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.

Fig. 1.

Virtual ligand screening of the NCI compound library against WNV NS3pro. The complete NCI database (∼275,000 compounds) was docked into the NS2B cofactor-binding cavity in the NS3pro domain. The initial screening led to identification of the top 80 hits, which were then re-docked under more stringent conditions and ranked according to their relative binding energy E. The top 50 predicted binders were ordered from NCI and their inhibitory potency was tested in the in vitro cleavage assays. A significant fraction of the tested ligands were true-positives (active, gray dots), whereas other ligands were false-positives (inactive, white dots). WNV, West Nile virus.

Fig. 2.

Predicted binding models of the WNV NS2B-NS3pro inhibitors. (A) Ligand 1; (B) ligand 2; (C) ligand 3. The NS3pro is rendered as a molecular surface, colored by its electrostatic potential. NS2B is transparent green. The approximate position of the active site is highlighted by a yellow oval. Hydrogen bonds are shown as red dotted lines. Refer to Table 1 for the ligand structures. The figure was generated using PyMol Molecular Graphics software.

Fig. 3.

Selected inhibitors efficiently inhibit the catalytic activity of WNV NS2B-NS3pro. Before the addition of the pyroglutamic acid Pyr-Arg-Thr-Lys-Arg-7-amino-4-methylcoumarin substrate (25 μM), the purified WNV proteinase (10 nM) was co-incubated for 30 min with increasing concentrations of the inhibitors. The residual activity was then monitored continuously at λ_ex = 360 nm and λ_em = 465 nm to determine the initial velocity of the reactions. The initial velocity was calculated as a percentage of residual activity versus the untreated proteinase (control). Refer to Table 1 for the ligand structures.