Identification of Novel Inhibitors of JEV RdRp as Potent Antiviral Drugs: Targeting NS5-NS3 Protein Interaction

Japanese Encephalitis Virus (JEV), a member of the Flavivirus family, relies on the RNA dependent RNA polymerase (RdRp) domain of non structural protein 5 (NS5) for de novo viral genome synthesis. The NS5 RdRp contains a conserved priming loop in its thumb domain that initiates de novo genome synthesis. The NS5 and NS3 proteins interact to initiate replication, and inhibiting this interaction directly disrupts viral replication.

In this study, researchers examined the priming loop function in the NS5-NS3 interaction and its effect on viral replication. Employing a structure-based drug design approach, they screened the Maybridge compound library against the RdRp priming loop, identifying 15 candidate compounds based on binding energy. Among these compounds, DSHS00151 demonstrated significant dose-dependent inhibition of the NS5-NS3 interaction.

It inhibited viral infectivity and reduced viral RNA load with IC50 values of 2.54 and 2.9 micromolar, respectively, surpassing CD10712. Molecular Dynamics (MD) simulations indicated that DSHS00151 had stronger and more stable binding to priming loop residues (790 to 812) compared to CD10712.

The docking of NS5 and NS3 proteins suggested that the priming loop tends to rotate towards the NS3 protein to stabilize the complex. However, binding of DSHS00151 restricted this loop rotation, compromising the stability of the NS5-NS3 complex and affecting viral replication. The study validated the NS5 priming loop as an allosteric site, with compounds binding to this site belonging to the Non Nucleoside Reverse Transcriptase Inhibitors (NNRTI) class of inhibitors. These inhibitors disrupt the NS5-NS3 interaction and hold potential as novel anti-JEV therapeutics.