Role of Nucleoside Diphosphate Kinase in the Activation of Anti-HIV Nucleoside Analogs

Abstract

Nucleoside analogs are currently used in antiretrovirus therapies. The best known example isAZT one of the first drug to be used for the treatment of AIDS. However, only the triphosphatederivatives of these compounds act as substrates of the viral reverse transcriptase. Since theydo not enter cells, nucleoside analogs are administered and phosphorylated by cellular kinases.The last step in this phosphorylation pathway is catalyzed by nucleoside diphosphate (NDP)kinase. The incorporation of the nucleoside triphosphates into nascent viral DNA chain resultsin termination of the elongation process. We have performed kinetics studies of thephosphorylation reaction by NDP kinase of dideoxynucleoside diphosphates such as 2′,3′-dideoxy-3′-azidothymidine diphosphate (AZT-DP) and 2′,3′-dideoxy-2′,3′-didehydrothymidinediphosphate (d4T-DP). We show that the catalytic efficiency is strongly decreased and, therefore,that the reaction step catalyzed by NDP kinase constitutes a bottleneck in the processingpathway of anti-HIV compounds. In addition, the affinity of the analogs in the absence ofcatalysis was determined using a catalytically inactive NDP kinase mutant, showing a reductionof affinity by a factor of 2 to 30, depending on the analog. The structure of NDP kinaseprovides a structural explanation for these results. Indeed, all nucleoside analogs acting aschain terminators must lack a 3′-OH in the nucleotide deoxyribose. Unfortunately, this samesubstitution is detrimental for their capacity to be phosphorylated by NDP kinase. This definesthe framework for the design of new nucleoside analogs with increased efficiency inantiretroviral therapies.