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Vol. 57, Issue 5, 948-953, May 2000

The Mechanism of Phosphorylation of Anti-HIV D4T by Nucleoside Diphosphate Kinase1

Benoit Schneider, Ricardo Biondi,2 Robert Sarfati, Fabrice Agou, Catherine Guerreiro, Dominique Deville-Bonne, and Michel Veron

Unité de Régulation Enzymatique des Activités Cellulaires, Centre National de la Recherche Scientifique, Unité de Recherche Associée 1773 (B.S., R.B., F.A., D.D-B., M.V.), and Unité de Chimie Organique, Centre National de la Recherche Scientifique, Unité de Recherche Associée 2128, 558 (R.S., C.G.), Institut Pasteur, Paris, France

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.

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.

Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics


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