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

We designed, synthesized, and characterized a novel nucleoside analog, (1S,3S,5S)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-hydroxy-1-(hydroxyl-methyl)-2-methylenecyclopentanecarbonitrile or 4′-cyano-methylenecarbocyclic-2′-deoxyguanosine (CMCdG) and evaluated its anti-HBV activity, safety, and related features. CMCdG9s in vitro activity was determined using qPCR and Southern blotting assays and its cytotoxicity with MTT assay, while that of in vivo activity and safety were determined in wild-type HBV genotype-Ce (HBV WT Ce )- and ETV-resistant HBV (HBV ETV-R L180M/S202G/M204V )-infected human-liver-chimeric mice. CMCdG potently inhibited HBV production in HepG2.2.15 cells (IC 50 ∼30 nM) and HBV WT Ce -plasmid-transfected Huh7 cells (IC 50 : 206 nM) and efficiently suppressed an ETV-resistant HBV ETV-R L180M/S202G/M204V (IC 50 : 2,657 nM), while it showed no or least cytotoxicity (CC 50 : >500 μM in most of hepatocytic cells examined). Two-week peroral administration of CMCdG (1 mg/kg/day, q.d. ) to HBV WT Ce -infected human-liver-chimeric mice reduced viremia by ∼2 logs. CMCdG also reduced HBV ETV-R L180M/S202G/M204V viremia by ∼1 log in HBV ETV-R L180M/S202G/M204V -infected human-liver-chimeric mice, while ETV (1 mg/kg/day, q.d. ) completely failed to reduce the viremia. None of the CMCdG-treated mice had significant drug-related changes in body-weights or serum human-albumin levels. Structural analyses using homology modeling, semi-empirical quantum methods, and molecular dynamics revealed that although ETV-triphosphates (TP) forms good van der Waals contacts with L180 and M204 of HBV WT Ce reverse transcriptase (RT), its contacts with M180 substitution are totally lost in the HBV ETV-R L180M/S202G/M204V -RT complex. However, CMCdG-TP retains good contacts with both HBV WT Ce - and HBV ETV-R L180M/S202G/M204V -RT complexes. The present data warrant further studies toward development of CMCdG as a potential therapeutic for infection with drug-resistant HBV and shed light on further development of more potent and safer anti-HBV agents.