CELLULAR PHOSPHORYLATION OF ANTI-HIV NUCLEOSIDES - ROLE OF NUCLEOSIDEDIPHOSPHATE KINASE

Nucleotide analogs are widely used in antiviral therapy and particular ly against AIDS. Delivered to the cell as nucleosides, they are phosph orylated into their active triphospho derivative form by cellular kina ses from the host. The last step in this series of phosphorylations is performed by nucleoside diphosphate (NDP) kinase, an enzyme that can use both purine or pyrimidine and oxy- or deoxynucleotides as substrat es. Using pure recombinant human NDP kinase type B (product of the gen e nm23-H2), we have characterized the kinetic parameters of several nu cleotide analogs for this enzyme. Contrary to what is generally assume d, diphospho- and triphospho- derivatives of azidothymidine as well as of dideoxyadenosine and dideoxythymidine are very poor substrates for NDP kinase. The rate of phosphorylation of these analogs varies betwe en 0.05% and 0.5%, as compared to the corresponding natural nucleotide , a result that is not due to the inability of the analogs to bind to the enzyme. Using the data from the high resolution crystal structure of NDP kinase, we provide an interpretation of these results based on the crucial role played by the 3'-OH moiety of the nucleotide in catal ysis.