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.

Inhibition of hepatitis B virus replication by quercetin in human hepatoma cell lines

“…HepG2.2.15 and pCH-9/3091-transfected HuH-7 cells treated with 50 μmol/L quercetin showed significant inhibition of viral DNA levels from day 1 and 2, respectively. In both cell models, 30 nmol/L ETV reduced HBV DNA levels by about 90% (Figure 5, 6), which was similar to previously reported studies (Seifer et al, 1998;Bader and Korba, 2010).…”

“…These oral agents selectively and potently inhibit HBV polymerase. For example, ETV blocks all three distinct phases of replication mediated by viral polymerase including priming, reverse transcription, and DNA-dependent DNA synthesis (Seifer et al, 1998). Therefore, it reduces the encapsulated HBV DNA level in host cells by 90% at a very low concentration (30 nmol/L, Figure 5, 6).…”

Management of hepatitis B in patients with HBeAg-negative disease

“…Entecavir is a deoxyguanosine analogue and is the most potent antiviral agent in the current arsenal against HBV [22]. In a pivotal trial, 48 weeks of therapy with entecavir at a dose of 0.5 mg/d in lamivudine-naive patients with HBeAg-negative CHB was superior to lamivudine monotherapy.…”

Management of hepatitis B virus co-infection on and off antiretroviral therapy

“…Entecavir (ETV) is a guanosine analogue that inhibits HBV replication at three different steps (priming, reverse transcriptase, and positive strand synthesis) [40]. ETV shows more potency in suppressing serum HBV-DNA than 3TC and ADV and is effective against wild-type and 3TC-and ADV-resistant HBV [41,42].…”