Showing papers by "Richard J. Colonno published in 1998"

In Vitro Inhibition of Hepadnavirus Polymerases by the Triphosphates of BMS-200475 and Lobucavir

Abstract: The guanosine analogs BMS-200475 and lobucavir have previously been shown to effectively suppress propagation of the human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) in 2.2.15 liver cells and in the woodchuck animal model system, respectively. This repression was presumed to occur via inhibition of the viral polymerase (Pol) by the triphosphate (TP) forms of BMS-200475 and lobucavir which are both produced in mammalian cells. To determine the exact mode of action, BMS-200475–TP and lobucavir-TP, along with several other guanosine analog-TPs and lamivudine-TP were tested against the HBV, WHV, and duck hepatitis B virus (DHBV) polymerases in vitro. Estimates of the 50% inhibitory concentrations revealed that BMS-200475–TP and lobucavir-TP inhibited HBV, WHV, and DHBV Pol comparably and were superior to the other nucleoside-TPs tested. More importantly, both analogs blocked the three distinct phases of hepadnaviral replication: priming, reverse transcription, and DNA-dependent DNA synthesis. These data suggest that the modest potency of lobucavir in 2.2.15 cells may be the result of poor phosphorylation in vivo. Kinetic studies revealed that BMS-200475–TP and lobucavir-TP competitively inhibit HBV Pol and WHV Pol with respect to the natural dGTP substrate and that both drugs appear to bind to Pol with very high affinities. Endogenous sequencing reactions conducted in replicative HBV nucleocapsids suggested that BMS-200475–TP and lobucavir-TP are nonobligate chain terminators that stall Pol at sites that are distinct yet characteristically two to three residues downstream from dG incorporation sites.

Efficacy of the Carbocyclic 2′-Deoxyguanosine Nucleoside BMS-200475 in the Woodchuck Model of Hepatitis B Virus Infection

Abstract: Human hepatitis B virus (HBV), the prototype member of a small family of hepadnaviruses, is an enveloped DNA virus with a partially double-stranded 3.2-kb circular genome. HBV infection is widespread and is a major cause of human liver disease (4). While primary human HBV infections are usually self-limiting, 6 to 10% of infected adults and a higher percentage of children become chronically infected carriers who face a high incidence of sequellae such as liver cirrhosis and hepatocellular carcinoma. Efforts to combat persistent HBV infection via antiviral chemotherapy (18, 29, 47) have focused on the viral replication cycle (36). Briefly, HBV replication uses a virally encoded polymerase with reverse transcriptase activity to convert a greater-than-genome-length 3.5-kb pregenomic RNA (pgRNA) intermediate into the characteristic double-stranded DNA genome. This process occurs inside cytoplasmic viral nucleocapsids. Most nucleocapsids bearing matured HBV DNA genomes then acquire an outer envelope coat at the endoplasmic reticulum to form virions which exit the cell via the secretory pathway. However, by mechanisms that remain to be clarified, a fraction of the newly replicated HBV genomes are recycled to the cell nucleus where they give rise to a small population of supercoiled covalently closed circular viral DNA (cccDNA) species (43). cccDNA serves as the template for the synthesis of further pgRNA transcripts (45), thus fueling additional cycles of hepadnaviral replication. So far, a few nucleoside analogs have emerged as the most promising inhibitors of HBV replication, most notably lamivudine (3TC) and famciclovir, a prodrug of penciclovir. These compounds are active against HBV both in cultured cells (10, 20, 25) and in ongoing clinical trials (1, 9, 33). Limitations of these nucleosides, however, include rapid rebounds in HBV viremia once therapy ends and the emergence of drug-resistant mutant viruses (2, 3, 28, 30, 42). It is also not clear that nucleosides can eliminate the reservoir of long-lived cccDNA (14, 32). Thus, the need remains for additional agents for the antiviral chemotherapy of HBV. BMS-200475 (formerly SQ 34676) (5) is a cyclopentyl 2′-deoxyguanosine nucleoside analog (Fig. ​(Fig.1)1) with excellent potency and selectivity against HBV (5, 20). In the stably transfected HBV-expressing HepG2.2.15 cell line, BMS-200475 inhibits the replication of HBV with a 50% effective concentration of 3.75 nM (20). This inhibition was reversed upon withdrawal of compound and was accompanied by marked reductions in the levels of intracellular HBV DNA genomes and viral replicative intermediates, suggesting that BMS-200475 is an inhibitor of the HBV polymerase. Mechanism-of-action studies confirmed (37) that the triphosphate form of BMS-200475 directly inhibits hepadnaviral polymerases in vitro. FIG. 1 Chemical structure of BMS-200475. The efficacy of BMS-200475 against hepadnavirus infection in woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus (WHV) was determined. This animal model (35, 40) of HBV infection is widely accepted and has proved to be useful for the evaluation of antiviral agents directed against HBV (7, 11, 13, 15, 19, 30, 34, 35, 39, 41). Daily oral administration of BMS-200475 at doses as low as 0.02 mg/kg of body weight markedly reduced serum WHV DNA levels in woodchucks with no evidence of toxicity at the dose levels used.