Fragment-based design of potent non-nucleoside inhibitors targeting the thumb domain of HCV NS5B

Hepatitis C virus (HCV) is a major global health problem because of the high reported infection rate and the absence of a reliable vaccine to prevent it. HCV NS5B (non-structural protein 5B) is an essential RNA-dependent RNA polymerase with no functional equivalent in mammalian cells, making it an attractive target for selective inhibition. This study aims to design potent non-nucleoside inhibitors that bind to the thumb domain allosteric site 2 of NS5B polymerase. To achieve this, fragment replacement methods were combined with ligand-based virtual screening techniques. New potential inhibitors were generated by employing fragment-based drug design and subsequently filtered through quantitative structure-activity relationship (QSAR) predictive models in order to predict their biological activities. Then, to select inhibitors that were both drug-like and synthetically feasible, compounds were screened based on Lipinski’s rule of five and synthetic accessibility. Promising candidates were thereafter screened for their binding affinity by molecular docking at the NS5B allosteric site. The best candidates were further validated with molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations to analyze the structural stability, interaction strength, and dynamic behavior of the target protein during the process. This study led to the identification of three lead compounds exhibiting high binding affinity, favorable structural rigidity, and promising pharmacokinetic profiles. These findings provide a solid foundation for future in vitro validation and further drug development efforts targeting HCV.