Computational discovery of DENV-3 RdRp allosteric inhibitors: biological evaluation and mechanistic studies.

The RNA-dependent RNA polymerase (RdRp) of Dengue virus (DENV) is a key antiviral target due to its essential and conserved role in viral replication. In this study, we have employed a similarity-guided virtual screening approach to the Maybridge compound library by combining molecular docking and molecular dynamics (MD) simulations with binding free energy calculations to identify and prioritize candidate allosteric site inhibitors for DENV-3 RdRp. A picogreen-based RdRp enzymatic assay was developed and validated for the biological evaluation of the prioritized compounds obtained from computational screening. GK00498 was identified as a moderately active molecule with an IC50 of 23.3 µM in the enzymatic assay. Further, experimental evaluation of GK00498-based molecules generated through the computational workflow reveals that GK00598, GK01138 and SPB05603 are potent inhibitors with IC50 values of 6.78, 7.59 and 7.74 µM, respectively. Mechanistic insights from structural and dynamical analyses of MD simulations suggested an allosteric mode of inhibition that orchestrated changes in RdRp functional motions and disrupted the conformational dynamics required for RNA synthesis. Overall, these findings provide a strong computational and experimental foundation for DENV-3 RdRp inhibitors that can be optimized and evaluated for anti-dengue activity in vitro and in vivo.