Development of a high-throughput screening assay for inhibitors of small ubiquitin-like modifier proteases.
Development of a high-throughput screening assay for inhibitors of small ubiquitin-like modifier proteases.
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DOI：
10.1177/1087057113479971
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发表时间：
2013-06
影响因子：
--
通讯作者：
Paschen W
中科院分区：
文献类型：
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作者：
Yang W;Wang L;Paschen W
小泛素样修饰物（SUMO1-3）是连接到靶蛋白赖氨酸残基的一小群蛋白质。SUMO偶联是一个高度动态的过程，因为SUMO蛋白酶会迅速解除SUMO蛋白的偶联。SUMO偶联/去偶联在主要细胞通路中起关键作用，并与许多病理条件有关。因此，开发新的策略来筛选特异性干扰SUMO偶联/去偶联的化合物具有重要的临床意义。在这里，我们描述了一种新的高通量筛选相容试验，以确定SUMO蛋白酶的抑制剂。该试验基于alphasgreen技术，使用his标记的SUMO2与strep标记的SUMO3结合作为SUMO蛋白酶底物。细菌SUMOylation系统被用来产生这种底物。采用三步纯化策略，得到高质量的底物。我们的数据表明，这种独特的底物可以很容易地在alphasgreen测定中以剂量依赖的方式检测到。基于alphasgreen信号监测有或无抑制剂的SUMO蛋白酶的裂解反应。此外，该分析适用于384孔格式，并在8个384孔板中评估板间和日间变异性。平均Z′因子为0.83±0.04，可用于高通量筛选。
Small ubiquitin-like modifier (SUMO1-3) is a small group of proteins that are ligated to lysine residues in target proteins. SUMO conjugation is a highly dynamic process, as SUMOylated proteins are rapidly deconjugated by SUMO proteases. SUMO conjugation/deconjugation plays pivotal roles in major cellular pathways, and is associated with a number of pathological conditions. It is therefore of significant clinical interest to develop new strategies to screen for compounds to specifically interfere with SUMO conjugation/deconjugation. Here, we describe a novel high throughput screening-compatible assay to identify inhibitors of SUMO proteases. The assay is based on AlphaScreen technology and uses His-tagged SUMO2 conjugated to Strep-tagged SUMO3 as a SUMO protease substrate. A bacterial SUMOylation system was used to generate this substrate. A three-step purification strategy was employed to yield substrate of high quality. Our data indicated that this unique substrate can be readily detected in the AlphaScreen assays in a dose-dependent manner. Cleavage reactions by SUMO protease with or without inhibitor were monitored based on AlphaScreen signals. Furthermore, the assay was adapted to a 384-well format, and the interplate and interday variability was evaluated in eight 384-well plates. The average Z’ factor was 0.83±0.04, confirming the suitability for high throughput screening applications.