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

Abstract

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.

The A–Z of Zika drug discovery

“…Brecher et al developed an assay to analyze the conformational changes in DENV NS2B-NS3 protease using luciferase [69]. They performed a VS assay using an allosteric site conformation that is present only in the enzyme's inactive state.…”

“…Only NSC135618 was able to inhibit the protease. The same compound also inhibited viral replication of DENV, ZIKV, WNV and YFV [69]. Crystal structure elucidations and MD studies have been undertaken to further understand the interactions and conformational changes of the protein bound to its inhibitors.…”

Trimethylsilyl tag for probing protein–ligand interactions by NMR

“…Compound 2, which is known as an active-site inhibitor of flaviviral proteases (de la Cruz et al 2011;Nitsche et al 2014;Brecher et al 2017), was chosen as a model ligand to assess the performance of the TMS tag 1 in a ligand titration experiment. Inhibition experiments revealed IC50 values of about 160 µM for the wild type and the tagged mutant V36C (Table 1).…”

Structure‐guided screening of chemical database to identify NS3‐NS2B inhibitors for effective therapeutic application in dengue infection

“…Hence, to overcome such situation presently, available more resources, much faster, and advance scientific techniques need to be adopted and explored. Earlier, a number of studies have proposed various traditional and specific methods for the identification of different chemical entities and demonstrated their selective inhibition mechanism and inhibitory efficacy against NS3‐NS2B protease complex at different levels …”