4′‐modified nucleoside analogs: Potent inhibitors active against entecavir‐resistant hepatitis B virus

Abstract

Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against HIV-1 and HBV. However, both viruses often acquire NRTI resistance, making it crucial to develop more potent agents that offer profound viral suppression. We report here that 4′-C-cyano-2-amino-2′-deoxyadenosine (CAdA) is a novel highly potent inhibitor of both HBV (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 μM. 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, while 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. Conclusion: 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.

Structural basis of HIV inhibition by translocation-defective RT inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)

“…Entecavir is the most potent anti-hepatitis B NRTI drug, and sofosbuvir is the most successful anti-hepatitis C virus nucleotide drug (1)(2)(3)(4)(5). 4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) extends this concept as an anti-HIV agent.…”

CMCdG, a Novel Nucleoside Analog with Favorable Safety Features, Exerts Potent Activity against Wild-Type and Entecavir-Resistant Hepatitis B Virus

“…We previously reported that CAdA and CdG highly potently suppressed the replication of a wild-type HBV genotype D (HBV WT D ) in HepG2.2.15 cells. CAdA and CdG also suppressed the production of wild-type HBV genotype Ce (HBV WT Ce ) in HBV WT Ce plasmid-transfected Huh7 cells and potently decreased the HBV WT Ce viremia level in HBV WT Ce -exposed human liver-chimeric mice (19). CAdA and CdG were also potent against a highly ETV-resistant HBV variant containing three critical amino acid substitutions: L180M, S202G, and M204V (HBV L180M/S202G/M204V ) as examined in HBV L180M/S202G/M204V plasmid-transfected Huh7 cells and HBV L180M/S202G/M204Vexposed human liver-chimeric mice.…”

“…CAdA and CdG were also potent against a highly ETV-resistant HBV variant containing three critical amino acid substitutions: L180M, S202G, and M204V (HBV L180M/S202G/M204V ) as examined in HBV L180M/S202G/M204V plasmid-transfected Huh7 cells and HBV L180M/S202G/M204Vexposed human liver-chimeric mice. However, both compounds proved to be much more cytotoxic than ETV, and they were dropped from further development (19). Thus, based on the structural findings that we had obtained (19,20), we continued the design and synthesis of approximately 220 novel nucleoside analogs containing a cyano moiety at the 4= position of the ribose and identified CMCdG.…”

“…However, both compounds proved to be much more cytotoxic than ETV, and they were dropped from further development (19). Thus, based on the structural findings that we had obtained (19,20), we continued the design and synthesis of approximately 220 novel nucleoside analogs containing a cyano moiety at the 4= position of the ribose and identified CMCdG. It is of note that CMCdG structurally resembles CdG, in that both compounds have a 4= cyano moiety, and it also resembles ETV, in that both CMCdG and ETV have an exocyclic double bond or methylidene in the 4= position of the cyclopentyl moiety ( Fig.…”

“…We have also previously reported that two EFdA congeners, 4=-C-cyano-2-amino-2=-deoxyadenosine (CAdA) and 4=-C-cyano-2=-deoxyguanosine (CdG; Fig. 1) exert potent activity against HBV WT and drug-resistant HBV variants, such as HBV ETV-R L180M/M204V and HBV ADV-R A181T/N236T , both in cell culture and in HBV-inoculated severe combined immunodeficiency (SCID) mice that were transgenic for the urokinase-type plasminogen activator (uPA) gene (uPA/SCID mice) and in which the liver was partly replaced with human hepatocytes (human liverchimeric mice) (19). However, both CAdA and CdG proved to be more cytotoxic than currently available anti-HBV therapeutics and were not pursued for further development.…”