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.

FIG. 1

Chemical structures of dG and dC analogs.

FIG. 2

Inhibitory effects of nucleoside analogs on HBV Pol-mediated viral minus-strand DNA synthesis. Recombinant HBV nucleocapsids were subjected to endogenous Pol assays in the presence of increasing (from left to right) concentrations of dG analog TPs or the dC analog 3TC-TP and constant concentrations of cold dNTPs and [α-³²P]dATP. (A) The radiolabeled Pol-linked minus-strand DNA products were extracted and analyzed through SDS–8% polyacrylamide gels. The position of HBV Pol (∼93 kDa) is indicated at the sides of the figure; an in vitro-translated ³⁵S-labeled Pol marker is shown in lane ³⁵S (authentic 93-kDa HBV Pol is the second largest species in this lane). Substr., substrate. (B) Inhibition curves were generated by phosphorimaging of the gels shown in panel A.

FIG. 3

Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α-³²P]dATP. The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.

FIG. 4

Effect of guanosine versus cytosine analogs on DHBV priming. In vitro-translated DHBV Pol was incubated with 250 nM [α-³²P]dGTP; 220 nM unlabeled dCTP, dATP, and TTP; and increasing concentrations of the indicated analog-TPs. (A) The radiolabeled Pol-oligonucleotide adducts were analyzed by conventional SDS-PAGE and autoradiography. The migration positions of the ³⁵S-labeled DHBV Pol (lane ³⁵S) are indicated at the sides. Substr., substrate. (B) Titration curves were generated by phosphorimaging.

FIG. 5

Effect of BMS-200475-TP versus 3TC-TP on the HBV priming and reverse transcription activities. Cores were subjected to endogenous Pol reactions in the presence of 50 μM BMS-200475–TP or 250 μM 3TC-TP. The product pattern generated in the absence of drug (lane Control) is shown for comparison. In vitro-translated [³⁵S]-labeled HBV pol (p93) served as size marker.

FIG. 6

Kinetic analyses of dGTP, BMS-200475–TP, and lobucavir-TP using WHV and HBV Pols. Lineweaver-Burk plots indicate that lobucavir-TP (A) and BMS-200475-TP (B) are competitive inhibitors of dGTP for WHV Pol (A) and HBV Pol (B). The velocities of the Pol reactions were measured as femtomoles of [α-³³P]dGMP incorporation in 120 min at 30°C. (C) Double-reciprocal plot to determine the K_m of dGTP with HBV Pol. The reaction velocity was measured as described above. (D) IC₅₀ titrations of BMS-200475–TP and lobucavir-TP. HBV nucleocapsids were incubated for 120 min at 30°C with dNTPs and serial dilutions of the indicated guanosine analog-TPs. The percentage of [α-³³P]dGTP incorporation relative to that for the no-drug control is plotted on a semilogarithmic scale (mean of triplicate samples). The values from panels C and D were used to calculate the K_is of the guanosine analog-TPs.

FIG. 7

(B) Endogenous sequencing of viral minus-strand DNA from replicative recombinant HBV nucleocapsids. Purified cores were incubated with 130 nM [α-³²P]dNTPs and either one of the four cold ddNTPs (26 μM) or unlabeled lobucavir-TP or BMS-200475–TP (2 and 10 μM), as indicated at the top. Deproteinized reaction products were electrophoresed through a urea–6% polyacrylamide gel alongside conventional dideoxynucleotide sequencing ladders (A and C). The incorporated radioactivity was visualized by scanning the dried gel with a phosphorimager. The corresponding sequence of HBV minus-strand DNA is depicted below the figure. Major drug-induced termination sites (sites 1 to 8), are indicated, as are their alignments relative to the G residues (+2 and +3). nts., nucleotides.