Eppley Institute for Cancer Research and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Anal Biochem. 2013 Sep 1;440(1):71-7. doi: 10.1016/j.ab.2013.05.017. Epub 2013 Jun 5.
Deubiquitinases (DUBs) play an important role in regulating the ubiquitin landscape of proteins. The DUB AMSH (associated molecule with the SH3 domain of STAM) has been shown to be involved in regulating the ubiquitin-dependent down-regulation of activated cell surface receptors via the endolysosomal degradative pathway. Therefore, small molecule AMSH inhibitors will be useful chemical probes to study the effect of AMSH DUB activity on cell surface receptor degradation. Currently, there are no known selective inhibitors of AMSH or high-throughput compatible assays for their identification. We report the development and optimization of a novel fluorescence resonance energy transfer (FRET)-based add-and-read AMSH DUB assay in a 384-well format. In this format, the optimal temperature for a high-throughput screen (HTS) was determined to be 30°C, the assay tolerates 5% dimethyl sulfoxide (DMSO), and it has a Z-score of 0.71, indicating HTS compatibility. The assay was used to show that AMSH selectively cleaves Lys63-linked diubiquitin over Lys48- and Lys11-linked diubiquitin. The IC50 value of the nonspecific small molecule DUB inhibitor N-ethylmaleimide was 16.2±3.2 μM and can be used as a qualitative positive control for the screen. We conclude that this assay is high-throughput compatible and can be used to identify novel small molecule inhibitors of AMSH.
去泛素化酶（DUBs）在调节蛋白质的泛素景观中发挥着重要作用。已经表明，DUB AMSH（与 STAM 的 SH3 结构域相关的分子）参与通过内体溶酶体降解途径调节激活的细胞表面受体的泛素依赖性下调。因此，小分子 AMSH 抑制剂将是研究 AMSH DUB 活性对细胞表面受体降解影响的有用化学探针。目前，尚无已知的 AMSH 选择性抑制剂或高通量兼容的鉴定方法。我们报告了在 384 孔格式中开发和优化一种新型荧光共振能量转移（FRET）基础的添加和读取 AMSH DUB 测定法。在这种格式中，确定了高通量筛选（HTS）的最佳温度为 30°C，该测定法可耐受 5％二甲基亚砜（DMSO），并且 Z 值为 0.71，表明 HTS 兼容性。该测定法用于表明 AMSH 选择性地切割 Lys63 连接的二泛素，而不是 Lys48 和 Lys11 连接的二泛素。非特异性小分子 DUB 抑制剂 N-乙基马来酰亚胺的 IC50 值为 16.2±3.2 μM，可以用作该筛选的定性阳性对照。我们得出结论，该测定法具有高通量兼容性，可用于鉴定新型 AMSH 小分子抑制剂。
