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The role of herpes simplex virus-1 thymidine kinase alanine 168 in substrate specificity.

Candice L W, Django S, Margaret E B - Open Biochem J (2008)

Bottom Line: After administration, the prodrug is selectively converted to a toxic drug by the suicide gene product thereby bringing about the eradication of the cancer cells.A major drawback to this therapy is the low activity the enzyme displays towards the prodrugs, requiring high prodrug doses that result in adverse side effects.While these mutants contain multiple amino acid substitutions, molecular modeling suggests that substitutions at alanine 168 (A168) may be responsible for the observed increase in prodrug sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Washington State University, Pullman, WA.

ABSTRACT
Herpes simplex virus type 1 (HSV) thymidine kinase (TK) has been widely used in suicide gene therapy for the treatment of cancer due to its broad substrate specificity and the inability of the endogenous human TK to phosphorylate guanosine analogs such as ganciclovir (GCV). The basis of suicide gene therapy is the introduction of a gene that encodes a prodrug-activating enzyme into tumor cells. After administration, the prodrug is selectively converted to a toxic drug by the suicide gene product thereby bringing about the eradication of the cancer cells. A major drawback to this therapy is the low activity the enzyme displays towards the prodrugs, requiring high prodrug doses that result in adverse side effects. Earlier studies revealed two HSV TK variants (SR39 and mutant 30) derived by random mutagenesis with enhanced activities towards GCV in vitro and in vivo. While these mutants contain multiple amino acid substitutions, molecular modeling suggests that substitutions at alanine 168 (A168) may be responsible for the observed increase in prodrug sensitivity. To evaluate this, site-directed mutagenesis was used to individually substitute A168 with phenylalanine or tyrosine to reflect the mutations found in SR39 and mutant 30, respectively. Additionally, kinetic parameters and the ability of these mutants to sensitize tumor cells to GCV in comparison to wild-type thymidine kinase were determined.

No MeSH data available.


Related in: MedlinePlus

Mutant enzyme relative specificity differences compared to wild-type enzyme for ganciclovir. Using the equation, (kcat/Km (GCV))/((kcat/Km (GCV)) + (kcat/Km (dT))),the relative specificities were calculated. This equation takes into account the presence of endogenous thymidine that could compete with prodrug for the active site. The data are plotted as the fold increase from wild-type enzyme. Mutant 30 and SR39values have been taken from Kokoris et al. [17] and [22], respectively.
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Figure 2: Mutant enzyme relative specificity differences compared to wild-type enzyme for ganciclovir. Using the equation, (kcat/Km (GCV))/((kcat/Km (GCV)) + (kcat/Km (dT))),the relative specificities were calculated. This equation takes into account the presence of endogenous thymidine that could compete with prodrug for the active site. The data are plotted as the fold increase from wild-type enzyme. Mutant 30 and SR39values have been taken from Kokoris et al. [17] and [22], respectively.

Mentions: Overall, the A168Y mutation in HSV TK causes a dramatic reduction in kcat values for both dT and GCV whereas the Km values remain unchanged. In comparison to kinetic values previously obtained for mutant 30 [17], the A168Y single mutation does not lead to as great an increase in activity towards GCV (Fig. 2). Mutant 30 has a 67-fold increase in relative specificity for GCV, while the A168Y mutant has only a 5.3-fold increase in relative specificity for GCV.


The role of herpes simplex virus-1 thymidine kinase alanine 168 in substrate specificity.

Candice L W, Django S, Margaret E B - Open Biochem J (2008)

Mutant enzyme relative specificity differences compared to wild-type enzyme for ganciclovir. Using the equation, (kcat/Km (GCV))/((kcat/Km (GCV)) + (kcat/Km (dT))),the relative specificities were calculated. This equation takes into account the presence of endogenous thymidine that could compete with prodrug for the active site. The data are plotted as the fold increase from wild-type enzyme. Mutant 30 and SR39values have been taken from Kokoris et al. [17] and [22], respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570551&req=5

Figure 2: Mutant enzyme relative specificity differences compared to wild-type enzyme for ganciclovir. Using the equation, (kcat/Km (GCV))/((kcat/Km (GCV)) + (kcat/Km (dT))),the relative specificities were calculated. This equation takes into account the presence of endogenous thymidine that could compete with prodrug for the active site. The data are plotted as the fold increase from wild-type enzyme. Mutant 30 and SR39values have been taken from Kokoris et al. [17] and [22], respectively.
Mentions: Overall, the A168Y mutation in HSV TK causes a dramatic reduction in kcat values for both dT and GCV whereas the Km values remain unchanged. In comparison to kinetic values previously obtained for mutant 30 [17], the A168Y single mutation does not lead to as great an increase in activity towards GCV (Fig. 2). Mutant 30 has a 67-fold increase in relative specificity for GCV, while the A168Y mutant has only a 5.3-fold increase in relative specificity for GCV.

Bottom Line: After administration, the prodrug is selectively converted to a toxic drug by the suicide gene product thereby bringing about the eradication of the cancer cells.A major drawback to this therapy is the low activity the enzyme displays towards the prodrugs, requiring high prodrug doses that result in adverse side effects.While these mutants contain multiple amino acid substitutions, molecular modeling suggests that substitutions at alanine 168 (A168) may be responsible for the observed increase in prodrug sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Washington State University, Pullman, WA.

ABSTRACT
Herpes simplex virus type 1 (HSV) thymidine kinase (TK) has been widely used in suicide gene therapy for the treatment of cancer due to its broad substrate specificity and the inability of the endogenous human TK to phosphorylate guanosine analogs such as ganciclovir (GCV). The basis of suicide gene therapy is the introduction of a gene that encodes a prodrug-activating enzyme into tumor cells. After administration, the prodrug is selectively converted to a toxic drug by the suicide gene product thereby bringing about the eradication of the cancer cells. A major drawback to this therapy is the low activity the enzyme displays towards the prodrugs, requiring high prodrug doses that result in adverse side effects. Earlier studies revealed two HSV TK variants (SR39 and mutant 30) derived by random mutagenesis with enhanced activities towards GCV in vitro and in vivo. While these mutants contain multiple amino acid substitutions, molecular modeling suggests that substitutions at alanine 168 (A168) may be responsible for the observed increase in prodrug sensitivity. To evaluate this, site-directed mutagenesis was used to individually substitute A168 with phenylalanine or tyrosine to reflect the mutations found in SR39 and mutant 30, respectively. Additionally, kinetic parameters and the ability of these mutants to sensitize tumor cells to GCV in comparison to wild-type thymidine kinase were determined.

No MeSH data available.


Related in: MedlinePlus