，我们报告了一个通用的，无底物的HTS平台，它通过使用荧光偏振来监测广谱的、基于活性的探针与酶的反应来克服这个问题。我们表明，该平台适用于来自多个机械类别的酶，无论它们的功能注释程度如何，并可以与基于二级竞争活性的蛋白质组分析相结合，以快速确定筛选HITS的特异性。利用这一平台，我们确定了生物活性生物碱埃米丁是一种选择性抑制未鉴定的癌症相关水解酶RBBP9。我们进一步证明了解毒酶GSTO1，也与癌症有关，被公共图书馆中发现的几种亲电化合物抑制，其中一些化合物对这种酶表现出高选择性。
Target-based high-throughput screening (HTS) is essential for the discovery of small-molecule modulators of proteins. Typical screening methods for enzymes rely on extensively tailored substrate assays, which are not available for targets of poorly characterized biochemical activity. Here, we report a general, substrate-free platform for HTS that overcomes this problem by monitoring the reaction of broad-spectrum, activity-based probes with enzymes using fluorescence polarization. We show that this platform is applicable to enzymes from multiple mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary competitive activity-based proteomic assays to rapidly determine the specificity of screening hits. Using this platform, we identified the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. We furthermore show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this enzyme.
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影响因子：
64.5
作者：
GUPTA, RS;SIMINOVITCH, L
通讯作者：
SIMINOVITCH, L
影响因子：
2.9
作者：
Board, Philip G.;Coggan, Marjorie;Anders, M. W.
通讯作者：
Anders, M. W.
影响因子：
56.9
作者：
Greenbaum, DC;Baruch, A;Bogyo, M
通讯作者：
Bogyo, M
影响因子：
2.7
作者：
Hoover, Heather S.;Blankman, Jacqueline L.;Cravatt, Benjamin F.
通讯作者：
Cravatt, Benjamin F.
影响因子：
--
作者：
Blankman, Jacqueline L.;Simon, Gabriel M.;Cravatt, Benjamin F.
通讯作者：
Cravatt, Benjamin F.