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

► 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. 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.