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Research ArticleArticle

The Mechanism of Phosphorylation of Anti-HIV D4T by Nucleoside Diphosphate Kinase

Benoit Schneider, Ricardo Biondi, Robert Sarfati, Fabrice Agou, Catherine Guerreiro, Dominique Deville-Bonne and Michel Veron
Molecular Pharmacology May 2000, 57 (5) 948-953;
Benoit Schneider
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Ricardo Biondi
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Robert Sarfati
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Fabrice Agou
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Catherine Guerreiro
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Dominique Deville-Bonne
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Michel Veron
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Abstract

The last step in the intracellular activation of antiviral nucleoside analogs is the addition of the third phosphate by nucleoside diphosphate (NDP) kinase resulting in the synthesis of the viral reverse transcriptase substrates. We have previously shown that dideoxynucleotide analogs and 3′-deoxy-3′-azidothymidine (AZT) as di- or triphosphate are poor substrates for NDP kinase. By use of protein fluorescence, we monitor the phosphotransfer between the enzyme and the nucleotide analog. Here, we have studied the reactivity of D4T (2′,3′-dideoxy-2′,3′-didehydrothymidine; stavudine) as di- (DP) or triphosphate (TP) at the pre-steady state. The catalytic efficiency of D4T-DP or -TP is increased by a factor of 10 compared with AZT-DP or -TP, respectively. We use an inactive mutant of NDP kinase to monitor the binding of a TP derivative, and show that the affinity for D4T-TP is in the same range as for the natural substrate deoxythymidine triphosphate, but is 30 times higher than for AZT-TP. Our results indicate that D4T should be efficiently phosphorylated after intracellular maturation of a prodrug into D4T-monophosphate.

Footnotes

  • Send reprint requests to: Dominique Deville-Bonne, Unité de Régulation Enzymatique des Activités Cellulaires, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. E-mail: ddeville{at}pasteur.fr

  • ↵1 This work was supported by funds from Agence Nationale de la Recherche contre le SIDA, the Association de la Recherche sur le Cancer, and from SIDACTION. R. B. was recipient of a fellowship from Fondation de la Recherche Médicale. This work has been presented as a poster presentation at Gordon Research Conferences, Purines, pyrimidines and related substances, Salve Regina University, University of Rhode Island, Kingston, RI, July 4–9,1999, and at the 7th Symposium of the European Society for the Study of Purine & Pyrimidine Metabolism in Man, Gdansk, Poland, September 15–19, 1999.

  • ↵2 Present address: Division of Signal Transduction Therapy, University of Dundee, UK.

  • Abbreviations:
    AZT
    3′-deoxy-3′-azidothymidine
    DP
    diphosphate
    MP
    monophosphate
    TP
    triphosphate
    Dd-NDPK
    Dictyostelium nucleoside diphosphate kinase
    D4T
    2′,3′-didehydro-2′,3′-dideoxythymidine
    ddTTP
    2′,3′-dideoxythymidine triphosphate
    ddNDP
    2′,3′-dideoxynucleotide diphosphate
    ddNTP
    2′,3′-dideoxynucleotide triphosphate
    NDP
    nucleoside diphosphate
    NDPK-A
    human nucleoside diphosphate kinase type A
    NTP
    nucleoside triphosphate
    PMSF
    phenylmethylsulfonyl fluoride, TK, thymidine kinase
    DTE
    dithioerythritol
    • Received September 29, 1999.
    • Accepted January 20, 2000.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 57 (5)
Molecular Pharmacology
Vol. 57, Issue 5
1 May 2000
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Research ArticleArticle

The Mechanism of Phosphorylation of Anti-HIV D4T by Nucleoside Diphosphate Kinase

Benoit Schneider, Ricardo Biondi, Robert Sarfati, Fabrice Agou, Catherine Guerreiro, Dominique Deville-Bonne and Michel Veron
Molecular Pharmacology May 1, 2000, 57 (5) 948-953;

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Research ArticleArticle

The Mechanism of Phosphorylation of Anti-HIV D4T by Nucleoside Diphosphate Kinase

Benoit Schneider, Ricardo Biondi, Robert Sarfati, Fabrice Agou, Catherine Guerreiro, Dominique Deville-Bonne and Michel Veron
Molecular Pharmacology May 1, 2000, 57 (5) 948-953;
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