Mechanistic Insights into Zika Virus NS3 Helicase Inhibition by Epigallocatechin-3-Gallate

Abstract

Since 2007, repeated outbreaks of Zika virus (ZIKV) have affected millions of people worldwide and created a global health concern with major complications like microcephaly and Guillain Barre's syndrome. To date, there is not a single Zika-specific licensed drug present in the market. However, in recent months, several antiviral molecules have been screened against ZIKV. Among those, (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, has shown great virucidal potential against flaviviruses including ZIKV. The mechanistic understanding of EGCG-targeting viral proteins is not yet entirely deciphered except that little is known about its interaction with viral envelope protein and viral protease. We designed our current study to find inhibitory actions of EGCG against ZIKV NS3 helicase. NS3 helicase performs a significant role in viral replication by unwinding RNA after hydrolyzing NTP. We employed molecular docking and simulation approach and found significant interactions at the ATPase site and also at the RNA binding site. Further, the enzymatic assay has shown significant inhibition of NTPase activity with an IC₅₀ value of 295.7 nM and Ki of 0.387 ± 0.034 μM. Our study suggests the possibility that EGCG could be considered as a prime backbone molecule for further broad-spectrum inhibitor development against ZIKV and other flaviviruses.

Figure 1

Extra precision (XP) docking of EGCG at the ATPase site of ZIKV NS3 helicase. (A) ZIKV NS3 helicase with PDB ID: 5GJC shows the ATP molecule bound at the NTPase site (red dotted circle) between domain 1 and domain 2. (B) After molecular docking, EGCG exhibits molecular interactions (3D view) by H-bonds (yellow dotted lines), π–cation interactions (green dotted lines), and salt bridges (pink dotted lines). (C) 2D interaction diagram illustrating EGCG binding interactions where interactions are represented as H-bonds (pink arrow), π–cation interactions (solid red line), and salt bridges (blue-red straight line). (D) NS3 helicase is represented as the solid grey surface where docked EGCG (green color) superimposes with the ATP molecule (red color) in the ATPase pocket.

Figure 2

EGCG molecular docking interactions at RNA binding cavity of ZIKV NS3 helicase. (A) NS3 helicase of ZIKV with PDB ID: 5GJB displays RNA (red color) bound at the interface between domain 1, domain 2, and domain 3 (red dotted square). (B) 3D view of EGCG showing molecular interactions at the RNA binding cavity by H-bonds (yellow dotted lines), π–π interactions (Cyan dotted lines), and π–cation interactions (green dotted lines). (C) 2D interaction diagram of EGCG showing significant interaction displayed as H-bonds (pink arrow), π–cation interactions (red solid line), and π–π stacking (green solid lines). (D) Solid grey surface represented by NS3 helicase and docked EGCG (green color) is superimposed with RNA (red color) bound at the helicase site.

Figure 3

Molecular dynamics simulation of the EGCG complex with the ATPase site of ZIKV helicase. (A) rmsd graph of the apo protein helicase and the helicase complex with EGCG at the NTPase site for the time period of 100 ns simulation. (B) Comparison of RMSF graph of Cα of the Apo protein helicase and helicase complex with EGCG at the NTPase site for the time period of 100 ns simulation. (C) Comparison plot of radius of gyration of apo-NS3 helicase and EGCG complex with helicase (D) histogram displaying different types of interaction fractions between EGCG and ATPase site of helicase during the simulation period.

Figure 4

Molecular dynamics simulation of the EGCG complex with the RNA binding site of ZIKV helicase. (A) rmsd graph of the apo protein helicase and helicase complex with EGCG at the RNA binding site for the time period of 100 ns simulation. (B) Comparison of RMSF graph of Cα of the Apo protein helicase and helicase complex with EGCG at the RNA binding site for the time period of 100 ns simulation. (C) Comparison plot of radius of gyration of apo-NS3 helicase and EGCG complex with helicase. (D) Histogram displaying different types of interaction fractions between EGCG and RNA binding site of helicase during the simulation period. (E) Comparison plot of RMSF was made between, EGCG-ATP site (red color), EGCG-RNA site (blue color), and apo-helicase (black color).

Figure 5

EGCG inhibits ATPase activity of NS3 helicase. (A) Purification of NS3 helicase by Ni-NTA affinity chromatography. A 10% SDS gel was run and stained with Coomassie dye where final his-tagged NS3 helicase fractions were pooled and concentrated by an Amicon (10 kDa) centrifugal filter. In this figure, M-protein ladder (Bio-Rad Precision Plus) and L1 contains purified concentrated his-tagged NS3 helicase protein of ZIKV. (B) Kinetic parameters (substrate–velocity curve) calculated for 80 nM NS3 helicase after varying the substrate (ATP) concentration ranging from 50 to 2500 μM. (C) IC₅₀ calculated for EGCG against the NTPase site by incubating 80 nM helicase with varying concentration of EGCG (serially diluted: 1200 nM: 23.40 nM). (D) Inhibition constant (K_i) was calculated for 80 nM helicase at different concentrations of ATP (100, 150, 250, 400, 600, and 1000 μM), and EGCG concentrations were kept at 0.5 and 0.25 μM.