Characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity.
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.
Department of Pharmaceutical Sciences, Washington State University, Pullman, Washington 99164-6534, USA.
Mark S
MS
Kokoris
Margaret E
ME
Black
D000212:Acyclovir / Q000378:metabolism*; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000378:metabolism; D000998:Antiviral Agents / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D015774:Ganciclovir / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D011355:Prodrugs / Q000378:metabolism; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D000998:Antiviral Agents; D011355:Prodrugs; D013937:Thymidine Kinase; D015774:Ganciclovir; D000212:Acyclovir