Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. 243 Feb 86

Zidovudine is a potent in vitro inhibitor of human immunodeficiency virus (HIV) with varying efficacy against other retroviruses. With the exception of Epstein-Barr virus, all non-retroviruses tested so far have been insensitive to inhibition by zidovudine. In vivo, efficacy of zidovudine was demonstrated against Rauscher murine leukemia virus and feline leukemia virus. In both experimental models, infections completely resolved in animals when the drug was administered soon after infection. These results suggest that prompt initiation of zidovudine therapy, following a known exposure to HIV, should be considered. Mechanism studies show that zidovudine is phosphorylated to the monophosphate and diphosphate derivatives by the host cell cytosolic thymidine kinase and thymidylate kinase, respectively. The identity of the enzyme that phosphorylates zidovudine diphosphate is not known, but is believed to be the cellular nucleoside diphosphate kinase. The triphosphate of zidovudine appears to be the active form of the drug. Zidovudine triphosphate competes well with thymidine 5'-triphosphate for binding to the HIV reverse transcriptase and also functions as an alternative substrate. Incorporation of zidovudine monophosphate results in chain termination. However, it is not clear which mechanism, chain termination or competition with thymidine 5'-triphosphate, or a combination of both, is responsible for the inhibition of HIV replication.
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PMID:Spectrum of antiviral activity and mechanism of action of zidovudine. An overview. 304 82

3'-Azido-2',3'-dideoxy-5-iodouridine (AzIdUrd) and 3'-azido-2',3'-dideoxy-5-bromouridine (AzBdUrd), previously shown to be potent and selective inhibitors of human immunodeficiency virus replication in vitro were minimally toxic to the uninfected human lymphoid cell line H9 (IC50 = 197 and 590 microM, respectively). Both compounds strongly inhibited the incorporation of [3H]thymidine but not [3H]deoxyadenosine into DNA, and we observed no significant inhibition of [3H]uridine incorporation into RNA or [3H]amino acid incorporation into protein. Exposure of H9 cells to AzIdUrd or AzBdUrd (100 microM, 24 hr) and pulse-labeling with [3H]thymidine resulted in approximately 80% reduction in levels of tritiated dTMP, dTDP, and dTTP relative to control. [125I]AzIdUrd was phosphorylated rapidly in H9 cells with the monophosphate accounting for over 90% of total soluble radioactivity. A relatively low but stable level of AzIdUTP was maintained over a 12-hr period. [125I]AzIdUrd was phosphorylated by a cell free extract of H9 cells at a rate approximately three times that of thymidine and its phosphorylation was inhibited by excess thymidine. AzIdUrd was found to be a competitive inhibitor of cytosolic thymidine kinase with a Ki of 2.63 microM and AzIdUMP a weak competitive inhibitor of thymidylate kinase with a Ki of 55.3 microM. Both AzIdUTP and AzBdUTP were potent competitive inhibitors of HIV-1 reverse transcriptase (Ki = 0.028 and 0.043 microM, respectively) and relatively poor inhibitors of H9 cell DNA polymerase alpha (Ki = 42.0 and 42.7 microM, respectively). Thus, the high therapeutic index of these compounds is due to the sensitivity of the viral reverse transcriptase, coupled with the relative insensitivity of the host cell DNA polymerase alpha.
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PMID:Metabolism and mode of selective inhibition of human immunodeficiency virus replication by 3'-azido-2',3'-dideoxy-5-iodouridine and 3'-azido-2',3'-dideoxy-5-bromouridine. 767 40

The T-cell line Jurkat E6-1 was rendered resistant to zidovudine (AZT) in vitro by exposure to low but gradually increased concentrations of the drug. Biochemical pharmacology studies of [3H]AZT in the AZT-resistant T-cell lines showed a significant reduction of AZT phosphorylation to the mono-, di-, and triphosphate anabolites. Peripheral blood mononuclear cells (PBMCs) from pediatric patients with human immunodeficiency virus type 1 (HIV-1) infection showed a similar pattern of decreased AZT anabolism. Enzymatic studies with purified thymidine kinase (TK) preparations from these cell lines showed a gradual decline in Vmax related to their level of resistance to AZT. The Jurkat/AZT-20 and Jurkat/AZT-100 cells were studied in greater detail with reverse transcriptase/polymerase chain reaction (RT/PCR) cloned probes to determine possible molecular mechanisms of resistance to AZT. TK mRNA was significantly decreased (approximately 5- to 10-fold) in the AZT-resistant T-cell lines. Southern blot analyses indicated that there were no major rearrangements or deletions of the TK gene, but the 5' end of the gene in the AZT-resistant cells is highly methylated when compared to wild-type cells. No apparent differences were seen in thymidylate kinase (dTMPk) mRNA levels in the same T-cell lines. Thus the decreased expression of TK mRNA and resultant TK enzymatic activity is responsible for the observed reduction in the AZT anabolism in the resistant T-cell lines. Decreased T-cell TK activity could allow wild-type, AZT-sensitive HIV-1 to replicate in the presence of subinhibitory AZT triphosphate (AZT-TP) cellular concentrations enabling a genetic variant with drug resistance to emerge and outgrow the AZT-sensitive, wild-type virus.
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PMID:Development of zidovudine (AZT) resistance in Jurkat T cells is associated with decreased expression of the thymidine kinase (TK) gene and hypermethylation of the 5' end of human TK gene. 854 39

