1887

Abstract

The triphosphorylated form of the nucleoside analogue AZT (AZTTP) acts as a chain terminator during reverse transcription of the human immunodeficiency virus (HIV) genome. The bottleneck in the conversion of AZT to AZTTP is the phosphorylation of AZT monophosphate (AZTMP) by cellular thymidylate kinase. Human thymidylate kinase was engineered to exhibit highly improved activity for AZTMP to AZTDP conversion. It was demonstrated here that genetically modified human cells transiently expressing these enzyme variants show more than 10-fold higher intracellular concentrations of AZTDP and AZTTP. Stable clones expressing these enzymes appear to phosphorylate AZTMP less efficiently, but first experiments indicate they are still more potent in HIV inhibition than the parental cells. It was proposed that the concept of introducing into human cells a catalytically improved human enzyme, rather than an enzyme of viral, bacterial or yeast origin, may serve as a paradigm for ameliorating the metabolic activation of an established drug.

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2005-03-01
2019-11-21
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References

  1. Balzarini, J., Degreve, B. & De Clercq, E. ( 1998; ). Improving AZT efficacy. Nat Med 4, 132.
    [Google Scholar]
  2. Bonini, C., Ferrari, G., Verzeletti, S. & 8 other authors ( 1997; ). HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 276, 1719–1724.[CrossRef]
    [Google Scholar]
  3. Brundiers, R., Lavie, A., Veit, T., Reinstein, J., Schlichting, I., Ostermann, N., Goody, R. S. & Konrad, M. ( 1999; ). Modifying human thymidylate kinase to potentiate azidothymidine activation. J Biol Chem 274, 35289–35292.[CrossRef]
    [Google Scholar]
  4. Caruso, M. & Klatzmann, D. ( 1994; ). Genetically controlled pharmacomodulation: application to the treatment of HIV infection. C R Acad Sci III 317, 1027–1030.
    [Google Scholar]
  5. Charneau, P., Alizon, M. & Clavel, F. ( 1992; ). A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication. J Virol 66, 2814–2820.
    [Google Scholar]
  6. Cohen, J. L., Boyer, O. & Klatzmann, D. ( 1999; ). Would suicide gene therapy solve the ‘T-cell dilemma’ of allogeneic bone marrow transplantation? Immunol Today 20, 172–176.[CrossRef]
    [Google Scholar]
  7. Cosset, F. L., Takeuchi, Y., Battini, J. L., Weiss, R. A. & Collins, M. K. ( 1995; ). High-titer packaging cells producing recombinant retroviruses resistant to human serum. J Virol 69, 7430–7436.
    [Google Scholar]
  8. Furman, P. A., Fyfe, J. A., St Clair, M. H. & 8 other authors ( 1986; ). Phosphorylation of 3′-azido-3′-deoxythymidine and selective interaction of the 5′-triphosphate with human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci U S A 83, 8333–8337.[CrossRef]
    [Google Scholar]
  9. Goldschmidt, V. & Marquet, R. ( 2004; ). Primer unblocking by HIV-1 reverse transcriptase and resistance to nucleoside RT inhibitors (NRTIs). Int J Biochem Cell Biol 36, 1687–1705.[CrossRef]
    [Google Scholar]
  10. Goody, R. S., Müller, B. & Restle, T. ( 1991; ). Factors contributing to the inhibition of HIV reverse transcriptase by chain-terminating nucleotides in vitro and in vivo. FEBS Lett 291, 1–5.[CrossRef]
    [Google Scholar]
  11. Guettari, N., Loubière, L., Brisson, E. & Klatzmann, D. ( 1997; ). Use of herpes simplex virus thymidine kinase to improve the antiviral activity of zidovudine. Virology 235, 398–405.[CrossRef]
    [Google Scholar]
  12. Huang, S. H., Tang, A., Drisco, B., Zhang, S. Q., Seeger, R., Li, C. & Jong, A. ( 1994; ). Human dTMP kinase: gene expression and enzymatic activity coinciding with cell cycle progression and cell growth. DNA Cell Biol 13, 461–471.[CrossRef]
    [Google Scholar]
  13. Kirn, D., Niculescu-Duvaz, I., Hallden, G. & Springer, C. J. ( 2002; ). The emerging fields of suicide gene therapy and virotherapy. Trends Mol Med 8, S68–S73.[CrossRef]
    [Google Scholar]
  14. Kremmer, T., Paulik, E., Boldizsar, M. & Holenyi, I. ( 1989; ). Application of the fast protein liquid chromatographic system and MonoQ HR 5/5 anion exchanger to the separation of nucleotides. J Chromatogr 493, 45–52.[CrossRef]
    [Google Scholar]
  15. Lavie, A., Schlichting, I., Vetter, I. R., Konrad, M., Reinstein, J. & Goody, R. S. ( 1997a; ). The bottleneck in AZT activation. Nat Med 3, 922–924.[CrossRef]
    [Google Scholar]
  16. Lavie, A., Vetter, I. R., Konrad, M., Goody, R. S., Reinstein, J. & Schlichting, I. ( 1997b; ). Structure of thymidylate kinase reveals the cause behind the limiting step in AZT activation. Nat Struct Biol 4, 601–604.[CrossRef]
    [Google Scholar]
  17. Lavie, A., Konrad, M., Brundiers, R., Goody, R. S., Schlichting, I. & Reinstein, J. ( 1998a; ). Crystal structure of yeast thymidylate kinase complexed with the bisubstrate inhibitor P1-(5′-adenosyl) P5-(5′-thymidyl) pentaphosphate (TP5A) at 2.0 A resolution: implications for catalysis and AZT activation. Biochemistry 37, 3677–3686.[CrossRef]
    [Google Scholar]
  18. Lavie, A., Ostermann, N., Brundiers, R., Goody, R. S., Reinstein, J., Konrad, M. & Schlichting, I. ( 1998b; ). Structural basis for efficient phosphorylation of 3′-azidothymidine monophosphate by Escherichia coli thymidylate kinase. Proc Natl Acad Sci U S A 95, 14045–14050.[CrossRef]
    [Google Scholar]
  19. Ostermann, N., Lavie, A., Padiyar, S., Brundiers, R., Veit, T., Reinstein, J., Goody, R. S., Konrad, M. & Schlichting, I. ( 2000a; ). Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP. J Mol Biol 304, 43–53.[CrossRef]
    [Google Scholar]
  20. Ostermann, N., Schlichting, I., Brundiers, R., Konrad, M., Reinstein, J., Veit, T., Goody, R. S. & Lavie, A. ( 2000b; ). Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate. StructureFold Des 8, 629–642.[CrossRef]
    [Google Scholar]
  21. Rossi, F. M. & Blau, H. M. ( 1998; ). Recent advances in inducible gene expression systems. Curr Opin Biotechnol 9, 451–456.[CrossRef]
    [Google Scholar]
  22. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual. 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  23. Zimmerman, T. P., Mahony, W. B. & Prus, K. L. ( 1987; ). 3′-azido-3′-deoxythymidine. An unusual nucleoside analogue that permeates the membrane of human erythrocytes and lymphocytes by nonfacilitated diffusion. J Biol Chem 262, 5748–5754.
    [Google Scholar]
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