Ligand screening using enzymatic assays.
Ligand screening using enzymatic assays.
复制标题
使用酶测定进行配体筛选。
DOI：
10.1007/978-1-4939-0354-2_21
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Lee,Hyun
中科院分区：
文献类型：
--
作者：
Ratia,Kiira;Mehboob,Shahila;Lee,Hyun
酶测定的一个重要方面是配体对酶活性的影响是显而易见且可量化的。因此，基于酶的测定非常适合高通量配体筛选，它可以描述一组小分子对指定靶标的作用。为了使酶测定提供有用的筛选数据，在筛选之前对测定的动力学、测定成分、读出信号和整体稳定性进行优化并适应设备。对于屏幕本身，要仔细考虑重复次数、板布局、化合物浓度和测定组装的细节。最后，在筛选后阶段，使用各种策略处理和分析配体筛选数据，并对所得的初步命中进行一系列二级和三级测定，以消除假阳性和质量差的命中。使用来自丙型肝炎病毒的病毒蛋白酶 NS3/4A 作为酶靶点的示例，描述了筛选的各个阶段。
An important aspect of enzymatic assays is that the effect of a ligand on enzyme activity is readily apparent and quantifiable. Enzyme-based assays are, therefore, highly amenable to high-throughput ligand screening, which profiles the effect of a panel of small molecules on a designated target. In order for enzyme assays to provide useful screening data, the kinetics, assay components, readout signal, and overall stability of the assay are optimized and adapted to the equipment prior to the screen. For the screen itself, careful consideration is given to the number of replicates, the plate layout, the compound concentration, and the details of assay assembly. Lastly, in the post-screen stages, the ligand screening data is processed and analyzed using various strategies, and the resulting preliminary hits are subjected to a series of secondary and tertiary assays to eliminate false positives and poor quality hits. The various stages of screening are described, using a viral protease, NS3/4A from Hepatitis C virus, as an example of an enzyme target.