Nucleoside analogues, used in HIV-therapy, need to be phosphorylated by cellular enzymes in order to become potential substrates for HIV reverse transcriptase. After incorporation into the viral DNA chain, because of lacking of their 3'-hydroxyl groups, they stop the elongation process and lead to the death of the virus. Phosphorylation of the HIV-drug derivative, tenofovir monophosphate was tested with the recombinant mammalian nucleoside diphosphate kinase (NDPK), 3-phosphoglycerate kinase (PGK), creatine kinase (CK) and pyruvate kinase (PK). Among them, only CK was found to phosphorylate tenofovir monophosphate with a reasonable rate (about 45-fold lower than with its natural substrate, ADP), while PK exhibits even lower, but still detectable activity (about 1000-fold lower compared to the value with ADP). On the other hand, neither NDPK nor PGK has any detectable activity on tenofovir monophosphate. The absence of activity with PGK is surprising, since the drug tenofovir competitively inhibits both CK and PGK towards their nucleotide substrates, with similar inhibitory constants, K(I) of 2.9 and 4.8mM, respectively. Computer modelling (docking) of tenofovir mono- or diphosphate forms to these four kinases suggests that the requirement of large-scale domain closure for functioning (as for PGK) may largely restrict their applicability for phosphorylation/activation of pro-drugs having a structure similar to tenofovir monophosphate.
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