A cell-based high-throughput screening method based on a ubiquitin-reference technique for identifying modulators of E3 ligases
A cell-based high-throughput screening method based on a ubiquitin-reference technique for identifying modulators of E3 ligases
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基于泛素参考技术的细胞高通量筛选方法，用于鉴定 E3 连接酶调节剂
DOI：
10.1074/jbc.ra118.003822
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发表时间：
2018-12
影响因子：
4.8
通讯作者：
Wang Hong Rui
中科院分区：
文献类型：
--
作者：
Tian Maoyuan;Zeng Taoling;Liu Mingdong;Han Shang;Lin Huayue;Lin Qi;Li Li;Jiang Tingting;Li Gao;Lin Hong;Zhang Ting;Kang Qiaofeng;Deng Xianming;Wang Hong Rui
越来越多的证据表明，E3泛素连接酶广泛参与多种人类疾病的发生发展。寻找这些E3泛素连接酶的小分子调节剂是一种很有前途的药物发现策略。在这里，我们报道了一种基于细胞的高通量筛选方法的发展，通过整合泛素参考技术(URT)，基于位于目的蛋白和参考蛋白部分之间的泛素融合蛋白，与双荧光素酶系统相结合来识别E3泛素连接酶的调节子。使用这种方法，我们筛选了SMAD泛素调节因子1(SMURF1)的小分子调节剂，SMURF1属于E3泛素连接酶NEDD4家族，由于其在肿瘤发生中的作用，是一个有吸引力的治疗靶点。在这个筛选中，我们以RAS同源家族成员B(RHOB)作为SMURF1底物，我们发现了一个有效的SMURF1抑制剂，并证实它也能抑制SMURF1依赖的SMAD家族成员1(Smad1)和RHOA的降解。体外自动泛素化实验表明，该化合物同时抑制SMURF1和SMURF2的活性，提示该化合物可能是SMURF1/2中Hect结构域催化活性的拮抗剂。此外，细胞功能分析表明，该化合物有效地抑制HEK293T细胞的突起活性，并阻断转化生长因子β(转化生长因子β)诱导的MDCK细胞上皮-间充质转化，与SMURF1缺失对上述过程的影响相似。综上所述，本文提出的筛选方法可能在鉴定E3泛素连接酶的调节子方面具有很大的实用潜力。
Accumulating evidence indicates that a wide range of E3 ubiquitin ligases are involved in the development of many human diseases. Searching for small-molecule modulators of these E3 ubiquitin ligases is emerging as a promising drug discovery strategy. Here, we report the development of a cell-based high-throughput screening method to identify modulators of E3 ubiquitin ligases by integrating the ubiquitin-reference technique (URT), based on a fusion protein of ubiquitin located between a protein of interest and a reference protein moiety, with a Dual-Luciferase system. Using this method, we screened for small-molecule modulators of SMAD ubiquitin regulatory factor 1 (SMURF1), which belongs to the NEDD4 family of E3 ubiquitin ligases and is an attractive therapeutic target because of its roles in tumorigenesis. Using RAS homolog family member B (RHOB) as a SMURF1 substrate in this screen, we identified a potent SMURF1 inhibitor and confirmed that it also blocks SMURF1-dependent degradation of SMAD family member 1 (SMAD1) and RHOA. An in vitro auto-ubiquitination assay indicated that this compound inhibits both SMURF1 and SMURF2 activities, indicating that it may be an antagonist of the catalytic activity of the HECT domain in SMURF1/2. Moreover, cell functional assays revealed that this compound effectively inhibits protrusive activity in HEK293T cells and blocks transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) in MDCK cells, similar to the effects on these processes caused by SMURF1 loss. In summary, the screening approach presented here may have great practical potential for identifying modulators of E3 ubiquitin ligases.
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蛋白酶体抑制剂在固体恶性肿瘤治疗中的定位。
影响因子：
3
作者：
Roeten MSF;Cloos J;Jansen G
通讯作者：
Jansen G
影响因子：
2.4
作者：
Adams, J;Kauffman, M
通讯作者：
Kauffman, M
影响因子：
3.3
作者：
Murakami, G;Watabe, T;Imamura, T
通讯作者：
Imamura, T
DOI：
10.1038/nrc.2017.105
发表时间：
2018-03
期刊：
Nature reviews. Cancer
影响因子：
--
作者：
Senft D;Qi J;Ronai ZA
通讯作者：
Ronai ZA
DOI：
10.1073/pnas.93.22.12142
发表时间：
1996-10-29
影响因子：
11.1
作者：
Varshavsky, A
通讯作者：
Varshavsky, A