Selective depletion of HBV-infected hepatocytes by class A capsid assembly modulators requires high levels of intrahepatic HBV core protein.

Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.

Nucleotide Prodrug Containing a Nonproteinogenic Amino Acid To Improve Oral Delivery of a Hepatitis C Virus Treatment.

Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2'-C-methyl-1'-cyano-7-deaza-adenosine C-nucleotide analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection. However, the prodrug selected for clinical development, GS-6620, required a high dose for meaningful efficacy and had unacceptable variability due to poor oral absorption as a result of suboptimal solubility, intestinal metabolism, and efflux transport. While obtaining clinical proof of concept for the nucleotide analog, a more effective prodrug strategy would be necessary for clinical utility. Here, we report an alternative prodrug of the same nucleoside analog identified to address liabilities of GS-6620. A phosphoramidate prodrug containing the nonproteinogenic amino acid methylalanine, an isopropyl ester and phenol in the (S) conformation at phosphorous, GS2, was found to have improved solubility, intestinal stability, and hepatic activation. GS2 is a more selective substrate for hepatically expressed carboxyl esterase 1 (CES1) and is resistant to hydrolysis by more widely expressed hydrolases, including cathepsin A (CatA) and CES2. Unlike GS-6620, GS2 was not cleaved by intestinally expressed CES2 and, as a result, was stable in intestinal extracts. Levels of liver triphosphate following oral administration of GS2 in animals were higher than those of GS-6620, even when administered under optimal conditions for GS-6620 absorption. Combined, these properties suggest that GS2 will have better oral absorption in the clinic when administered in a solid dosage form and the potential to extend the clinical proof of concept obtained with GS-6620.

Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation.

UNLABELLED: Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors. IMPORTANCE: Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.

Inhibition of hepatitis C virus replication by GS-6620, a potent C-nucleoside monophosphate prodrug.

As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 µM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 µM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

Preclinical characterization of GS-9669, a thumb site II inhibitor of the hepatitis C virus NS5B polymerase.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC(50)) of ≤ 11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.).

Woolly Monkey-HBV Infection in Squirrel Monkeys as a Surrogate Nonhuman Primate Model of HBV Infection.

Development of curative therapies for chronic hepatitis B virus (HBV) infection will likely require new animal models. Here, we evaluate HBV infection in squirrel monkeys based on the high-sequence homology of the HBV receptor, Na+/taurocholate co-transporting peptide (NTCP), between humans and squirrel monkeys. HBV PreS1 peptide was examined for binding human and squirrel monkey NTCP. Immunodeficient Fah -/- , NOD, Rag1 -/- , Il2Rg null (FNRG) mice engrafted with human or squirrel monkey hepatocytes were challenged with HBV or Woolly Monkey HBV (WMHBV). In addition, adult squirrel monkeys were inoculated with HBV, WMHBV, adeno-associated virus containing an infectious genome of HBV (AAV-HBV), and AAV-WMHBV. Finally, neonate squirrel monkeys were assessed for the potential of chronic infection with WMHBV. PreS1 peptide efficiently bound to human and squirrel monkey NTCP but not to mouse or capuchin NTCP. FNRG mice engrafted with squirrel monkey hepatocytes were susceptible to infection by WMHBV but not human HBV. Similarly, adult squirrel monkeys could be infected with WMHBV but not human HBV, whereas chimeric mice engrafted with human hepatocytes were susceptible to HBV but not WMHBV. Infection of squirrel monkeys with AAV-WMHBV yielded maximum viremia of 108 genomes/mL with detectable virus for up to 8 months. Notably, covalently closed circular DNA was detected in the liver of these animals. Infection of neonates with WMHBV led to detectable viremia for up to 6 months. Conclusions: Adult and neonate squirrel monkeys exhibited prolonged WMHBV viremia lasting 6-8 months. This is greater than twice the duration of viremia achieved in other nonhuman primates and suggests that squirrel monkeys may be a suitable model for testing HBV therapeutics.