Use of Herpes Simplex Virus Thymidine Kinase to Improve the Antiviral Activity of Zidovudine

Guettari, N.; Loubière, Laurence; Brisson, E.; Klatzmann, David

doi:10.1006/viro.1997.8706

Cited by 22 publications

(16 citation statements)

References 25 publications

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“…Some viral TKs (e.g., varicella zoster and herpes simplex virus type 2) also possess a TMPK activity as part of the same TK protein or have a TMPK enzyme with broader substrate specificity than that of human TMPK (44,45 ). AZTMP generated in this manner can be further phosphorylated to form AZT 5Ј-diphosphate, which does not significantly bind to the anti-AZTMP antibody (33 ).…”

Section: Discussionmentioning

confidence: 99%

Sensitive Nonradiometric Method for Determining Thymidine Kinase 1 Activity

Öhrvik¹,

Lindh²,

Einarsson³

et al. 2004

Clinical Chemistry

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Section: Discussionmentioning

confidence: 99%

Sensitive Nonradiometric Method for Determining Thymidine Kinase 1 Activity

Öhrvik¹,

Lindh²,

Einarsson³

et al. 2004

Clinical Chemistry

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“…that can phosphorylate AZT-MP but not dTMP. Earlier attempts to improve the inhibitory effect of AZT on HIV replication (13), by transfecting human cells with the herpes simplex virus thymidine kinase (which is several orders of magnitude slower in AZT-MP phosphorylation than our best human TmpK variant), have been partially successful, demonstrating the feasibility of this approach.…”

Section: Improving Azt-mp Phosphorylation By Thymidylate Kinase 35290mentioning

confidence: 99%

Modifying Human Thymidylate Kinase to Potentiate Azidothymidine Activation

Brundiers¹,

Lavie²,

Veit³

et al. 1999

Journal of Biological Chemistry

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Based on the knowledge of the crystal structures of yeast and Escherichia coli thymidylate kinases (TmpKs) and the observation that TmpK from E. coli can phosphorylate azidothymidine monophosphate (AZT-MP) much more efficiently than either the yeast or the highly homologous human enzyme, we have engineered yeast and human TmpKs to obtain enzymes that have dramatically improved AZT-MP phosphorylation properties. These modified enzymes have properties that make them attractive candidates for gene therapeutic approaches to potentiating the action of AZT as an inhibitor of human immunodeficiency virus (HIV) replication. In particular, insertion of the lid domain of the bacterial TmpK into the human enzyme results in a pronounced change of the acceptance of AZT-MP such that it is now phosphorylated even faster than TMP.

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“…Cells treated with AZT accumulate the toxic AZT-MP (1-3) in millimolar concentration (4,5), and thus the active AZT triphosphorylated metabolite is at very low concentration. This situation is only slightly improved upon coinfection with herpes simplex virus I thymidine kinase-carrying vectors that efficiently phosphorylate the antiviral drugs acyclovir or ganciclovir but not AZT or AZT-MP (6,7).…”

mentioning

confidence: 99%

Structural basis for efficient phosphorylation of 3′-azidothymidine monophosphate by Escherichia coli thymidylate kinase

Lavie

Ostermann

Brundiers

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

100

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The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P 1 -(5-adenosyl)-P 5 -(5-thymidyl)pentaphosphate and P 1 -(5-adenosyl)P 5 -[5-(3-azido-3-deoxythymidine)] pentaphosphate have been solved to 2.0-Å and 2.2-Å resolution, respectively. The overall structure of the bacterial TmpK is very similar to that of yeast TmpK. In contrast to the human and yeast TmpKs, which phosphorylate 3-azido-3-deoxythymidine 5-monophosphate (AZT-MP) at a 200-fold reduced turnover number (k cat ) in comparison to the physiological substrate dTMP, reduction of k cat is only 2-fold for the bacterial enzyme. The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and E. coli TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate. Yeast TmpK interacts with the 3-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent. In E. coli TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3-hydroxyl of dTMP. This different mode of interaction between the P loop carboxylic acid with the 3 substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the E. coli enzyme without significant P loop movement. In addition, although the yeast enzyme uses Arg-15 (a glycine in E. coli) to stabilize the transition state, E. coli seems to use Arg-153 from a region termed Lid instead. Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase.Phosphorylation of AZT-MP by thymidylate kinase (TmpK; EC 2.7.4.9, ATP:dTMP phosphotransferase) has been implicated as the rate-limiting step in the activation pathway of the anti-HIV prodrug 3Ј-azido-3Ј-deoxythymidine (AZT). AZT must be phosphorylated by cellular enzymes to azidothymidine triphosphate before it can exert its antiviral effect by inhibiting HIV reverse transcriptase and acting as a chain terminator of nascent DNA strands. The bottleneck of AZT activation lies in the second phosphorylation step, the step that adds a phosphate group to azidothymidine monophosphate (AZT-MP) to yield azidothymidine diphosphate and is catalyzed by TmpK. Cells treated with AZT accumulate the toxic AZT-MP (1-3) in millimolar concentration (4, 5), and thus the active AZT triphosphorylated metabolite is at very low concentration. This situation is only slightly improved upon coinfection with herpes simplex virus I thymidine kinase-carrying vectors that efficiently phosphorylate the antiviral drugs acyclovir or ganciclovir but not AZT or AZT-MP (6, 7).Therefore, we set out to understand the structural basis for the slow activation of AZT-MP. The structures of the yeast TmpK (TmpK yeast ) complexed with either the physiological substrate d...

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Use of Herpes Simplex Virus Thymidine Kinase to Improve the Antiviral Activity of Zidovudine

Cited by 22 publications

References 25 publications

Sensitive Nonradiometric Method for Determining Thymidine Kinase 1 Activity

Sensitive Nonradiometric Method for Determining Thymidine Kinase 1 Activity

Modifying Human Thymidylate Kinase to Potentiate Azidothymidine Activation

Structural basis for efficient phosphorylation of 3′-azidothymidine monophosphate by Escherichia coli thymidylate kinase

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