Abstract
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.
Highlights
Persistent or chronic infection with the hepatitis B virus (HBV) represents one of the most common viral diseases in humans
Hepatitis B X protein acts as a multifunctional regulator to induce hepatitis B virus X protein (HBx)-responsive transcription factors like NF-B and NF-AT [17, 18], interacts with transcriptional activator CREB/ ATF [19, 20], modulates intracellular calcium signaling, and stimulates signal transduction pathways resulting in initiation of tyrosine kinases following downstream activation of mitogen-activated protein kinases [21,22,23]
Because HBx has been described as being important for HBV replication and functions as an RNAi suppressor protein, we aimed at identifying small molecules that bind HBx and block its activity
Summary
Role of HBx by targeting it using either short hairpin RNAs (shRNAs) [27, 28] or siRNA for cessation of HBV replication [29]. Molecular mechanisms of HBx for in vivo replication in woodchuck hepatitis virus as well as in human HBV have garnered interest, highlighting the crucial function of this protein during viral replication [30, 31]. We customized an assay by measuring the quantitative expression of the reporter as a readout for high-throughput screening of pharmacologically active compounds from the Maybridge library for their ability to bind HBx and inhibit its RNAi suppressor activity. Our results provide evidence for antiviral function of host RNAi machinery in HBV replication and identify a pharmacophore that binds and blocks HBx activity
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