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. The discovery of safe and effective antiviral drugs continues to present considerable challenges. The rapid emergence of resistance to antiviral drugs is a major problem, and combinations of a limited repertoire of antiviral drugs often need to be employed as a therapeutic strategy. The major stumbling block in antiviral drug development is the limited structural, mechanistic, and functional information on many virus-specific molecular targets. Consequently, the design of target-specific compounds that interfere with viral life cycles is a difficult challenge. There continues to be a substantial unmet clinical need for antiviral drugs with different structures and unique mechanisms of action, other than those conferred by conventional nucleoside analogs (2).Given a lack of sufficient structural information on new targets, an approach that seems appropriate for antiviral drug discovery is the screening of structurally diverse compounds, generated using combinatorial approaches that would modulate biological pathways without regard to specific molecular targets. In theory, this approach would allow simultaneous functional validation of a target, as well as the discovery of a lead structure that modulates the function of the target. This strategy has been variously referred to as "diversity-oriented organic synthesis for therapeutic target validation" or "combinatorial target-guided ligand assembly" (6, 20).We describe here the application of this concept for the discovery of anti-hepatitis B virus (HBV) agents. In recent publications, we have reported methods of assembling various classes of di-and trinucleotide libraries (7,8,18). Through screening, as well as lead optimization work, and in conjunction with cell-based assays, we have discovered that certain molecules show very promising anti-HBV activity. Furthermore, some of these molecules display synergistic activity when used in combination with lamivudine (3TC) and adefovir dipivoxil (ADV), the two nucleoside analogs currently licensed for the treatment of chronic HBV infection. This work summarizes the in vitro analysis and preliminary toxicity evaluation of these compounds.(A preliminary report of this work was presented at the 14th International Conference for Antiviral Research, Seattle, Washington, 8 to 12 April 2001.) MATERIALS AND METHODSTest compounds. Nucleotide libraries were synthesized ...

Small-Molecule Effectors of Hepatitis B Virus Capsid Assembly Give Insight into Virus Life Cycle

“…Assays for antiviral activity were conducted as previously described (12,13). In general, secreted virus was assayed by testing culture medium for HBV DNA, intracellular virus was assayed by quantitative Southern blotting for the DNA replicative intermediate, and cell viability was tested by uptake of neutral red (13).…”

“…Cytotoxicity was assessed by uptake of neutral red dye 24 h following the last treatment. Activity against lamivudineresistant (12, 13) and adefovir dipivoxil-resistant (1) HBV mutants was tested in a 5-day assay using a transient-transfection method as previously described (12). Antiviral activity against the drug-resistant panel was determined by quantitative Southern blot hybridization of intracellular HBV DNA replication intermediates.…”

Alkoxyalkyl Esters of 9-( S )-(3-Hydroxy-2-Phosphonomethoxypropyl) Adenine Are Potent and Selective Inhibitors of Hepatitis B Virus (HBV) Replication In Vitro and in HBV Transgenic Mice In Vivo

“…Cytotoxicity was assessed by measurement of the uptake of neutral red dye and quantitative analysis of the absorbance of the internalized dye at 510 nM 24 h following the last treatment (three cultures per test concentration). The activities of the compounds against lamivudineresistant and adefovir-resistant HBV mutants were determined in a 5-day assay by use of a transient transfection method with Huh7 cells, as described previously (16).…”

Metabolism, Pharmacokinetics, Tissue Distribution, and Stability Studies of the Prodrug Analog of an Anti-Hepatitis B Virus Dinucleoside Phosphorothioate

“…In contrast, in vitro studies using Caco-2 cell lines and in vivo studies in rats revealed that none of the SMNH analogs has potential for oral bioavailability, presumably because of the negative charge(s) on their backbone that impedes their passive diffusion across the cellular lipid bilayer (Iyer et al, 2004aCoughlin et al, 2010).…”

“…In previous studies, we have shown that in general, the SMNH class of molecules has unique metabolic and pharmacokinetic properties that are distinct from traditional small molecule drugs (Iyer et al, 2004a. Thus, in contrast to the hydrophobic small molecule compounds, which are subject to cytochrome P450 (P450)-mediated Phase I oxidative processes and Phase II conjugation reactions, SMNH analogs are not metabolized by P450 enzyme systems.…”