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FOR THE RECORD

Characterization of Herpes Simplex Virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity

¹ Department of Pharmaceutical Sciences, Washington State University, Pullman, Washington 99164-6534, USA
² Chiroscience R&D, Inc., Bothell, Washington 98021, USA

Reprint requests to: Margaret E. Black, Department of Pharmaceutical Sciences, P.O. Box 646534, Washington State University, Pullman, WA 99164-6534, USA; e-mail: blackm{at}mail.wsu.edu; fax: (509) 335-5902.

Herpes Simplex Virus type 1 (HSV-1) thymidine kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer. Tumor cells that express HSV-1 thymidine kinase are rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme. Although ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug. From a random mutagenesis library, seven thymidine kinase variants containing multiple amino acid substitutions were identified on the basis of activity towards ganciclovir and acyclovir based on negative selection in Escherichia coli. Using a novel affinity chromatography column, three mutant enzymes and the wild-type TK were purified to homogeneity and their kinetic parameters for thymidine, ganciclovir, and acyclovir determined. With ganciclovir as the substrate, one mutant (mutant SR39) demonstrated a 14-fold decrease in K_m compared to the wild-type enzyme. The most dramatic change is displayed by mutant SR26, with a 124-fold decrease in K_m with acyclovir as the substrate. Such new "prodrug kinases" could provide benefit to ablative gene therapy by now making it feasible to use the relatively nontoxic acyclovir at nanomolar concentrations or ganciclovir at lower, less immunosuppressive doses.

Keywords: Herpes Simplex Virus type 1 thymidine kinase; ganciclovir; acyclovir; random sequence mutagenesis

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