Nucleoside-based inhibitors of reverse transcriptase were the first drugs to be used in the chemotherapy of AIDS. After entering the cell, these substances are activated to their triphosphate form by cellular kinases, after which they are potent chain terminators for the growing viral DNA. The two main factors limiting their efficacy are probably interrelated. These are the insufficient degree of reduction of viral load at the commencement of treatment and the emergence of resistant variants of the virus. The reason for the relatively poor suppression of viral replication appears to be inefficient metabolic activation. Thus, for the most extensively used drug, 3'-azido-3'-deoxythymidine (AZT), whereas phosphorylation to the monophosphate is facile, the product is a very poor substrate for the next kinase in the cascade, thymidylate kinase. Because of this, although high concentrations of the monophosphate can be reached in the cell, the achievable concentration of the active triphosphate is several orders of magnitude lower. Determination of the structure of thymidylate kinase as a complex with AZT monophosphate (AZTMP) together with studies on the kinetics of its phosphorylation have now led to a detailed understanding of the reasons for and consequences of the poor substrate properties.
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PMID:The bottleneck in AZT activation. 946 Nov 64

Many antiviral drugs must be metabolized to their active form by cellular enzymes. Their antiviral activity may therefore be limited by an inefficient metabolism, leading to low intracellular concentration of the active form or to the accumulation of toxic intermediate metabolites. Gene transfer might be used to overcome such limitations by transducing a gene able to increase intracellular drug metabolism. To prove such a concept, we chose the well-studied paradigm of zidovudine (AZT) metabolism and anti-HIV activity. AZT-triphosphate is the active form of AZT, acting through inhibition of HIV reverse transcription. In human cells, the rate-limiting step for AZT phosphorylation is catalyzed by the thymidylate kinase. We thus tested the capacity of herpes simplex virus type 1 thymidine kinase, which possesses a thymidylate kinase activity, to improve AZT metabolism and antiviral activity. Our results show enhanced AZT phosphorylation in HSV-1 TK-expressing lymphoid and monoblastoid cells, which correlated with significantly improved antiviral activity against different strains of HIV-1. The antiviral activity of Foscarnet, another reverse transcriptase inhibitor that does not require phosphorylation, remained unchanged. These results suggest that gene transfer might be envisioned for genetic pharmacomodulation of antiviral drugs.
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PMID:Use of herpes simplex virus thymidine kinase to improve the antiviral activity of zidovudine. 928 20

Emergence of antiviral drug resistance is a major challenge to human immunodeficiency virus (HIV) therapy. The archetypal example of this problem is loss of antiviral activity of the nucleoside analogue 3'-azido-3'-deoxythymidine (AZT), caused by mutations in reverse transcriptase (RT), the viral polymerase. AZT resistance results from an imbalance between rates of AZT-induced proviral DNA chain termination and RT-induced excision of the chain-terminating nucleotide. Conversion of the AZT prodrug from its monophosphorylated to diphosphorylated form by human thymidylate kinase (TMPK) is inefficient, resulting in accumulation of the monophosphorylated AZT metabolite (AZT-MP) and a low concentration of the active triphosphorylated metabolite (AZT-TP). We reasoned that introduction of an engineered, highly active TMPK into T cells would overcome this functional bottleneck in AZT activation and thereby shift the balance of AZT activity sufficiently to block replication of formerly AZT-resistant HIV. Molecular engineering was used to link highly active, engineered TMPKs to the protein transduction domain of Tat for direct cell delivery. Combined treatment of HIV-infected T cells with AZT and these cell-permeable, engineered TMPKs restored AZT-induced repression of viral production. These results provide an experimental basis for the development of new strategies to therapeutically increase the intracellular concentrations of active nucleoside analogue metabolites as a means to overcome emerging drug resistance.
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PMID:Restoration of the antiviral activity of 3'-azido-3'-deoxythymidine (AZT) against AZT-resistant human immunodeficiency virus by delivery of engineered thymidylate kinase to T cells. 1855 37