An RNAi based high-throughput screening assay to identify small molecule inhibitors of Hepatitis B Virus replication

  1. Raj K. Bhatnagar1*
  1. 1 International Centre for Genetic Engineering and Biotechnology, India;
  2. 2 Institute of Liver and Biliary Sciences, India;
  3. 3 National Institute of Plant Genome Research, India;
  4. 4 North Eastern Region-Biotechnology Programme Management Cell; Indian Agriculture Research Institute, India
  1. ↵* Corresponding author; email: raj{at}icgeb.res.in

  1. Author contributions: RKB, SKM and PM designed and conceived the study. SG performed the experiments. AK and DG performed and analysed the bioinformatics data for molecular modelling and docking experiment. SK and PD performed the SPR experiment. AV provided assistance for HBsAg and HBeAg measurement. AKS and SKS provided valuable suggestions for the antigen kinetics experiments. JKT provided SPR instrumentation facility with experimental assistance. SG, PM, SKM and RKB analysed the data and prepared the manuscript. All authors read and approved the final version of the paper.

Abstract

Persistent or chronic infection with the hepatitis B virus 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 HBxs 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.

  • Received January 3, 2017.
  • Accepted June 5, 2017.

This Article

  1. First Published on June 5, 2017 doi: 10.1074/jbc.M117.775155 jbc.M117.775155.
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