AZT Monophosphate Research Articles

Interactions of azidothymidine triphosphate with the cellular DNA polymerases alpha, delta, and epsilon and with DNA primase.

The interactions of azidothymidine triphosphate, the metabolically active form of the anti-AIDS drug azidothymidine (zidovudine), with the cellular DNA polymerases alpha, delta, and epsilon, as well as with the RNA primer-forming enzyme DNA primase were studied in vitro. DNA polymerase alpha was shown to incorporate azidothymidine monophosphate into a growing polynucleotide chain. This occurred 2000-fold slower than the incorporation of natural dTTP. Despite the ability of polymerase alpha to use azidothymidine triphosphate as an alternate substrate, this compound was only marginally inhibitory to the enzyme (Ki greater than 1 mM). Furthermore, the DNA primase activity associated with DNA polymerase alpha was barely inhibited by azidothymidine triphosphate (Ki greater than 1 mM). Inhibition was more pronounced for DNA polymerases delta and epsilon. The type of inhibition was competitive with respect to dTTP, with Ki values of 250 and 320 microM, respectively. No incorporation of azidothymidine monophosphate was detectable with these two DNA polymerases because their associated 3'- to 5'-exonuclease activities degraded primer molecules prior to any measurable elongation. Template-primer systems with a preformed 3'-azidothymidine-containing primer terminus inhibited the three replicative polymerases rather potently. DNA polymerase alpha was inhibited with a Ki of 150 nM and polymerases delta and epsilon with Ki values of 25 and 20 nM, respectively. The type of inhibition was competitive with respect to the unmodified substrate poly(dA).oligo(dT) for all DNA polymerases tested. Performed 3'-azidothymidine-containing primers hybridized to poly(dA) were rather resistant to degradation by the 3'- to 5'-exonuclease of DNA polymerases epsilon and more susceptible to the analogous activity that copurified with DNA polymerase delta. It is proposed that the repair of 3'-azidothymidine-containing primers might become rate-limiting for the process of DNA replication in cells that have been treated with azidothymidine triphosphate.

Intracellular Metabolism of 3′-Azidothymidine in Isolated Human Peripheral Blood Mononuclear Cells

Azidothymidine (AZT) inhibits the replication of human immunodeficiency virus and it is the only drug so far licensed for treatment of acquired immunodeficiency syndrome (AIDS). A prerequisite for its antiviral activity is phosphorylation by cellular nucleoside kinases to the mono-, di-, and triphosphate levels. This study determined the capacity of isolated peripheral blood mononuclear cells (PBMC), resting or mitogen stimulated, from 36 different people of whom 5 were HIV+, to phosphorylate and accumulate intracellular AZT nucleotides. We found a large variation in the amount of products formed between PBMC treated at different times from the same individual as well as between the PBMC from different individuals. Resting PBMC showed the largest interindividual variation and their levels of AZT nucleotides were about 60-150-fold lower than in activated PBMC. The intracellular half lives of azidothymidine mono-, di-, and triphosphates, constituting, on the average, 96-99.2, 0.7-1.8, and 0.4-2.7% of total nucleotides at 0.08-1.6 microM AZT, respectively, were also determined. In mitogen-stimulated PBMC it was approximately 2.5 +/- 0.6 h for all the azidothymidine metabolites. The half-life for intracellular azidothymidine monophosphate in resting PBMC from two individuals was determined to 1.5 +/- 0.2 h. There appeared to be no significant difference in the AZT metabolism in PBMC from HIV-positive or-negative persons. A relative decrease in the intracellular formation of AZTDP and AZTTP from AZTMP was observed at concentrations of AZTMP above 1 microM. This fact may explain why lowering the doses of AZT still gives therapeutically efficient levels of the active metabolite AZTTP.

Synthesis and anti-HIV Activity of Some Novel Phosphorodiamidate Derivatives of 3′-azido-3′-deoxythymidine (AZT)

The reaction of 3′-azido-3′-deoxythymidine (AZT) with phosphoryl chloride followed by amino acid methyl esters gave novel diamidate derivatives of AZT 5′-monophosphate (AZTMP). It was hoped that the 5′-phosphorodiamidates might act as membrane-soluble prodrugs of the bio-active free nucleotides of AZT. Five different amino acids were employed, covering a range of structures and polarities. The reaction was also conducted with propylamine, and with diethylamine. The derivatives were tested for their inhibitory effect on human immunodeficiency virus type 1 (HIV-1) proliferation in a human lymphoblastoid cell line. The amino acid derivatives were potent inhibitors of viral proliferation, small changes in structure leading to marked changes in activity.

Inhibition of human immunodeficiency virus 1 reverse transcriptase by 3′‐azidothymidine triphosphate

In the presence of oligo(dT).poly(rA) as primer-template, 3'-azidothymidine triphosphate (N3'(3)-ddTTP) is a substrate for human immunodeficiency virus 1 reverse transcriptase with an apparent Km value of 3.0 microM. This compares with an apparent Km for thymidine monophosphate (dTMP) incorporation of 2.5 microM. The apparent Vmax value for 3'-azidothymidine monophosphate (N3'(3)-ddTMP) incorporation is 50 times lower than that of dTMP incorporation. Kinetic analysis of the inhibition of reverse transcriptase by N3'(3)-ddTTP shows competitive inhibition with thymidine triphosphate (dTTP) with a Ki of 41 nM and an uncompetitive pattern of inhibition with template-primer having a Ki of 140 nM. This indicates incorporation of the analogue into the primer and inhibition of the enzyme by formation of a dead-end complex. The 3'-azidothymidine-terminated primer-template [N3'(3)-ddT-(dT)15.poly(rA)] is a potent competitive inhibitor versus primer-template with a Ki of 2.4 nM and shows mixed-type inhibition against dTTP with a Ki of 8 nM. The low inhibition constant for this chain-terminated primer suggests that such oligonucleotides can act as potent inhibitors of enzyme catalysis.

Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase.

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. & Broder, S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentrations above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5'-mono-, di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di- and triphosphate were low (less than or equal to 5 microM and less than or equal to 2 microM, respectively). Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent Km values of 2.9 microM and 3.0 microM. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 microM vs. 4.1 microM), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. IC50 (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase alpha. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase alpha. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.