Inhibitory effects of selected antiviral compounds on human hepatitis B virus DNA synthesis

Abstract

By using an assay system based on a human hepatoblastoma cell line (HB611) that continuously synthesizes hepatitis B virus DNA, the following compounds were found to inhibit hepatitis B virus DNA synthesis at concentrations that were significantly lower than their minimum cytotoxic concentrations: 9-(2-phosphonylmethoxyethyl)-2,6-dianiinopurine (PMEDAP), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, 9-(phosphonylmethoxyethyl)adenine, 2',3'-dideoxy-2',3'-didehydrocytidine, and 2',3'-dideoxycytidine. The most potent compound was PMEDAP (50% effective concentration, 0.02 pg/ml). The Recently, a number of assay systems have been described which seem suitable for evaluation of the inhibitory effects of antiviral compounds against hepadnaviruses, including HBV and duck hepatitis B virus (DHBV) (5, 9, 11, 13, 15). DHBV is able to propagate in primary duck hepatocytes in vitro (12). Selective inhibition of the synthesis of DHBV DNA and core antigen in primary duck hepatocytes can be used as a marker for anti-DHBV activity. Using this assay system, we examined a variety of purine and pyrimidine nucleoside analogs for their inhibitory effects on DHBV replication (15).Ueda et al. (13) have recently described a human hepatoblastoma cell line (HB611) that contains an integrated HBV genome and that accumulates a significant amount of HBV DNA replicative intermediates. Some of these DNA molecules are packed and released as Dane particles into the cell culture medium. Selective inhibition of the synthesis of these replicative intermediates could serve as a marker for anti-HBV activity. Using the HB611 cell line, we examined the inhibitory effects of selected antiviral compounds on HBV DNA synthesis. The compounds which we evaluated for their inhibitory effect on HBV DNA synthesis were previously examined for their inhibitory effects on DHBV DNA and core antigen synthesis in primary duck hepatocytes (15).The compounds fell into two categories: phosphonylmethoxyalkylpurines and -pyrimidines and 2',3'-dideoxynucleoside analogs. Their origins were as follows. Test compounds were assayed by a modification of the method described by Ueda et al. (13). The HB611 cell line was generously provided by K. Matsubara, Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan. HB611 cells were seeded in tissue culture trays (diameter, 35 mm) at a density of 5 x 104 cells per well by using Dulbecco modified minimum essential medium supplemented with 10% fetal bovine serum, 100 ,ug of streptomycin per ml, 100 IU of penicillin G per ml, and 0.2 mg of Geneticin R (GIBCO) per ml. After 2 days of incubation, when the cells were grown to confluency, the growth medium was withdrawn. Then, the cells were incubated for 12 days, during which time the medium containing the compounds was exchanged every 3 days. The cells were then harvested and total DNA was prepared upon lysis of the cells in 0.5 ml of 10 mM Tris hydrochloride (pH 7.8)-S5 mM EDTA-150 mM NaCI-1% sodium dodecyl sulfate-0.1 mg of proteinase K (Sigma) per ml. After...

Colorimetric Assay System for Screening Antiviral Compounds against Hepatitis B Virus

“…However, the EC50 values that we obtained for PMEG and PMEGEE were much lower than those obtained by others, and the EC50 value of PMPA was higher than that of the Southern blot analysis (18). The selective indexes of (S)-HPMPC and araA estimated by this assay system were higher than previous results (17,18). However, these differences should pose no problem in the screening of anti-HBV compounds.…”

“…Yokota et al (17,18) was also similar to previously reported data (18). However, the EC50 values that we obtained for PMEG and PMEGEE were much lower than those obtained by others, and the EC50 value of PMPA was higher than that of the Southern blot analysis (18).…”

“…This colorimetric method allows for the quantitative detection of HBV DNA and can be partially automated. The use of HB611 cells for analysis of the anti-HBV compounds has been reported previously (4,8,14,17,18). However, we have developed a rapid and accurate colorimetric assay system for anti-HBV agents using HB611 cells, immunoaffinity purification, PCR, and hybrid-capture detection.…”

“…However, human hepatoma cell lines transfected with HBV DNA have also been developed (HB611 and HepG2 2.2.15 cells (11,13)) and used for the screening of antiviral drugs (3,4,8,14,17,18). In these studies, drug efficacy was determined by measuring HBV DNA levels using Southern blot analyses with 9)-labeled HBV DNA as the probe.…”

The clinical potential of the acyclic (and cyclic) nucleoside phosphonates. The magic of the phosphonate bond

“…Addressing these questions will open new perspectives for the clinical usefulness of these nucleotide analogues, thereby pertaining to the importance (or "magic") of the phosphonate bound. 7 therefore, does not exist of (R)-and (S)-enantiomers, were first mentioned by De Clercq et al [1]. From a mechanistic viewpoint, all acyclic nucleoside phosphonates (ANPs) need two consecutive intracellular phosphorylations, to their diphosphate form, before they can interact with their target enzyme (DNA polymerase for DNA viruses, reverse transcriptase for retroviruses) as alternate substrates (with respect to dATP, for PMEA diphosphate or (S)-…”