Discovery of Small-Molecule Inhibitors Against Norovirus 3CLpro Using Structure-Based Virtual Screening and FlipGFP Assay.

Norovirus, a major cause of acute gastroenteritis, possesses a single-stranded positive-sense RNA genome. The viral 3C-like cysteine protease (3CLpro) plays a critical role in processing the viral polyprotein into mature non-structural proteins, a step essential for viral replication. Targeting 3CLpro has emerged as a promising strategy for developing small-molecule inhibitors against Norovirus. In this study, we employed a combination of virtual screening and the FlipGFP assay to identify potential inhibitors targeting the 3CLpro of Norovirus genotype GII.4. A library of approximately 58,800 compounds was screened using AutoDock Vina tool, yielding 20 candidate compounds based on their Max Affinity scores. These compounds were subsequently evaluated using a cell-based FlipGFP assay. Among them, eight compounds demonstrated significant inhibitory activity against 3CLpro, with Gedatolisib showing the most potent effect (IC50 = 0.06 ± 0.01 μM). Molecular docking and molecular dynamics simulations were conducted to explore the binding mechanisms and structural stability of the inhibitor-3CLpro complexes. Our findings provide valuable insights into the development of antiviral drugs targeting Norovirus 3CLpro, offering potential therapeutic strategies to combat Norovirus infections.