USP28 and USP25 are downregulated by Vismodegib in vitro and in colorectal cancer cell lines
USP28 and USP25 are downregulated by Vismodegib in vitro and in colorectal cancer cell lines
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USP28 和 USP25 在体外和结直肠癌细胞系中被 Vismodegib 下调
DOI：
10.1111/febs.15461
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发表时间：
2020-07-20
期刊：
影响因子：
5.4
通讯作者：
Zhang, Naixia
中科院分区：
文献类型：
--
作者：
Wang, Hui;Meng, Qian;Zhang, Naixia
去泛素化酶USP 28通过增强多种癌症相关蛋白（包括c-Myc、Notch 1和LSD 1）的稳定性在肿瘤发生中起关键作用，并且已成为抗癌药物开发的有吸引力的靶点。然而，到目前为止，只有少数的USP 28靶向活性化合物已被开发出来，并在USP 28中的活性化合物结合口袋尚未被实验揭示。在这项研究中，基于生物测定的高通量筛选应用于从市售药物库中发现USP 28靶向抑制剂。Vismodegib是一种Hedgehog信号通路抑制剂，FDA批准用于治疗基底细胞癌，发现其对USP 28具有抑制活性（IC 50：4.41 +/- 1.08 μ m）。然后使用多种生物物理和生物化学技术，包括NMR、ITC、热位移测定、HDX-MS和定点诱变分析来表征维莫德吉和USP 28之间的相互作用。揭示了USP 28中维莫德吉的结合口袋，其主要由跨越D255-N278和N286-Y293的两个螺旋结构组成。根据HDX-MS数据定义的分子对接产生的可能结合位姿，USP 28中维莫德吉占据的结合腔与USP 7中其抑制剂的一个标记结合口袋很好地对齐。此外，进行细胞测定以证实Vismodegib可以与进化相关的去泛素化酶USP 28和USP 25相互作用，并下调两种酶的底物蛋白c-Myc、Notch 1和端锚聚合酶-1/2的水平。
Deubiquitinase USP28 plays a crucial role in tumorigenesis by enhancing the stabilities of multiple cancer-related proteins including c-Myc, Notch1, and LSD1, and has become an attractive target for anticancer drug development. However, to date, only a few of USP28-targeted active compounds have been developed, and the active compound-binding pocket in USP28 has not been experimentally revealed yet. In this study, bioassay-based high-throughput screening was applied to discover USP28-targeted inhibitors from the commercially available drug library. Vismodegib, an inhibitor of Hedgehog signaling pathway and FDA-approved drug for the treatment of basal cell carcinoma, was found to exhibit inhibition activity against USP28 (IC50: 4.41 +/- 1.08 mu m). Multiple biophysical and biochemical techniques including NMR, ITC, thermal shift assay, HDX-MS, and site-directed mutagenesis analysis were then used to characterize the interaction between Vismodegib and USP28. The binding pocket in USP28 for Vismodegib, which is mainly composed of two helical structures spanning D255-N278 and N286-Y293, was revealed. According to the possible binding pose generated by HDX-MS data-defined molecular docking, the binding cavity occupied by Vismodegib in USP28 aligns well with one of the reported-binding pockets in USP7 for its inhibitors. Furthermore, cellular assays were conducted to confirm that Vismodegib could interact with the evolutionarily related deubiquitinases USP28 and USP25 and downregulate the levels of the two enzymes' substrate proteins c-Myc, Notch1, and Tankyrase-1/2.