Virtual Ligand Screening of the National Cancer Institute (NCI) Compound Library Leads to the Allosteric Inhibitory Scaffolds of the West Nile Virus NS3 Proteinase

Abstract

Viruses of the genus Flavivirus are responsible for significant human disease and mortality. The N-terminal domain of the flaviviral nonstructural (NS)3 protein codes for the serine, chymotrypsin-fold proteinase (NS3pro). The presence of the nonstructural (NS)2B cofactor, which is encoded by the upstream gene in the flaviviral genome, is necessary for NS3pro to exhibit its proteolytic activity. The two-component NS2B-NS3pro functional activity is essential for the viral polyprotein processing and replication. Both the structure and the function of NS2B-NS3pro are conserved in the Flavivirus family. Because of its essential function in the posttranslational processing of the viral polyprotein precursor, NS2B-NS3pro is a promising target for anti-flavivirus drugs. To identify selective inhibitors with the reduced cross-reactivity and off-target effects, we focused our strategy on the allosteric inhibitors capable of targeting the NS2B-NS3pro interface rather than the NS3pro active site. Using virtual ligand screening of the diverse, ∼275,000-compound library and the catalytic domain of the two-component West Nile virus (WNV) NS2B-NS3pro as a receptor, we identified a limited subset of the novel inhibitory scaffolds. Several of the discovered compounds performed as allosteric inhibitors and exhibited a nanomolar range potency in the in vitro cleavage assays. The inhibitors were also potent in cell-based assays employing the sub-genomic, luciferase-tagged WNV and Dengue viral replicons. The selectivity of the inhibitors was confirmed using the in vitro cleavage assays with furin, a human serine proteinase, the substrate preferences of which are similar to those of WNV NS2B-NS3pro. Conceptually, the similar in silico drug discovery strategy may be readily employed for the identification of inhibitors of other flaviviruses.

Characterization of the Zika virus two-component NS2B-NS3 protease and structure-assisted identification of allosteric small-molecule antagonists

“…Therefore, we focused our search for NS2B-NS3pro inhibitors on compounds that bind to viral protease exosites (Johnston et al, 2007; Shiryaev et al, 2011; Shiryaev and Strongin, 2010; Sidique et al, 2009). We tested a focused sub-library of allosteric exosite-targeting inhibitors previously shown to inhibit the structurally homologous WNV NS2B-NS3pro.…”

“…NSC157058, NSC86314, and NSC716903 have been shown to bind to the WNV NS3pro domain exosite, which is distant from the active site cavity (Shiryaev et al, 2011). However, we considered that exosite binding might still interfere with the positioning of the NS2B cofactor relative to the NS3pro active site, thereby inactivating the protease activity.…”

“…These inhibitors appeared to interfere with the productive fold of the NS2B cofactor relative to the NS3pro domain (Shiryaev et al, 2011). In the present study, we found that several of the WNV inhibitors were potent inhibitors of ZIKV NS2B-NS3pro, suggesting that some of the WNV and DENV inhibitors could be re-purposed to develop ZIKV inhibitors.…”

Integrated Approach to Structure-Based Enzymatic Drug Design: Molecular Modeling, Spectroscopy, and Experimental Bioactivity

“…820,821 In 2009, 820 nonstructural 3 protease (NS3pro) inhibitors of the WNV were identified using automatic high-throughput fragment-based docking of about 12 000 compounds to the active site of the WNV protease. 15 N-HSQC spectra of the corresponding inhibitor−enzyme complexes were recorded and the K d value derived from the fitting curves.…”

Zika Virus Serene Protease Complex (Ns2b-Ns3) Inhibition by 2-Amino-5-{[(1z)-Amino({[(z)-Benzoyl]imino})methyl]amino}-N-(5-Amino-7-{[Carbamoyl(phenyl)methyl]amino}-6-Oxoheptyl)pentanamide, in Silico Studies

“…NS3 is a multi-domain protein with RNA triphosphatase (NS3 RTpase) and RNA helicase (NS3 hell) activities while NS5 consist of an N-terminal methyltransferase domain and RNA polymerase (NS5 RdRp) [16][17][18]. According to Potapova et al (2012) and Shiryaev et al (2011) [19,20], the pathogenic properties of encephalitis virus are due to NS3 polymerase. ZIKV protease consists of the N-terminal domain of NS3, which is a chymotrypsin-like serine protease carrying an absolutely conserved triad His51, Asp75 and Ser135, NS2B is membrane bound and serves as a cofactor essential for folding and catalysis [21,22].…”