Chronic hepatitis B virus infection cannot be cured by current therapies, so new treatments are urgently needed. We recently identified novel inhibitors of the hepatitis B virus ribonuclease H that suppress viral replication in cell culture. Here, we employed immunodeficient FRG KO mice whose livers had been engrafted with primary human hepatocytes to ask whether ribonuclease H inhibitors can suppress hepatitis B virus replication in vivo. Humanized FRG KO mice infected with hepatitis B virus were treated for two weeks with the ribonuclease H inhibitors #110, an α-hydroxytropolone, and #208, an N-hydroxypyridinedione. Hepatitis B virus viral titers and S and e antigen plasma levels were measured. Treatment with #110 and #208 caused significant reductions in plasma viremia without affecting hepatitis B virus S or e antigen levels, and viral titers rebounded following treatment cessation. This is the expected pattern for inhibitors of viral DNA synthesis. Compound #208 suppressed viral titers of both hepatitis B virus genotype A and C isolates. These data indicate that Hepatitis B virus replication can be suppressed during infection in an animal by inhibiting the viral ribonuclease H, validating the ribonuclease H as a novel target for antiviral drug development.
Declaration of interest J Tavis holds awarded and pending patents associated with the subject matter and materials reported in this manuscript. Some of the intellectual property underlying the work reported in this article has been licensed to Casterbridge Pharmaceuticals, Inc, MO, USA. J Tavis is a stockholder and scientific advisor to Casterbridge Pharmaceuticals. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Hepatitis B virus (HBV) chronically infects >250 million people. It replicates by a unique protein‐primed reverse transcription mechanism, and the primary anti‐HBV drugs are nucleos(t)ide analogs targeting the viral polymerase (P). P has four domains compared to only two in most reverse transcriptases: the terminal protein (TP) that primes DNA synthesis, a spacer, the reverse transcriptase (RT), and the ribonuclease H (RNase H). Despite being a major drug target and catalyzing a reverse transcription pathway very different from the retroviruses, HBV P has resisted structural analysis for decades. Here, we exploited computational advances to model P. The TP wrapped around the RT domain rather than forming the anticipated globular domain, with the priming tyrosine poised over the RT active site. The orientation of the RT and RNase H domains resembled that of the retroviral enzymes despite the lack of sequences analogous to the retroviral linker region. The model was validated by mapping residues with known surface exposures, docking nucleic acids, mechanistically interpreting mutations with strong phenotypes, and docking inhibitors into the RT and RNase H active sites. The HBV P fold, including the orientation of the TP domain, was conserved among hepadnaviruses infecting rodent to fish hosts and a nackednavirus, but not in other non‐retroviral RTs. Therefore, this protein fold has persisted since the hepadnaviruses diverged from nackednaviruses >400 million years ago. This model will advance mechanistic analyses into the poorly understood enzymology of HBV reverse transcription and will enable drug development against non‐active site targets for the first time.
The α-hydroxytropolones (αHT) are troponoid inhibitors of hepatitis B virus (HBV) replication that can target the HBV ribonuclease H (RNase H) with sub-micromolar efficacies. αHTs and related troponoids (tropones and tropolones) can be cytotoxic in cell lines as measured by MTS assays that assesses mitochondrial function. Earlier studies suggest that tropolones induce cytotoxicity through inhibition of mitochondrial respiration. Therefore, we screened 35 diverse troponoids for effects on mitochondrial function, mitochondrial:nuclear genome ratio, cytotoxicity, and reactive oxygen species (ROS) production. Troponoids as a class did not inhibit respiration or glycolysis, although the α-ketotropolone subclass did interfere with these processes. The troponoids had no impact on the mitochondrial DNA to nuclear DNA ratio after three days of compound exposure. Patterns of troponoid-induced cytotoxicity among three hepatic cell lines were similar for all compounds, but three potent HBV RNase H inhibitors were not cytotoxic in primary human hepatocytes. Tropolones and αHTs increased ROS production in cells at cytotoxic concentrations but had no effect at lower concentrations that efficiently inhibit HBV replication. Troponoid-mediated cytotoxicity was significantly decreased upon addition of the ROS scavenger N-acetylcysteine. These studies show that troponoids can increase ROS production at high concentrations within cell lines leading to cytotoxicity, but are not be cytotoxic in primary hepatocytes. Future development of αHTs as potential therapeutics against HBV may need to mitigate ROS production by altering compound design and/or by co-administration with ROS antagonists to ameliorate increased ROS levels.
