Review
The hepatitis B virus ribonuclease H as a drug target
John E. Tavis, , Elena Lomonosova
Department of Molecular Microbiology and Immunology, Saint Louis University Liver Center, United States
Received 18 February 2015, Revised 30 March 2015, Accepted 2 April 2015, Available online 8 April 2015
Highlights
• The HBV ribonuclease H is a promising but unexploited drug target.
• Recent technical advances permit low-throughput screening for ribonuclease H inhibitors.
• Some of the known HIV ribonuclease H inhibitors also inhibit HBV.
• Combining ribonuclease H inhibitors with existing or novel drugs may significantly improve treatment efficacy for HBV.
Abstract
Chronic hepatitis B virus (HBV) infection is a leading cause of hepatitis, liver failure, and hepatocellular carcinoma. An outstanding vaccine is available; however, the number of infections remains high. Current anti-HBV treatments with interferon α and nucleos(t)ide analogs clear the infection in only a small minority of patients, and either induce serious side-effects or are of very long duration. HBV is a small, enveloped DNA virus that replicates by reverse transcription via an RNA intermediate. The HBV ribonuclease H (RNaseH) is essential for viral replication, but it has not been exploited as a drug target. Recent low-throughput screening of compound classes with anti-Human Immunodeficiency Virus RNaseH activity led to identification of HBV RNaseH inhibitors in three different chemical families that block HBV replication. These inhibitors are promising candidates for development into new anti-HBV drugs. The RNaseH inhibitors may help improve treatment efficacy enough to clear the virus from the liver when used in combination with existing anti-HBV drugs and/or with other novel inhibitors under development. This article forms part of a symposium in Antiviral Research on “An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B.”
Keywords
Hepatitis B virus; Ribonuclease H; Reverse transcription; Inhibitors
Over 190 compounds in the αHT, N-hydroxyisoquinolinedione, napthyridinone, dioxobutanoic acid, hydroxychromenone, Elvitegravir, Raltegravir, aminocyanothiophene, hydroxyxanthenone, cyanopyran, and thienopyrimidinone chemical families have been screened against the HBV RNaseH. Nineteen compounds inhibited the HBV RNaseH at ⩽20 μM, with the best hits having low micromolar IC50 values [(Cai et al., 2014, Hu et al., 2013, Lu et al., 2015 and Tavis et al., 2013) and unpublished]. Strong hits were observed among the αHTs, N-hydroxyisoquinolinediones, napthyridinones, and hydroxychomenones. In all cases, the compounds had similar activities against HBV genotypes C, D, and/or H. The inhibitors appear to act by chelating the divalent cations in the RNaseH active site because removing one of the three Lewis basic moieties or altering their angles relative to each other ablates inhibition [(Cai et al., 2014 and Lu et al., 2015) and unpublished]. Counter-screening against human RNaseH1 revealed that most of these screening hits were also active against the human enzyme, but differences in the sensitivity of the two enzymes were apparent (Lu et al., 2015). This indicates that increasing selectivity for the HBV enzyme must be a major focus during chemical optimization.
Thirty-seven compounds have been tested for ability to suppress HBV replication in culture; 10 inhibited HBV replication at ⩽20 μM (Table 1). Preferential suppression of the viral plus-polarity DNA strand confirmed that inhibition was due to blocking the viral RNaseH activity for all compounds except #56. Moderate cytotoxicity was observed for all active compounds, with CC50 values ranging from 6.1 to 79 μM. Therefore, it will be essential to minimize cytotoxicity during development of RNaseH inhibitors. The EC50 values for most of these inhibitors was substantially lower than their IC50 values. The cause for this quantitative discrepancy is unknown, but it could be due to accumulation of the compounds in cells, metabolism of the inhibitors to more active forms, or most likely, that the recombinant RNaseH does not recapitulate all aspects of the enzyme in its natural context as part of the full-length HBV polymerase protein. Nevertheless, strong inhibition of the recombinant enzyme is an excellent predictor of a compound’s potential to inhibit HBV replication in cells.
7. Outlook
The HBV RNaseH is a promising but unexploited target for antiviral drug development. Anti-RNaseH drugs are envisioned to be used in combination with the existing nucleos(t)ide analogs to suppress HBV replication much further than is currently possible. The goal will be to suppress viral replication enough to block replenishment of the cccDNA in the liver, permitting turnover of the cccDNA and elimination of infected cells by the immune system to clear the infection. The long apparent halflife of the cccDNA implies that therapy will be prolonged and may well benefit from co-administration with novel immune-stimulating therapies that are under development (Bertoletti and Gehring, 2013 and Gehring et al., 2014). Therefore, minimizing toxicity and optimizing specificity and pharmacokinetic properties of potential RNaseH drugs will be especially important.
Acknowledgements
Writing of this review was supported in part by R01 AI104494 to J.T. and an ancillary study within the NIDDK-sponsored Hepatitis B Virus Research Network (U01 DK082871 to Adrian Di Bisceglie and J.T.). We thank our collaborators Drs. Stuart Le Grice, Ryan Murelli, Fabrice Bailly, Philippe Cotelle, and Marvin Meyers for their many contributions to this RNaseH project. Use of some of these compounds against HBV is covered by U.S. Patent Application 13/072201 (Pending).作者: StephenW 时间: 2015-7-29 15:54