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Characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or
Mark S Kokoris1, Margaret E Black
1Department of Pharmaceutical Sciences, Washington State University, Pullman, Washington 99164-6534, USA.
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.
Insights
Researchers engineered novel Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) variants. These enhanced "prodrug kinases" improve cancer gene therapy by enabling lower, less toxic prodrug doses.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Gene Therapy
Background:
- Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) is a key enzyme in cancer gene therapy, activating prodrugs.
- Current prodrugs like ganciclovir (GCV) have limitations due to toxicity and efficacy.
- Developing improved TK enzymes is crucial for enhancing gene therapy outcomes.
Purpose of the Study:
- To engineer novel HSV-1 TK variants with altered substrate specificities and kinetic properties.
- To identify mutant TK enzymes that can efficiently phosphorylate less toxic prodrugs at lower concentrations.
- To enhance the efficacy and safety of cancer gene therapy strategies.
Main Methods:
- Random mutagenesis of HSV-1 TK was performed.
- Negative selection in Escherichia coli identified active TK variants against ganciclovir and acyclovir.
- Novel affinity chromatography was used to purify wild-type and mutant TK enzymes.
- Kinetic parameters (Km) for thymidine, ganciclovir, and acyclovir were determined for purified enzymes.
Main Results:
- Seven HSV-1 TK variants with multiple amino acid substitutions were identified.
- Mutant SR39 showed a 14-fold decrease in Km for ganciclovir.
- Mutant SR26 exhibited a significant 124-fold decrease in Km for acyclovir.
- Purified mutant enzymes demonstrated altered substrate affinities.
Conclusions:
- Engineered HSV-1 TK variants, termed "prodrug kinases," show enhanced activity towards acyclovir and ganciclovir.
- These novel enzymes facilitate the use of less toxic prodrugs at significantly lower concentrations.
- The improved TK variants hold promise for advancing cancer gene therapy by increasing efficacy and reducing side effects.