From the State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Fujian 361102, China.
From the State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Fujian 361102, China
J Biol Chem. 2019 Feb 22;294(8):2880-2891. doi: 10.1074/jbc.RA118.003822. Epub 2018 Dec 26.
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 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.
越来越多的证据表明，广泛的 E3 泛素连接酶参与了许多人类疾病的发展。寻找这些 E3 泛素连接酶的小分子调节剂正成为一种很有前途的药物发现策略。在这里，我们报告了一种基于泛素参考技术（URT）的细胞高通量筛选方法的开发，该方法基于泛素与感兴趣的蛋白质和参考蛋白质部分之间的融合蛋白，与双荧光素酶系统相结合，用于鉴定 E3 泛素连接酶的调节剂。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 NEDD4 家族的 E3 泛素连接酶，是一种有吸引力的治疗靶点，因为它在肿瘤发生中发挥作用。在这个筛选中，我们使用 RAS 同源家族成员 B（RHOB）作为 SMURF1 的底物，鉴定出一种有效的 SMURF1 抑制剂，并证实它还阻断了 SMURF1 依赖的 SMAD 家族成员 1（SMAD1）和 RHOA 的降解。自动泛素化测定表明，该化合物抑制 SMURF1 和 SMURF2 的活性，表明它可能是 SMURF1/2 中 HECT 结构域催化活性的拮抗剂。此外，细胞功能测定表明，该化合物有效抑制 HEK293T 细胞的突起活性，并阻断 MDCK 细胞中转化生长因子 β（TGFβ）诱导的上皮-间充质转化（EMT），类似于 SMURF1 缺失对这些过程的影响。总之，这里提出的筛选方法对于鉴定 E3 泛素连接酶的调节剂可能具有很大的实际潜力。
