Phosphorothioate di- and trinucleotides as a novel class of anti-hepatitis B virus agents

Abstract

Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di- and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15. Through an iterative process of synthesis, lead optimization, and screening, three analogs were identified as potent inhibitors of HBV replication: dinucleotides ORI-7246 (drug concentration at which a 10-fold reduction of HBV DNA was observed [EC(90)], 1.4 microM) and ORI-9020 (EC(90), 1.2 microM) and trinucleotide ORI-7170 (EC(90), 7.2 microM). These analogs inhibited the replication of both strands of HBV DNA. No suppression of HBV protein synthesis or intracellular core particle formation by these analogs was observed. No inhibition of HBV DNA strand elongation by the analogs or their 5'-triphosphate versions was apparent in in vitro polymerase assays. Although the exact mechanism of action is not yet identified, present data are consistent with an inhibition of the HBV reverse transcriptase-directed priming step prior to elongation of the first viral DNA strand. In transient-transfection assays, these analogs inhibited the replication of 3TC-resistant HBV. Synergistic interactions in combination treatments between the analogs and either 3TC or ADV were observed. These compounds represent a novel class of anti-HBV molecules and warrant further investigation as potential therapeutic agents.

FIG. 1.

Structures of active compounds from this study.

FIG. 2.

Effect of analog ORI-7246 on intracellular HBV replication in 2.2.15 cells. Cultures were exposed to the indicated concentrations of agents for 9 consecutive days. DNA was extracted 24 h following the final addition of compounds and analyzed by Southern blot hybridization for HBV DNA.

FIG. 3.

Examples of the analysis of compound interactions during combination treatments. The interactions between ORI-9020 and either 3TC (A to C) or ADV (D to F) by the CALCUSYN software program (Biosoft, Inc., Cambridge, United Kingdom) are displayed. Three types of drug interaction analyses are displayed: drug-effect plots (A and D), FA-CI plots (with Monte Carlo analysis) (B and E), and conservative isobolograms (C and F). For the FA-CI plot analysis, a combination index (CI) greater than 1.0 indicates antagonism and a CI less than 1.0 indicates synergism. Evaluations of synergy, additivity (summation), or antagonism at different levels of virus inhibition (e.g., 5% [FA = 0.5] to 99% [FA = 0.99]) are provided. The triple lines on these plots, i.e., median values ±1.96 standard deviations, were calculated by Monte Carlo analysis (3). For the isobolograms, ED50, ED75, and ED90 values (ED, effective antiviral dose) for the combination treatments are displayed as single points. Values that fall to the left of the lines (representing values for additive interactions based on the monotherapies) indicate synergy, and values to the right indicate antagonism. For the dose-effect and median-effect plots, plotted values for the combination treatments that fall above both sets of lines for the monotherapies generally indicate synergism.