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

PLoS Pathogens

Laura D. Kramer¹  Jing Zhang²  Matthew Brecher³  Adham Alifarag³  Binbin Liu³  Zhong Li³  Qishan Lin³  Hongmin Li³  Cheri A. Koetzner³  Susan A. Jones³ 

[1] Center for Functional Genomics, University at Albany, Rensselaer, New York, United States of America;Department of food science, College of food science and technology, Guangdong Ocean University, Zhanjiang, Guangdong, People’s Republic of China;Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany NY, United States of America

关键词: Proteases;    Luciferase;    Library screening;    Zika virus;    West Nile virus;    Dengue virus;    Flaviviruses;    Luciferase assay;   

DOI  :  10.1371/journal.ppat.1006411

学科分类：生物科学（综合）

来源: Public Library of Science

【 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 μM, 11.4 μM, and 4.8 μM, 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 μM 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.

【 授权许可】

CC BY