Role of nucleoside diphosphate kinase in the activation of anti-HIV nucleoside analogs

Nucleoside analogs are currently used in antiretrovirus therapies. The best known example is AZT one of the first drug to be used for the treatment of AIDS. However, only the triphosphate derivatives of these compounds act as substrates of the viral reverse transcriptase. Since they do not enter cel ls, nucleoside analogs are administered and phosphorylated by cellular kina ses. The last step in this phosphorylation pathway is catalyzed by nucleosi de diphosphate (NDP) kinase. The incorporation of the nucleoside triphospha tes into nascent viral DNA chain results in termination of the elongation p rocess. We have performed kinetics studies of the phosphorylation reaction by NDP kinase of dideoxynucleoside diphosphates such as 2',3'-dideoxy-3'-az idothymidine diphosphate (AZT-DP) and 2',3'-dideoxy-2',3'-didehydrothymidin e diphosphate (d4T-DP). We show that the catalytic efficiency is strongly d ecreased and, therefore, that the reaction step catalyzed by NDP kinase con stitutes a bottleneck in the processing pathway of anti-HIV compounds. In a ddition, the affinity of the analogs in the absence of catalysis was determ ined using a catalytically inactive NDP kinase mutant, showing a reduction of affinity by a factor of 2 to 30, depending on the analog. The structure of NDP kinase provides a structural explanation for these results. Indeed, all nucleoside analogs acting as chain terminators must lack a 3'-OH in the nucleotide deoxyribose. Unfortunately this same substitution is detrimenta l for their capacity to be phosphorylated by NDP kinase. This defines the f ramework for the design of new nucleoside analogs with increased efficiency in antiretroviral therapies.