文献类型：期刊文献

英文题名：A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

作者：Brecher, Matthew[1];Li, Zhong[1];Liu, Binbin[1,2];Zhang, Jing[1];Koetzner, Cheri A.[1];Alifarag, Adham[1];Jones, Susan A.[1];Lin, Qishan[3];Kramer, Laura D.[1,4];Li, Hongmin[1,4]

机构：[1]New York State Dept Hlth, Wadsworth Ctr, 120 New Scotland Ave, Albany, NY 12203 USA;[2]Guangdong Ocean Univ, Dept Food Sci, Coll Food Sci & Technol, Zhanjiang, Guangdong, Peoples R China;[3]SUNY Albany, Ctr Funct Genom, Rensselaer, NY USA;[4]SUNY Albany, Sch Publ Hlth, Dept Biomed Sci, Albany, NY USA

年份：2017

卷号：13

期号：5

外文期刊名：PLOS PATHOGENS

收录：SCI-EXPANDED(收录号：WOS:000402877700052)、、Scopus(收录号：2-s2.0-85020219820)、WOS

基金：This research was partially supported by the Wadsworth Center flavivirus drug discovery initiative fund. MB was partially supported by the NIH Biodefense and Emerging Infectious Disease training grant AI055429. AA was supported by the National Science Foundation REU Program Grant DBI1062963. BL was partially supported by a visiting scholarship sponsored by Guangdong Ocean University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

语种：英文

外文摘要：The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 mu M, 11.4 mu M, and 4.8 mu M, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 mu M on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.