Orcutt Steven J, Wu Jian, Eddins Michael J, Leach Craig A, Strickler James E
LifeSensors, Inc., Malvern, PA 19355, USA.
Biochim Biophys Acta. 2012 Nov;1823(11):2079-86. doi: 10.1016/j.bbamcr.2012.06.004. Epub 2012 Jun 15.
As the importance of ubiquitylation in certain disease states becomes increasingly apparent, the enzymes responsible for removal of ubiquitin (Ub) from target proteins, deubiquitylases (DUBs), are becoming attractive targets for drug discovery. For rapid identification of compounds that alter DUB function, in vitro assays must be able to provide statistically robust data over a wide dynamic range of both substrate and enzyme concentrations during high throughput screening (HTS). The most established reagents for HTS are Ubs with a quenched fluorophore conjugated to the C-terminus; however, a luciferase-based strategy for detecting DUB activity (DUB-Glo™, Promega) provides a wider dynamic range than traditional fluorogenic reagents. Unfortunately, this assay requires high enzyme concentrations and lacks specificity for DUBs over other isopeptidases (e.g. desumoylases), as it is based on an aminoluciferin (AML) derivative of a peptide derived from the C-terminus of Ub (Z-RLRGG-). Conjugation of aminoluciferin to a full-length Ub (Ub-AML) yields a substrate that has a wide dynamic range, yet displays detection limits for DUBs 100- to 1000-fold lower than observed with DUB-Glo™. Ub-AML was even a sensitive substrate for DUBs (e.g. JosD1 and USP14) that do not show appreciable activity with DUB-Glo™. Aminoluciferin derivatives of hSUMO2 and NEDD8 were also shown to be sensitive substrates for desumoylases and deneddylases, respectively. Ub/Ubl-AML substrates are amenable to HTS (Z'=0.67) yielding robust signal, and providing an alternative drug discovery platform for Ub/Ubl isopeptidases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
随着泛素化在某些疾病状态中的重要性日益明显，负责从靶蛋白上去除泛素（Ub）的酶，即去泛素化酶（DUBs），正成为药物研发的有吸引力的靶点。为了快速鉴定改变DUB功能的化合物，体外测定必须能够在高通量筛选（HTS）期间，在底物和酶浓度的广泛动态范围内提供具有统计学稳健性的数据。用于HTS的最成熟的试剂是在C末端缀合有淬灭荧光团的Ub；然而，基于荧光素酶的检测DUB活性的策略（DUB-Glo™，Promega）比传统的荧光试剂提供更宽的动态范围。不幸的是，该测定需要高酶浓度，并且对DUBs的特异性低于其他异肽酶（例如去SUMO化酶），因为它基于源自Ub C末端（Z-RLRGG-）的肽的氨基荧光素（AML）衍生物。将氨基荧光素与全长Ub（Ub-AML）缀合产生具有宽动态范围的底物，但对DUBs的检测限比对DUB-Glo™观察到的低100至1000倍。Ub-AML甚至是对DUB-Glo™没有明显活性的DUBs（例如JosD1和USP14）的敏感底物。hSUMO2和NEDD8的氨基荧光素衍生物也分别被证明是去SUMO化酶和去NEDD化酶的敏感底物。Ub/Ubl-AML底物适用于HTS（Z'=0.67），产生稳健的信号，并为Ub/Ubl异肽酶提供替代的药物研发平台。本文是名为：泛素药物研发与诊断的特刊的一部分。
