Cell Lysate-Based AlphaLISA Deubiquitinase Assay Platform for Identification of Small Molecule Inhibitors.
Cell Lysate-Based AlphaLISA Deubiquitinase Assay Platform for Identification of Small Molecule Inhibitors.
复制标题
DOI：
10.1021/acschembio.7b00543
复制
发表时间：
2017-09-15
影响因子：
4
通讯作者：
Zhuang Z
中科院分区：
文献类型：
--
作者：
Ott CA;Baljinnyam B;Zakharov AV;Jadhav A;Simeonov A;Zhuang Z
去泛素酶（dub）与多种人类疾病有关，包括神经系统疾病、癌症和病毒感染，使其成为药物干预的绝佳候选者。针对DUBs的药物发现活动需要适合高通量筛选（HTS）的酶去泛素化测定。虽然近年来已经开发了几种DUB底物和测定方法，但它们在很大程度上仅限于重组纯化的DUB。许多dub是大的多结构域蛋白，难以重组获得足够数量的HTS。因此，一种不需要重组蛋白生成，同时又能概括出生理相关环境的检测方法是非常可取的。这种检测将为针对许多治疗相关但目前无法获得的dub的药物发现打开大门。在这里，我们报告了一种基于AlphaLISA技术的细胞裂解物DUB检测，用于高通量筛选。该检测平台使用生物素标记的泛素探针和ha标记的在人细胞中表达的DUB。该分析经过验证并适应1536孔格式，这使得使用15,000个化合物库筛选UCHL1作为原理证明。我们期望新的平台可以很容易地适应于其他dub，以允许识别更有效和选择性的小分子抑制剂和化学探针。
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 multi-domain 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 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 fifteen thousand 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.
登录
查看更多内容
影响因子：
7.3
作者：
Jadhav A;Ferreira RS;Klumpp C;Mott BT;Austin CP;Inglese J;Thomas CJ;Maloney DJ;Shoichet BK;Simeonov A
通讯作者：
Simeonov A
影响因子：
14.8
作者：
Liang, Qin;Dexheimer, Thomas S.;Zhang, Ping;Rosenthal, Andrew S.;Villamil, Mark A.;You, Changjun;Zhang, Qiuting;Chen, Junjun;Ott, Christine A.;Sun, Hongmao;Luci, Diane K.;Yuan, Bifeng;Simeonov, Anton;Jadhav, Ajit;Xiao, Hui;Wang, Yinsheng;Maloney, David J.;Zhuang, Zhihao
通讯作者：
Zhuang, Zhihao
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
5.2
作者：
Haj-Yahya, Najat;Hemantha, Hosahalli P.;Brik, Ashraf
通讯作者：
Brik, Ashraf
影响因子：
--
作者：
Edgington, Laura E.;van Raam, Bram J.;Bogyo, Matthew
通讯作者：
Bogyo, Matthew