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-(hydroxymethyl)-2-methylene-cyclopentanecarbonitrile, or 4'-cyano-methylenecarbocyclic-2'-deoxyguanosine (CMCdG), and evaluated its anti-hepatitis B virus (anti-HBV) activity, safety, and related features. CMCdG's in vitro activity was determined using quantitative PCR and Southern blotting assays, and its cytotoxicity was determined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, while its in vivo activity and safety were determined in human liver-chimeric mice infected with wild-type HBV genotype Ce (HBV_WT^Ce) and an entecavir (ETV)-resistant HBV variant containing the amino acid substitutions L180M, S202G, and M204V (HBV_ETV-R^{L180M/S202G/M204V}). CMCdG potently inhibited HBV production in HepG2.2.15 cells (50% inhibitory concentration [IC₅₀], ∼30 nM) and HBV_WT^Ce plasmid-transfected Huh7 cells (IC₅₀, 206 nM) and efficiently suppressed ETV-resistant HBV_ETV-R^{L180M/S202G/M204V} (IC₅₀, 2,657 nM), while it showed no or little cytotoxicity (50% cytotoxic concentration, >500 μM in most hepatocytic cells examined). Two-week peroral administration of CMCdG (1 mg/kg of body weight/day once a day [q.d.]) to HBV_WT^Ce-infected human liver-chimeric mice reduced the level of viremia by ∼2 logs. CMCdG also reduced the level of 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, semiempirical quantum methods, and molecular dynamics revealed that although ETV triphosphate (TP) forms good van der Waals contacts with L180 and M204 of HBV_WT^Ce Reverse Transcriptase (RT), its contacts with the M180 substitution are totally lost in the HBV_ETV-R^{L180M/S202G/M204V} RT complex. However, CMCdG-TP retains good contacts with both the HBV_WT^Ce RT and HBV_ETV-R^{L180M/S202G/M204V} RT complexes. The present data warrant further studies toward the development of CMCdG as a potential therapeutic for patients infected with drug-resistant HBV and shed LIGHT on the further development of more potent and safer anti-HBV agents.