Elsevier

Antiviral Research

Volume 92, Issue 2, November 2011, Pages 271-276
Antiviral Research

The main Hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro to non-nucleoside inhibitors of HBV replication

https://doi.org/10.1016/j.antiviral.2011.08.012Get rights and content

Abstract

Long-term treatment of chronic hepatitis B with nucleos(t)ide analogs can lead to the emergence of HBV resistant mutants of the polymerase gene. The development of drugs with a different mode of action is warranted to prevent antiviral drug resistance. Only a few non-nucleosidic molecules belonging to the family of phenylpropenamides (AT-61 & AT-130) and heteroaryldihydropyrimidines (BAY41-4109) can prevent RNA encapsidation or destabilize nucleocapsids, respectively. The sensitivity of the main nucleos(t)ide analog- resistant mutants to these inhibitors was evaluated in vitro. HepG2 stable cell lines permanently expressing wild type (WT) HBV or the main HBV mutants resistant to lamivudine and/or adefovir (rtL180M + rtM204V, rtV173L + rtL180M + rtM204V, rtM204I, rtL180M + rtM204I, rtN236T, rtA181V, rtA181V + rtN236T, rtA181T, rtA181T + rtN236T) were treated with AT-61, AT-130 or BAY-41 4109. Analysis of intracellular encapsidated viral DNA showed that all mutants were almost as sensitive to these molecules as WT HBV; indeed, the fold-resistance ranged between 0.7 and 2.3. Furthermore, the effect of a combination of either AT-61 or AT-130 with BAY41-4109, and the combination of these compounds with tenofovir was studied on wild type HBV as well as on a lamivudine and an adefovir-resistant mutant (rtL180M + M204V and rtN236T, respectively). These combinations of compounds resulted in inhibition of viral replication but showed slight antagonistic effects on the three HBV species. Based on this in vitro study, BAY-41 4109, AT-61 and AT-130 molecules that interfere with capsid morphogenesis are active against the main lamivudine- and adefovir-resistant mutants. These results suggest that targeting nucleocapsid functions may represent an interesting approach to the development of novel HBV inhibitors to prevent and combat drug resistance.

Highlights

► Non-nucleosidic compounds inhibit the main resistant mutants replication. ► Non-nucleosidic compounds acting against capsid morphogenesis are not cytotoxic. ► Combination of non-nucleosidic compounds results in inhibition of resistant mutant replication. ► Compounds targeting nucleocapsid assembly could be relevant to decrease resistance to polymerase inhibitors.

Introduction

Despite the availability of an effective vaccine, 350 million people around the world are chronically infected by HBV (Wright and Lau, 1993), and are exposed to major complications including cirrhosis and hepatocellular carcinoma, leading to the death of 600,000 patients per year according to the World Health Organization.

Alpha interferon, followed by its pegylated form, was the first treatment accepted against HBV infection but only one third of treated patients show a sustained virologic response. Furthermore, the beneficial antiviral effects of interferon administration have to be weighed against numerous adverse side effects (Zoulim, 2006). Over the last decade, specific inhibitors of the viral polymerase activity that belong to the family of nucleos(t)ide analogs (NA) and include lamivudine, adefovir, telbivudine, entecavir and tenofovir have been approved (Bhattacharya and Thio, 2010, Zoulim, 2006). In spite of their high efficiency in reducing viremia levels, eradication of HBV genomes from the infected liver cannot be achieved. Long term antiviral therapy is therefore necessary to control viral replication. Because of viral persistence in the liver and the error rate of the viral polymerase, prolonged antiviral administration can lead to the emergence and selection of drug-resistant mutants harboring amino acid substitutions in the viral polymerase (Zoulim and Locarnini, 2009). Because of the overlap of the surface and polymerase genes, these resistant mutants may also harbor envelope protein modifications that can result in immune escape or modification of viral fitness (Torresi et al., 2002, Villet et al., 2009). The management of antiviral drug resistance remains a clinical challenge, especially in countries where patients have been exposed to sequential therapy with antivirals having a low barrier to resistance. In these patients, the risk of multiple drug resistance is significant as well as the risk of suboptimal response to drugs with a high barrier to resistance, i.e. entecavir and tenofovir (Zoulim and Locarnini, 2009).

