4'-modified nucleoside analogs: potent inhibitors active against entecavir-resistant hepatitis B virus

Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). However, both viruses often acquire NRTI resistance, making it crucial to develop more-potent agents that offer profound viral suppression. Here, we report that 4'-C-cyano-2-amino-2'-deoxyadenosine (CAdA) is a novel, highly potent inhibitor of both HBV (half maximal inhibitory concentration [IC50 ] = 0.4 nM) and HIV-1 (IC50  = 0.4 nM). In contrast, the approved anti-HBV NRTI, entecavir (ETV), potently inhibits HBV (IC50  = 0.7 nM), but is much less active against HIV-1 (IC50  = 1,000 nM). Similarly, the highly potent HIV-1 inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA; IC50  = 0.3 nM) is less active against HBV (IC50  = 160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50  = 7.2 nM) and ETV-resistant HBV (IC50  = 69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 microM. Once-daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (P = 0.0005) in human-liver-chimeric/ HBVETV-RL180M/S202G/M204V-infected mice, whereas ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4'-cyano of CAdA-triphosphate (TP), but not the longer 4'-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4'-substitutions of both NRTIs. The ETV-TP's cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket.

These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients.