Structural Modifications Introduced by NS2B Cofactor Binding to the NS3 Protease of the Kyasanur Forest Disease Virus

Abstract

Kyasanur Forest Disease virus (KFDV), a neglected human pathogenic virus, is a Flavivirus that causes severe hemorrhagic fever in humans. KFDV is transmitted to humans by the bite of the hard tick (Haemaphysalis spinigera), which acts as a reservoir of KFDV. The recent expansion of the endemic area of KFDV is of concern and requires the development of new preventive measures against KFDV. Currently, there is no antiviral therapy against KFDV, and the existing vaccine has limited efficacy. To develop a new antiviral therapy against KFDV, we focused on the nonstructural proteins NS2B and NS3 of KFDV, which are responsible for serine protease activity. Viral proteases have shown to be suitable therapeutic targets in the development of antiviral drugs against many diseases. However, success has been limited in flaviviruses, mainly because of the important features of the active site, which is flat and highly charged. In this context, the present study focuses on the dynamics of NS2B and NS3 to identify potential allosteric sites in the NS2B/NS3 protease of KDFV. To our knowledge, there are no reports on the dynamics of NS2B and NS3 in KFDV, and the crystal structure of the NS2B/NS3 protease of KFDV has not yet been solved. Overall, we created the structure of the NS2B/NS3 protease of KFDV using AlphaFold and performed molecular dynamics simulations with and without NS2B cofactor to investigate structural rearrangements due to cofactor binding and to identify alternative allosteric sites. The identified allosteric site is promising due to its geometric and physicochemical properties and druggability and can be used for new drug development. The applicability of the proposed allosteric binding sites was verified for the best-hit molecules from the virtual screening and MD simulations.

Keywords: AlphaFold; KFDV; MD; NS2B; NS3; PCA; allosteric binding site; clustering; conformational change.

Figure 1

MD trajectory analysis. Backbone RMSD with conformer population along trajectory for KFDV NS3 (A) and NS2B/NS3 (B) proteins. Radius of gyration (C). Root mean square fluctuations per residue ((D), upper panel). Difference of RMSF values for KFDV NS3 protein for naked and NS2B-complexed NS3 protein ((D), lower panel).

Figure 2

Superposition of the structures of the representative conformations (A—blue; B—red; initial structure—yellow) of the NS2B/NS3 complex extracted from molecular dynamics simulation. (A) NS2B/NS3 complex, (B) NS3.

Figure 3

PCA analysis of trajectories from MD simulations. NS3 (left) and NS2B/NS3 (right). Two-dimensional subspace spawned by the first two principal components PC1 and PC2 (top, conformation A—blue; B—red). Visualization of molecular motion associated to the first (middle) and the second (bottom) PCs. The degree of motion is depicted by the length of the arrow.

Figure 4

Representative conformations with substrate binding site (or catalytic triad) highlighted in yellow. The representative conformations (A—blue; B—red) of the NS2B/NS3 complex extracted from molecular dynamics simulation (A). The NS2B cofactor is highlighted in light pink and predicted BP1 allosteric site highlighted in orange red (depicted in surface view) (B).

Figure 5

Analysis of the MD trajectories of conformations A of the NS2B/NS3 protease with selected potential allosteric inhibitors. RMSD (A) and RMSF (B).

Figure 6

Comparison of structures from the initial (blue) and the last conformations (red) from 100 ns molecular dynamics simulations run for the NS2B/NS3 protease with potential allosteric inhibitor (DB06997), with encircled areas (yellow and black circles) with the greatest structural rearrangements.