Inefficient Phosphorylation of 3′-fluoro-2′-dideoxythymidine in Liver Cells May Explain its Lack of Inhibition of Duck Hepatitis B virus Replication in vivo

Duck hepatitis B virus (DHBV) was used as a model in the search for antiviral compounds against hepatitis B. The triphosphate of 3′-fluoro-2′,3′-dideoxythymidine (FLT) inhibits DHBV DNA polymerase in vitro. The aim of this investigation was to evaluate the in vivo inhibitory effect of FLT on DHBV in ducks. Four DHBV-positive ducklings were treated with FLT by intraperitoneal injections of 25 mg kg−1, two times daily for 17 days. No decrease of DHBV DNA in serum was observed. Therefore, it was investigated if FLT could be efficiently phosphorylated in duck liver. From DHBV-infected and non-infected duck livers protein extracts were prepared and partially purified by ion-exchange chromatography. Only one enzyme with thymidine (and deoxycytidine) kinase activity was found, corresponding to thymidine kinase 2 (i.e. the mitochondrial type of deoxynucleoside kinase). In contrast to extracts from duck spleen, the liver extracts showed a very low capacity to phosphorylate FLT, and this may explain the failure of FLT to prevent DHBV replication in vivo. These results suggest that new nucleoside analogues designed for treatment of hepatitis B should be evaluated with respect to phosphorylation by enzymes from non-dividing liver cells.

[1]  D. Lorne,et al.  Inhibition of duck hepatitis B virus replication in vitro by 2',3'-dideoxy-3'-azidothymidine and related compounds. , 1991, Acta virologica.

[2]  B. Oberg,et al.  Antiviral effects of 3'-fluorothymidine and 3'-azidothymidine in cynomolgus monkeys infected with simian immunodeficiency virus. , 1991, Journal of acquired immune deficiency syndromes.

[3]  B. Oberg,et al.  Inhibition of RNA- and DNA-dependent duck hepatitis B virus DNA polymerase activity by nucleoside and pyrophosphate analogs. , 1989, Antiviral research.

[4]  A. Karlsson,et al.  Hydroxyurea increases the phosphorylation of 3'-fluorothymidine and 3'-azidothymidine in CEM cells. , 1989, European journal of biochemistry.

[5]  R. Miller,et al.  Inhibition of duck hepatitis B virus replication by 2',3'-dideoxycytidine. A potent inhibitor of reverse transcriptase. , 1989, Gastroenterology.

[6]  D. Tyrrell,et al.  In vitro and in vivo comparison of the abilities of purine and pyrimidine 2',3'-dideoxynucleosides to inhibit duck hepadnavirus , 1989, Antimicrobial Agents and Chemotherapy.

[7]  Elias S. J. Arnér,et al.  Deoxycytidine kinase is constitutively expressed in human lymphocytes: consequences for compartmentation effects, unscheduled DNA synthesis, and viral replication in resting cells. , 1988, Experimental cell research.

[8]  S. Eriksson,et al.  Deoxycytidine kinase from human leukemic spleen: preparation and characterization of the homogeneous enzyme , 1988 .

[9]  A. Widell,et al.  Hepatitis B virus‐DNA in the serum of patients followed‐up longitudinally with acute and chronic hepatitis B , 1985, Journal of medical virology.

[10]  H. Thomas,et al.  A randomized study of the effects of adenine arabinoside 5′‐monophosphate (short or long courses) and lymphoblastoid interferon on hepatitis B virus replication , 1985, Hepatology.

[11]  P. H. Ellims,et al.  Human liver thymidine kinase. Purification and some properties of the enzyme. , 1980, Journal of Biological Chemistry.

[12]  J. Summers,et al.  Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus , 1980, Journal of virology.

[13]  Y. Cheng,et al.  Human deoxythymidine kinase. I. Purification and general properties of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia. , 1976, The Journal of biological chemistry.

[14]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[15]  W P Havens,et al.  Viral hepatitis. , 1970, The Medical clinics of North America.

[16]  D. Ives,et al.  Rapid determination of nucleoside kinase and nucleotidase activities with tritium-labeled substrates. , 1969, Analytical biochemistry.

[17]  G. Maley,et al.  Nucleotide interconversions. X. Deoxyribo- and ribonucleoside 5'-phosphate synthesis via a phosphotransferase reaction in chick embryo extracts. , 1963, Archives of biochemistry and biophysics.