Steuer Christian, Heinonen Karl H, Kattner Lars, Klein Christian D
Institut für Pharmazie und Molekulare Biotechnologie, Universität Heidelberg, Heidelberg, Germany.
J Biomol Screen. 2009 Oct;14(9):1102-8. doi: 10.1177/1087057109344115. Epub 2009 Sep 2.
The aim of this work was to perform a systematic study of the effect of nonionic detergents on the activity of the dengue virus NS2B-NS3 protease. To ensure a high activity of the protease, the assay procedures for the dengue virus and other flaviviral proteases published to date are performed in the presence of up to 35% glycerol, which does not represent the cellular physicochemical environment. In addition, the high viscosity of glycerol-containing solutions leads to various experimental problems in miniaturized assays. Using an internally quenched peptide substrate, the authors show that glycerol is not essential for enzymatic activity if certain nonionic detergents are added to the assay buffer. In addition, nonionic detergents may help to avoid false-positive screening results caused by "promiscuous" inhibitors. Other polyalcohols can substitute glycerol and have less effect on the viscosity of the assay buffer. The assay was used to screen a compound library and allowed the identification of small-molecular nonpeptidic inhibitors of dengue NS3 protease. Finally, the authors discuss the mode of action of nonionic detergents and the influence that they may have on the conformational properties of the NS2B-NS3 protease.
这项工作的目的是对非离子去污剂对登革病毒NS2B - NS3蛋白酶活性的影响进行系统研究。为确保蛋白酶具有高活性，迄今为止发表的登革病毒和其他黄病毒蛋白酶的检测程序是在高达35%甘油存在的情况下进行的，而这并不代表细胞的物理化学环境。此外，含甘油溶液的高粘度在小型化检测中会导致各种实验问题。作者使用一种内部淬灭的肽底物表明，如果在检测缓冲液中添加某些非离子去污剂，甘油对于酶活性并非必不可少。此外，非离子去污剂可能有助于避免由“混杂”抑制剂导致的假阳性筛选结果。其他多元醇可以替代甘油，并且对检测缓冲液的粘度影响较小。该检测方法用于筛选化合物库，并鉴定出了登革病毒NS3蛋白酶的小分子非肽类抑制剂。最后，作者讨论了非离子去污剂的作用模式以及它们可能对NS2B - NS3蛋白酶构象性质产生的影响。
