Identifying Allosteric Small-Molecule Binding Sites of Inactive NS2B-NS3 Proteases of Pathogenic Flaviviridae

Abstract

Dengue, West Nile, Zika, Yellow fever, and Japanese encephalitis viruses persist as significant global health threats. The development of new therapeutic strategies based on inhibiting essential viral enzymes or viral-host protein interactions is problematic due to the fast mutation rate and rapid emergence of drug resistance. This study focuses on the NS2B-NS3 protease as a promising target for antiviral drug development. Promising allosteric binding sites were identified in two conformationally distinct inactive states and characterized for five flaviviruses and four Dengue virus subtypes. Their shapes, druggability, inter-viral similarity, sequence variation, and susceptibility to drug-resistant mutations have been studied. Two identified allosteric inactive state pockets appear to be feasible alternatives to a larger closed pocket near the active site, and they can be targeted with specific drug-like small-molecule inhibitors. Virus-specific sequence and structure implications and the feasibility of multi-viral inhibitors are discussed.

Keywords: Dengue; Japanese encephalitis; NS2B; NS3; Yellow Fever; Zika virus; allosteric druggable pockets; mutation rates; protease inhibitors.

Figure 1

Domain organization of the polyprotein precursor encoded by different flaviviruses. Red arrows indicate the cleavage by the NS2B-NS3 protease, and blue arrows indicate the cleavage by the host cell proteases. A green box shows the two domains of the protease.

Figure 2

The mobile structural determinants of the three states in existing crystallographic structures of NS2B-NS3 protease domains are shown by blue and orange backbone ribbons. The closed state (a) is exemplified by PDB ID 5YOF, the transient state (b) by 2FOM, and the fully opened (c) by 7M1V. The backbone ribbon is colored as follows: blue ribbon—mobile NS2B, gray ribbon—static NS3 core, orange ribbon—mobile NS3-pro-C-terminal hairpin. The catalytic triad is shown and labeled for reference.

Figure 3

Structural variations within each of the closed (a), transient (b), and fully opened (c) states of the flaviviral NS2B-NS3-pro domains for all five flaviviruses and four Dengue subtypes under study. Some differences in residue numbers (YFV S136 instead of S135 for other viruses) are due to sequence length variations in both NS2B and NS3-pro subunits (a). The NS2B loop in one of the closed-state DENV-2 constructs was conformationally shifted due to an inserted linker in a crystallized construct (see a green backbone fragment in (a)).

Figure 4

Allosteric pockets (AP1, AP2, and AP3) on flavivirus NS2B-NS3-pro proteins (gray surfaces) highlight conserved and variable residues. AP1 (a): Residues across DENV2 (black), ZIKV (green), and JEV (magenta) include highly variable sites like K74, E86, and L85. AP2 (b): Conserved residues in ZIKV (green) include L85, with surrounding residues contributing to structural integrity. AP3 (c): Variability across WNV (purple), ZIKV (green), YFV (dark yellow), and DENV3 (dark cyan) is shown, with key residues like W89, V166, and Q167. Bubble sizes reflect residue contribution to the pocket surface, with colors denoting specific viruses.

Figure 5

Sequence conservation (%) analysis of (a) active site pocket (closed state), (b) allosteric pocket 1 (transient), and (c) allosteric pocket 2 (fully opened state) within various flaviviruses. The letter “P” denotes the amino acid position within the pocket. The asterisk indicates the flaviviruses used to define the amino acids in the three pockets (closed—5IDK (WNV), transient—6MO2 (DENV-2), and fully opened—7M1V (ZIKV)).

Figure 6

Amino acid sequence conservation analysis of (a) active site pocket (closed state), (b) allosteric pocket 1 (transient), and (c) allosteric pocket 2 (fully opened state) among various flaviviruses. The letter “P” denotes the amino acid position within the pocket. The asterisk indicates the flaviviruses used to define the amino acids in the three pockets (closed—5IDK (WNV), transient—6MO2 (DENV-2), and fully opened—7M1V (ZIKV)). To simplify the analysis, we focused on these specific flaviviruses as representatives for the determination of amino acid residues within the pockets. Amino acids identical among all flaviviruses are outlined in black rectangles. Each flavivirus has its own local numbering for NS2B and NS3.