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Selective abolishment of pyrimidine nucleoside kinase activity of herpes simplex virus type 1 thymidine kinase by mutation of alanine-167 to tyrosine.

Abstract
Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that markedly differs from mammalian nucleoside kinases in terms of substrate specificity. It recognizes both pyrimidine 2'-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an attempt to modify this specificity, an HSV-1 TK mutant enzyme containing an alanine-to-tyrosine mutation at amino acid position 167 was constructed. Compared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme was heavily compromised in phosphorylating pyrimidine nucleosides such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV) and lobucavir was only reduced approximately 2-fold. Moreover, a markedly decreased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was observed. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene were extremely sensitive to the cytostatic effects of antiherpetic pyrimidine [i.e., (E)-5-(2-bromovinyl)-2'-deoxyuridine] and purine (i.e., GCV) nucleoside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the osteosarcoma cells to a variety of purine nucleoside analogs, whereas there was no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a therapeutic advantage in an in vivo setting due to the markedly reduced dThd competition with GCV for phosphorylation by the HSV-1 TK.
AuthorsB Degrève, R Esnouf, E De Clercq, J Balzarini
JournalMolecular pharmacology (Mol Pharmacol) Vol. 58 Issue 6 Pg. 1326-32 (Dec 2000) ISSN: 0026-895X [Print] United States
PMID11093770 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Antiviral Agents
  • Purine Nucleosides
  • Pyrimidine Nucleosides
  • Tyrosine
  • Thymidine Kinase
  • Alanine
  • Ganciclovir
Topics
  • Alanine (genetics, metabolism)
  • Amino Acid Substitution
  • Antiviral Agents (metabolism, pharmacology)
  • Bone Neoplasms
  • Cell Division (drug effects)
  • Ganciclovir (metabolism, pharmacology)
  • Herpesvirus 1, Human (drug effects, enzymology, genetics)
  • Humans
  • Osteosarcoma
  • Phosphorylation
  • Point Mutation
  • Purine Nucleosides (metabolism)
  • Pyrimidine Nucleosides (metabolism)
  • Thymidine Kinase (genetics, metabolism)
  • Transduction, Genetic
  • Tumor Cells, Cultured
  • Tyrosine (genetics, metabolism)

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