An RNAi-based high-throughput screening assay to identify small molecule inhibitors of hepatitis B virus replication.

Persistent or chronic infection with the hepatitis B virus (HBV) represents one of the most common viral diseases in humans. The hepatitis B virus deploys the hepatitis B virus X protein (HBx) as a suppressor of host defenses consisting of RNAi-based silencing of viral genes. Because of its critical role in countering host defenses, HBx represents an attractive target for antiviral drugs. Here, we developed and optimized a loss-of-function screening procedure, which identified a potential pharmacophore that abrogated HBx RNAi suppression activity. In a survey of 14,400 compounds in the Maybridge Screening Collection, we prioritized candidate compounds via high-throughput screening based on reversal of green fluorescent protein (GFP)-reported, RNAi-mediated silencing in a HepG2/GFP-shRNA RNAi sensor line. The screening yielded a pharmacologically active compound, N-(2,4-difluorophenyl)-N'-[3-(1H-imidazol-1-yl) propyl] thiourea (IR415), which blocked HBx-mediated RNAi suppression indicated by the GFP reporter assay. We also found that IR415 reversed the inhibitory effect of HBx protein on activity of the Dicer endoribonuclease. We further confirmed the results of the primary screen in IR415-treated, HBV-infected HepG2 cells, which exhibited a marked depletion of HBV core protein synthesis and down-regulation of pre-genomic HBV RNA. Using a molecular interaction analysis system, we confirmed that IR415 selectively targets HBx in a concentration-dependent manner. The screening assay presented here allows rapid and improved detection of small-molecule inhibitors of HBx and related viral proteins. The assay may therefore potentiate the development of next-generation RNAi pathway-based therapeutics and promises to accelerate our search for novel and effective drugs in antiviral research.