Hepatitis B virus (HBV) replicates by protein-primed reverse transcription. It chronically infects >250 million people, and the dominant anti-HBV drugs are nucleos(t)ide analogs targeting the viral polymerase (P). P has four domains, the terminal protein (TP) that primes DNA synthesis, a spacer, the reverse transcriptase (RT), and the ribonuclease H (RNaseH). Despite being a major drug target and catalyzing a reverse transcription pathway very different from the retroviral pathway, HBV P has resisted structural analysis for decades. Here, we exploited advances in protein structure prediction to model the structure of P. The predicted HBV RT and RNaseH domains aligned to the HIV RT-RNaseH at 3.75 A RMSD, had a nucleic acid binding groove spanning the two active sites, had DNA polymerase active site motifs in their expected positions, and accommodated two Mg++ ions in both active sites. Surprisingly, the TP domain wrapped around the RT domain, with the priming tyrosine poised over the RT active site. This model was validated using published mutational analyses, and by docking RT and RNaseH inhibitors, RNA:DNA heteroduplexes, and the HBV e RNA stem-loop that templates DNA priming into the model. The HBV P fold, including the orientation of the TP domain, was conserved among hepadnaviruses from rodents to fish and in P from a fish nackednavirus, but not in other non-retroviral RTs. Therefore, this protein fold has persisted since the hepadnaviruses diverged from nackednaviruses >400 million years ago. This model will guide drug development and mechanistic studies into P function.
Human immunodeficiency virus (HIV) and Hepatitis B virus (HBV) ribonucleases H (RNase H) are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. RNases H cleave RNA in RNA/DNA hybrids. Eukaryotic RNase H1 is an essential protein and probable off‐target enzyme for viral RNase H inhibitors. α‐hydroxytropolones (αHTs) comprise an anti‐RNase H inhibitor class that can inhibit the HIV, HBV, and human RNases H1. These compounds work by binding the RNase H active site by chelating the catalytic divalent metal cofactors. We hypothesized that a better understanding of RNase H1 inhibition will help development of compounds selective for the viral RNases H. To this end, we expressed and purified recombinant human RNase H1 and determined its inhibition mechanism(s) in steady‐state kinetics by two αHTs, 110 and 404 (Fig. 1). Inhibition was not competitive with a 12‐mer RNA/DNA substrate, but the turnover rate was reduced despite inhibitor binding to the active site (Fig. 2). 110 and 404displayed inhibition constants of 9 μM and 3 μM in saturating substrate concentrations, respectively, and these values were elevated 2‐3‐fold in very low substrate. Saturating 110and 404concentrations modestly reduced the apparent substrate binding constant (KM) from 90 nM to ~30 nM, while reducing the turnover rate (kcat = 0.17 s‐1) ~20‐fold. We found that 110enhanced affinity of RNase H1 for substrate by 4‐fold using a fluorescence polarization (FP) substrate binding assay with Ca2+ instead of Mg2+ to prevent RNA cleavage. 404, on the other hand, competed with substrate in binding assays, raising the substrate's KD~7‐fold from 24 nM without compound to ~150 nM. Induced fit docking studies in the Schrödinger suite suggest 110 binds to the active site metals as expected, while the substrate is still capable of binding via RNase H1’s high‐affinity auxiliary RNA/DNA hybrid binding domain (HBD) and substrate binding groove within the RNase H domain. 110 made favorable contacts with both enzyme and substrate, stabilizing an ESI complex. 404, on the other hand, occupies much of the substrate binding groove as well as the active site due to its larger structure. This would explain why 404 competes with substrate binding, while 110enhances substrate binding. The reason the KM decreased with 404 despite its competitive behavior in substrate binding assays is not clear. However, we hypothesize that 404locally competes with the substrate for the RNase H1 active site and the substrate binding groove within the RNase H domain without interfering with the HBD:substrate interface. This could lower the overall ES affinity. However, we speculate that if substrate release is slow relative to RNA hydrolysis and product release, the compound could behave uncompetitively in kinetics assays by inhibiting the breakdown of the ES complex through catalysis, only permitting enzyme‐substrate dissociation via the putatively slow substrate release pathway. Thus, these results illustrate that non‐competitive steady‐st...
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