Hepatitis B virus genetic diversity has minimal impact on sensitivity of the viral ribonuclease H to inhibitors

Abstract

Hepatitis B virus (HBV) causes hepatitis, cirrhosis, liver failure, and liver cancer, but the current therapies that employ either nucelos(t)ide analogs or (pegylated)interferon α do not clear the infection in the large majority of patients. Inhibitors of the HBV ribonuclease H (RNaseH) that are being developed with the goal of producing anti-HBV drugs are promising candidates for use in combination with the nucleos(t)ide analogs to improve therapeutic efficacy. HBV is genetically very diverse, with at least 8 genotypes that differ by ≥8% at the sequence level. This diversity is reflected in the viral RNaseH enzyme, raising the possibility that divergent HBV genotypes or isolates may have varying sensitivity to RNaseH inhibitors. To evaluate this possibility, we expressed and purified 18 patient-derived RNaseHs from genotypes B, C, and D. Basal RNaseH activity and sensitivity to three novel RNaseH inhibitors from three different chemotypes were assessed. We also evaluated four consensus HBV RNaseHs to determine if such sequences would be suitable for use in antiviral drug screening. The patient-derived enzymes varied by over 10-fold in their basal RNaseH activities, but they were equivalently sensitive to each of the three inhibitors. Similarly, all four consensus HBV RNaseH enzymes were active and were equally sensitive to an RNaseH inhibitor. These data indicate that a wide range of RNaseH sequences would be suitable for use in antiviral drug screening, and that genotype- or isolate-specific genetic variations are unlikely to present a barrier during antiviral drug development against the HBV RNaseH.

Efficacy of hepatitis B virus ribonuclease H inhibitors, a new class of replication antagonists, in FRG human liver chimeric mice

“…There were no differences in pretreatment human albumin levels among the four groups by a one-way ANOVA with the Dunnets’ post-hoc test. Genotype C was employed in this study rather than genotype A which was used in the preliminary study because we had preliminary data indicating that RNaseH inhibitors from these chemical classes could inhibit HBV from multiple genotypes (Lu et al, 2016). Using genotype A in the preliminary experiment and genotype C in the primary efficacy study allowed us to directly test that hypothesis in vivo .…”

“…Recently we identified HBV RNaseH inhibitors in three chemical families that block HBV replication in cell culture (Cai et al, 2014; Edwards et al, 2017; Lomonosova et al, 2017a; Lu et al, 2015; Tavis et al, 2013a; Tavis and Lomonosova, 2015). We found that these inhibitors are equally effective against RNaseH enzymes from multiple isolates of HBV genotypes B, C, and D, implying that HBV’s high genetic diversity is unlikely to be a barrier to drug development (Lu et al, 2016). We also found that combinations of two RNaseH inhibitors from different chemical classes (α-hydroxytropolones (αHTs) and N-hydroxyisoquinolinediones (HIDs)) with the NA lamivudine or with each other synergistically inhibited HBV replication in cell culture (Lomonosova et al, 2017b).…”

Synergistic Interactions between Hepatitis B Virus RNase H Antagonists and Other Inhibitors

“…HBV is a genetically diverse virus, with 8 to 10 genotypes that differ by Ն8% at the nucleotide level (51). Our studies were done with a genotype D isolate, but it is probable that our conclusions will apply to other genotypes as well for two reasons.…”

Shedding light on RNaseH: a promising target for hepatitis B virus (HBV)

“…These compounds inhibit the HBV RNaseH in biochemical assays and cause accumulation of RNA:DNA heteroduplexes in viral capsids, confirming they target the RNaseH [6,9]. The RNaseH inhibitors repress HBV RNaseHs from three genotypes, indicating that HBV's high genetic diversity is unlikely to complicate drug development [13]. The inhibitors are synergistic with the nucleoside analog drug Lamivudine and additive with the experimental HBV capsid protein assembly modifier Hap12 without enhancing cytotoxicity [10,14].…”