To counter resistance to antiviral drug directed against the viral polymerase, there is a major need to develop novel compounds with a different mode of action. In this respect, one of the potentially interesting targets is nucleocapsid assembly (Prevelige, 1998). This includes the formation and stability of viral capsids as well as the encapsidation of viral pregenomic RNA (pgRNA). Capsid sub-units interact to form a stable icosahedral structure (Seifer and Standring, 1995). Only a few compounds have been shown to target efficiently HBV nucleocapsid formation and stabilization (Weber et al., 2002). BAY-41 4109 is a heteroaryldihydropyrimidine known to reduce extra- and intra- cellular HBV DNA by impairing the formation of normal icosahedral capsids in vitro (Deres et al., 2003, Stray et al., 2005). Its antiviral activity was further confirmed in vivo in transgenic mice (Shi et al., 2007, Weber et al., 2002). Other compounds, AT-61 or AT-130 that belong to the family of phenylpropenamides, were shown to affect pgRNA encapsidation, leading to the production of empty capsids (Feld et al., 2007). It has recently been shown that by favoring protein-protein interactions to the detriment of pgRNA encapsidation and by trapping HBV capsid assembly intermediates, the administration of phenylpropenamides accelerates the assembly of empty capsids not containing the viral polymerase/pgRNA complex (Katen et al., 2010).

The inhibitory activity and the mechanism of action of phenylpropenamides and heteroaryldihydropyrimidines on the replication of WT HBV have been studied in detail, but only against a few mutants, mainly lamivudine resistant mutants (Delaney et al., 2002). The objective of our study was therefore to determine the antiviral activity of these compounds against a series of lamivudine, adefovir, and multi- resistant mutants in cell lines expressing these viral genomes. Our results showed a comparable activity against these major resistant mutants as with WT HBV. The combination of these compounds as well as their combination with tenofovir showed inhibitory activities against HBV replication but did not result in a synergistic effect in our experimental conditions. However, because of their different modes of action/non-overlapping resistance profiles, these combinations may represent a novel antiviral strategy that deserves further evaluation in study models where cccDNA formation and several rounds of infection can be observed.

Section snippets

Mutant cell lines

Stable cell lines were obtained by transfection of HepG2 cells with plasmids encoding HBV mutants made by site-directed mutagenesis as described (Qi et al., 2007, Yang et al., 2005). HepG2 stable cell lines permanently expressing the main HBV mutants resistant to lamivudine and adefovir were as follows: rtL180M + rtM204V, rtV173L + rtL180M + rtM204V, rtM204I, rtL180M + rtM204I, rtN236T, rtA181V, rtA181V + rtN236T, rtA181T, rtA181T + rtN236T (Table 1). The rtA181V + rtN236T and the rtA181T + rtN236T are

The main nucleoside analog resistant mutants are susceptible to inhibition by phenylpropenamides and heteroaryldihydropyrimidines

Results of MTT assay performed in all cell lines expressing either WT HBV or the lamivudine and adefovir resistant mutants and treated with increasing concentrations of AT-61, AT-130 and BAY-41 4109 showed the absence of cytotoxic effects, with CC50 > 25μM for AT-61, > 1μM for AT-130, and >2 μM for BAY-41 4109.

The inhibitory effect of phenylpropenamides and heteroaryldihydropyrimidines on viral DNA synthesis was determined in cell lines expressing the main nucleoside analog-resistant mutants (Table 1

Discussion

Currently, nucleos(t)ide analogs (NA) are the only virus specific drugs approved for the treatment of chronic HBV infection (Jafri and Lok, 2010). However, the administration of viral polymerase inhibitors can select for drug-resistant mutants that can lead to treatment failure (Zoulim and Locarnini, 2009). The search for new antiviral targets with a different mode of action is therefore warranted to develop combination strategies to combat NA drug resistance not only when it is already

Acknowledgements

This was work was funded in part by grants from the French agency for research against AIDS and hepatitis (ANRS), and Finovi foundation. Gaëtan Billioud received a fellowship from the French Ministry of Research (MRT). Special thanks to Katyna Boroto-Esoda (Gilead Sciences) for providing HepG2 cell lines permanently expressing HBV resistant mutants, and to Alan Kay (INSERM) for critically reviewing the manuscript.

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