Azidothymidine triphosphate is an inhibitor of both human immunodeficiency virus type 1 reverse transcriptase and DNA polymerase gamma

Abstract

The reverse transcriptase from human immunodeficiency virus type 1 was purified from the virus to near homogeneity. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA-synthesizing activity. Activated DNA as a heteropolymeric substrate was used as efficiently as was the homopolymeric substrate poly(rA)-oligo(dT). The Michaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Azidothymidine triphosphate, a well-known inhibitor of the enzyme, inhibited the enzyme competitively with respect to dTTP and noncompetitively with respect to the other nucleotides. Azidothymidine triphosphate acted as an efficient inhibitor of cellular DNA polymerase gamma, whereas other enzymes of eucaryotic DNA metabolism, namely, DNA polymerase alpha-primase and DNA polymerase beta, were not inhibited. This finding may explain why some acquired immunodeficiency syndrome patients suffer side effects during azidothymidine therapy.

Mitochondrial DNA Depletion, Oxidative Stress, and Mutation: Mechanisms 0f Dysfunction from Nucleoside Reverse Transcriptase Inhibitors

“…Incubation of AZT triphosphate with DNA pol-␥ in vitro resulted in mixed kinetics with a competitive K i of 1.8 Ϯ 0.2 M and a noncompetitive K i of 6.8 Ϯ 1.7 M. In studies by others, AZT triphosphate inhibited DNA pol-␥ activity by approximately 30% at 4 M, compared with 80% inhibition of reverse transcriptase activity at the same concentration in vitro (Konig et al, 1989). A mixed inhibition pattern also was determined for inhibition of cardiac DNA pol-␥ by D4T triphosphate, but with a much lower K i s (Lewis and Tankersley, 1995).…”

Simian immunodeficiency virus reverse transcriptase

“…It should be remembered at this point that the reverse transcriptase not only transcribes the viral RNA into DNA, but also replicates this DNA (after removal of the RNA by its accompanying RNase H) to yield double-stranded DNA which then can be integrated into the host cell genome. Our finding that the SIV,,, reverse transcriptase prefers magnesium over manganese as a cofactor for both RNA-and DNA-dependent DNA synthesis was not surprising since the same preference has been reported for the HIV-1 enzyme [23,28,291. It is not clear, however, why the optimal concentrations of this cofactor differ by a factor of five with the two different template primers.…”

“…First we investigated the influence of different pH values in the range 7.0-9.0 because the pH optimum of the HIV-1 enzyme differed from the pH normally chosen when measuring reverse transcriptases [23,24]. From the results illustrated in Fig.…”

“…Starting with assay conditions found to be optimal in our laboratory for assaying HIV-1 reverse transcriptase activity [23], we reexamined these assay conditions with purified SIV,,, reverse transcriptase in order to optimize the reaction parameters for this particular enzyme. First we investigated the influence of different pH values in the range 7.0-9.0 because the pH optimum of the HIV-1 enzyme differed from the pH normally chosen when measuring reverse transcriptases [23,24].…”

Inhibitory effect of 4‐(2,6‐dichlorophenyl)‐1,2,5‐thiadiazol‐3‐yl‐N‐methyl, N‐ethylcarbamate on replication of human immunodeficiency virus type 1 and the mechanism of action

“…Various compounds have been shown to selectively inhibit the activity of this enzyme (1) Iriphosphates act as DNA chain terminators and suppress the enzyme activity (3). However, they interact not only with HIV-1 RT but also with other retroviral RTs (4,5) and even with cellular DNA polymerases (6). Thus, serious side effects are often associated with a long-term administration of these compounds (7).…”