Characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity

Abstract

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.

Fig. 1.

Chemical structures of thymidine, ganciclovir (GCV) and acyclovir (ACV).

Fig. 2.

Deduced amino acid sequence of three HSV-1 TK mutants derived from the semirandom library. The top line shows the amino acid sequence of the wild-type HSV-1 thymidine kinase (residues 158–174) with the deduced amino acid substitutions of mutants SR11, SR26, and SR39 shown underneath. The underlined residues represent the codons that were randomized in the creation of the library. All Herpeviridae thymidine kinases contain six highly conserved motifs; two such motifs are boxed (Balsubramaniam et al. 1990). All mutants were identified on the basis of increased sensitivity to GCV and/or ACV. Standard single letter amino acid nomenclature is used. The (-) denotes identity with the wild-type amino acid sequence.