RT Journal Article
SR Electronic
T1 The Mechanism of Phosphorylation of Anti-HIV D4T by Nucleoside Diphosphate Kinase
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 948
OP 953
VO 57
IS 5
A1 Benoit Schneider
A1 Ricardo Biondi
A1 Robert Sarfati
A1 Fabrice Agou
A1 Catherine Guerreiro
A1 Dominique Deville-Bonne
A1 Michel Veron
YR 2000
UL http://molpharm.aspetjournals.org/content/57/5/948.abstract
AB 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. The American Society for Pharmacology and Experimental Therapeutics