Ott Christine A, Baljinnyam Bolormaa, Zakharov Alexey V, Jadhav Ajit, Simeonov Anton, Zhuang Zhihao
Department of Chemistry and Biochemistry, University of Delaware , 214A Drake Hall, Newark, Delaware 19716, United States.
National Center for Advancing Translational Sciences, NIH , Bethesda, Maryland 20892, United States.
ACS Chem Biol. 2017 Sep 15;12(9):2399-2407. doi: 10.1021/acschembio.7b00543. Epub 2017 Aug 24.
The deubiquitinases, or DUBs, are associated with various human diseases, including neurological disorders, cancer, and viral infection, making them excellent candidates for pharmacological intervention. Drug discovery campaigns against DUBs require enzymatic deubiquitination assays amenable for high-throughput screening (HTS). Although several DUB substrates and assays have been developed in recent years, they are largely limited to recombinantly purified DUBs. Many DUBs are large multidomain proteins that are difficult to obtain recombinantly in sufficient quantities for HTS. Therefore, an assay that obviates the need of recombinant protein generation and also recapitulates a physiologically relevant environment is highly desirable. Such an assay will open doors for drug discovery against many therapeutically relevant, but currently inaccessible, DUBs. Here, we report a cell lysate DUB assay based on AlphaLISA technology for high throughput screening. This assay platform uses a biotin-tagged ubiquitin probe and a HA-tagged DUB expressed in human cells. The assay was validated and adapted to a 1536-well format, which enabled a screening against UCHL1 as proof of principle using a library of 15 000 compounds. We expect that the new platform can be readily adapted to other DUBs to allow the identification of more potent and selective small molecule inhibitors and chemical probes.
去泛素化酶（DUBs）与多种人类疾病相关，包括神经紊乱、癌症和病毒感染，这使得它们成为药物干预的理想靶点。针对DUBs的药物研发需要适用于高通量筛选（HTS）的酶促去泛素化检测方法。尽管近年来已经开发了几种DUB底物和检测方法，但它们大多局限于重组纯化的DUBs。许多DUBs是大型多结构域蛋白，难以大量重组获得用于HTS。因此，迫切需要一种无需生成重组蛋白且能重现生理相关环境的检测方法。这样的检测方法将为针对许多具有治疗相关性但目前难以攻克的DUBs的药物研发打开大门。在此，我们报告一种基于AlphaLISA技术的用于高通量筛选的细胞裂解液DUB检测方法。该检测平台使用生物素标记的泛素探针和在人细胞中表达的HA标记的DUB。该检测方法经过验证并适用于1536孔板形式，以此作为原理验证，使用一个包含15000种化合物的文库对UCHL1进行筛选。我们期望这个新平台能够很容易地应用于其他DUBs，以鉴定出更有效和更具选择性的小分子抑制剂及化学探针。
