Potent inhibition of hepatitis B virus production in vitro by modified pyrimidine nucleosides

Abstract

2',3'-Dideoxy-3'-fluorothymidine (FddThd), 2',3'-didehydro-2',3'-dideoxythymidine (ddeThd), and 3'-fluoro-5-methyl-deoxycytidine (FddMeCyt) are, in their triphosphate forms, selective inhibitors of human immunodeficiency virus type 1 reverse transcriptase. We report that 0.3 ,uM FddThd, FddMeCyt, or ddeThd as well as 3'-chloro-5-methyl-deoxycytidine (ClddMeCyt) or 3'-amino-5-methyl-deoxycytidine (AddMeCyt) almost completely blocked production of hepatitis B virus (HBV) particles by HBV DNA-transfected cell line 2.2.15 in vitro. Only at an at least 10-fold-higher concentration was a cytotoxic effect observed. These results indicate that FddThd, FddMeCyt, ClddMeCyt, AddMeCyt, and ddeThd are potent anti-HBV agents in vitro.Hepatitis B is a disease with continuously increasing occurrence and is caused by the hepatitis B virus (HBV) (11). Vaccination against hepatitis B is one way of effectively preventing HBV infection (2). Two lines of treatment of hepatitis B infection have been followed: (i) treatment with cytokines (8) to substitute the impaired production of peripheral blood mononuclear cells (7, 32) and (ii) treatment with antiviral agents, e.g., adenine arabinoside (18,21,29). The rationale for a chemotherapeutic treatment for hepatitis B is the inhibition of the viral DNA polymerase (4).Recently, we found that the triphosphate of 2',3'-dideoxy-3'-fluorothymidine (FddThd) is a potent inhibitor of reverse transcriptase of human immunodeficiency virus, a weak inhibitor of cellular DNA polymerase ,, and not effective at all against DNA polymerase a (12). In addition, we established that FddThd is well phosphorylated to the 5'-triphosphate in relevant cell lines (13). Moreover, it was found that the triphosphate of FddThd and the triphosphates of 2',3'-didehydro-2',3'-dideoxythymidine (ddeThd) and 3'-fluoro-5-methyl-deoxycytidine (FddMeCyt) FddThd, FddMeCyt, ClddMeCyt, AddMeCyt, and ddeThd were synthesized as described previously (6, 9, 30; E. Matthes, C. Lehmann, D. Scholz, M. von Janta-Lipinski, K. Gaertner, P. Langen, and H. A. Rosenthal, European Patent 0254268A2, 1987).In vitro assay for antiviral activity. The human hepatoblastoma cell line HepG2 was transfected with pDolTHBV-1, a vector which contains HBV (27). The clonal line of cells was designated 2.2.15 and was found to secrete both hepatitis B surface antigen (HBsAg) and HBV DNA (27). The 2.2.15 cells were kept in RPMI 1640 medium supplemented with 2 mM glutamine and 10% (vol/vol) fetal bovine serum. The cultures were incubated at 37°C in 5% CO2 in air.Cells were planted at a density of 5 x 105/ml in plastic flasks. One hour later the compounds were added and incubation was continued for 4 days; the cultures reached confluence on day 5. On day 2 fresh serum was added to the culture medium and the respective compound concentration was renewed.Cell growth was determined by two methods. First, after the 4-day incubation period, the cultures were incubated for an additional 24 h with 3 pXCi of [3H]dAdo per ml. In order to avoid direct interference of th...

Inhibition of the replication of hepatitis B virus in vitro by 2',3'-dideoxy-3'-thiacytidine and related analogues.

“…By using computer-assisted DNA and protein sequence analyses, HBV DNA polymerase was shown to share homologies with the reverse transcriptase from retroviruses (10). Though HBV DNA polymerase has not yet been purified, a number of studies (11,12) have indicated that inhibitors for reverse transcriptase of oncogenic RNA viruses may suppress HBV DNA replication. 2',3'-Dideoxycytidine (ddC) has been shown to be a potent inhibitor of human immunodeficiency virus replication in cell culture (13).…”

The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine

“…This process is catalyzed by a polymerase that shares significant sequence homology with the reverse transcriptase from retroviruses (17). As a consequence, it has been demonstrated that a number of compounds that inhibit human immunodeficiency virus (HIV) replication in vitro (for example, 2',3'-dideoxycytidine) also inhibit HBV replication in vitro (4,14,15,18,24). These agents await further study to determine their usefulness as therapeutic agents for the treatment of HBV infections.…”

Partial selective inhibition of HIV-1 reverse transcriptase and human DNA polymerases γ and β by thiated 3′-fluorothymidine analogue 5′-triphosphates

“…Enzyme kinetic studies with HIV-1 RT (Lineweaver-Burk plots) showed that both 4-SFLTTP and 2-SFLTTP were, similarly to the parent compound and to AZTTP, competitive inhibitors of this enzyme with respect to dTMP incorporation into poly(rA)·p(dT) [12][13][14][15][16][17][18] . K app i values for both control inhibitors for HIV-1 RT inhibition with poly(rA)·p(dT) [12][13][14][15][16][17][18] were within the range of values reported in the literature (Cheng et al, 1987;Hart et al, 1992;Matthes et al, 1990;Reardon and Miller, 1990). 4-SFLTTP was about three and four times less inhibitory to HIV-1 RT than FLTTP and AZTTP, respectively (Table 1).…”