Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota - Twin Cities, Minneapolis, MN 55455, USA.
Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA; Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio, TX 78229, USA.
J Mol Biol. 2023 Dec 1;435(23):168316. doi: 10.1016/j.jmb.2023.168316. Epub 2023 Oct 17.
Deubiquitinases (DUBs) are proteolytic enzymes that catalyze the removal of ubiquitin from protein substrates. The critical role of DUBs in regulating protein ubiquitination makes them attractive drug targets in oncology, neurodegenerative disease, and antiviral development. Biochemical assays for quantifying DUB activity have enabled characterization of substrate preferences and discovery of small molecule inhibitors. However, assessing the efficacy of these inhibitors in cellular contexts to support clinical drug development has been limited by a lack of tractable cell-based assays. To address this gap, we developed a two-color flow cytometry-based assay that allows for sensitive quantification of DUB activity and inhibition in living cells. The utility of this system was demonstrated by quantifying the potency of GRL0617 against the viral DUB SARS-CoV-2 PL, identifying potential GRL0617 resistance mutations, and performing structure-function analysis of the vOTU domain from the recently emerged Yezo virus. In addition, the system was optimized for cellular DUBs by modifying a GFP-targeting nanobody to recruit USP7 and USP28 to benchmark a panel of reported inhibitors and assess inhibition kinetics. Together, these results demonstrate the utility of these assays for studying DUB biology in a cellular context with potential to aid in inhibitor discovery and development.
去泛素化酶（DUBs）是一种蛋白水解酶，可催化从蛋白底物上移除泛素。DUBs 在调节蛋白泛素化方面的关键作用使它们成为肿瘤学、神经退行性疾病和抗病毒药物开发中的有吸引力的药物靶点。用于定量 DUB 活性的生化测定法使得能够对底物偏好进行特征描述并发现小分子抑制剂。然而，由于缺乏可行的基于细胞的测定法，评估这些抑制剂在细胞环境中的功效以支持临床药物开发受到了限制。为了解决这一差距，我们开发了一种双色流式细胞术测定法，可在活细胞中灵敏地定量 DUB 活性和抑制作用。通过定量 GRL0617 对病毒 DUB SARS-CoV-2 PL 的效力、鉴定潜在的 GRL0617 耐药突变体以及对最近出现的 Yezo 病毒的 vOTU 结构域进行结构功能分析，证明了该系统的实用性。此外，通过修饰靶向 GFP 的纳米抗体来招募 USP7 和 USP28，该系统针对细胞 DUB 进行了优化，以对一组报道的抑制剂进行基准测试，并评估抑制动力学。总之，这些结果表明，这些测定法可用于在细胞环境中研究 DUB 生物学，具有辅助抑制剂发现和开发的潜力。
