The Mechanism of Phosphorylation of Anti-HIV D4T by Nucleoside Diphosphate Kinase1
- Benoit Schneider1,
- Ricardo Biondi2,1,
- Robert Sarfati2,
- Fabrice Agou1,
- Catherine Guerreiro2,
- Dominique Deville-Bonne1 and
- Michel Veron1
- 1Unité de Régulation Enzymatique des Activités Cellulaires, Centre National de la Recherche Scientifique, Unitéde Recherche Associée 1773 (B.S., R.B., F.A., D.D-B., M.V.), and2Unité de Chimie Organique, Centre National de la Recherche Scientifique, Unité de Recherche Associée 2128, 558 (R.S., C.G.), Institut Pasteur, Paris, France
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
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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
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↵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.
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↵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
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- Received September 29, 1999.
- Accepted January 20, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
