Modulation of the phosphorylation of 3'-azidothymidine by the human thymidine kinase by 5'-aminothymidine and thymidine triphosphate.

Important note: Information in this article was accurate in 1995. The state of the art may have changed since the publication date.

Modulation of the phosphorylation of 3'-azidothymidine by the human thymidine kinase by 5'-aminothymidine and thymidine triphosphate.

Natl Conf Hum Retroviruses Relat Infect (2nd). 1995 Jan 29-Feb 2;:145. Unique Identifier : AIDSLINE AIDS/95920516
Vazquez-Padua MA; Gonzalez MT; Universidad Central del Caribe School of Medicine, Bayamon,; Puerto Rico.

Abstract: Thymidine (dThd) kinase is responsible for the initial phosphorylation and, thus, the activation of several anticancer and antiviral drugs, including 3'-azidothymidine (AZT, Zidovudine). It is regulated by a feedback inhibitory mechanism which involves the direct interaction of the end- product of the pathway; thymidine triphosphate (dTTP). Therefore, the activation of AZT could be regulated by the intracellular pools of dTTP. These have been reported to be elevated in dividing, activated cells. 5'-Aminothymidine (5'-AdThd) is a dThd analogue capable of antagonizing the dTTP-mediated feedback inhibition of the enzyme which could be used to augment the degree of activation of AZT in HIV- infected cells. To test this biochemical approach, we studied the interactions of AZT, 5'-AdThd and dTTP with preparations of thymidine kinase derived from U937 cells, a human monocytoid cell line. Exponentially growing cells were pelleted, homogenized and ultracentrifuged to obtain a clear supernatant with cytosolic thymidine kinase activity. In these preparations, 10 micromolar dTTP inhibited dThd kinase activity by greater than 90% when using either dThd or AZT as substrate. In the absence of dTTP, 5'-AdThd inhibited dThd (10 micromolar) or AZT (10 micromolar) phosphorylation with an IC50 of 10 and 5 micromolar, respectively. However, in the presence of 10 micromolar dTTP, 5'-AdThd enhanced the phosphorylation of both dThd and AZT compared to controls (10 micromolar dTTP alone) with maximum of 210% and 230% of control, respectively. Therefore, 5'-AdThd can stimulate the phosphorylation of AZT under conditions in which thymidine kinase could be significantly feedback inhibited.

Keywords: Cell Line Cytosol/ENZYMOLOGY Drug Interactions Human HIV Infections/METABOLISM Phosphorylation Substrate Specificity Thymidine/ANALOGS & DERIVATIVES/*PHARMACOLOGY Thymidine Kinase/ANTAGONISTS & INHIB/*METABOLISM Thymine Nucleotides/METABOLISM/*PHARMACOLOGY Zidovudine/*METABOLISM ABSTRACT
951230
M95C2623

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