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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

Sensitivity of TK-expressing rat C6 constructs to GCV. Pools of stable transfectants containing vector only (pUB), wild-type TK, A168F, A168Y, Mutant 30 and SR39 were constructed and transfected in rat C6 glioma cells and evaluated for prodrug sensitivity as described in Materials and Methods. After six days of prodrug treatment, the growth inhibition was determined by staining with Alamar Blue and fluorescence recorded at 530/590 nm. Each data point (mean ± SEM, n=3 performed with at least fifteen replicates) is expressed as a percentage of the value for control wells with no prodrug treatment.
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Figure 3: Sensitivity of TK-expressing rat C6 constructs to GCV. Pools of stable transfectants containing vector only (pUB), wild-type TK, A168F, A168Y, Mutant 30 and SR39 were constructed and transfected in rat C6 glioma cells and evaluated for prodrug sensitivity as described in Materials and Methods. After six days of prodrug treatment, the growth inhibition was determined by staining with Alamar Blue and fluorescence recorded at 530/590 nm. Each data point (mean ± SEM, n=3 performed with at least fifteen replicates) is expressed as a percentage of the value for control wells with no prodrug treatment.

Mentions: To determine the prodrug activity of these mutants in vitro, mammalian expression vectors encoding the TK variants were constructed and used to transfect rat C6 glioma cells (see Materials and Methods). Immunoblot analyses of lysates from the pools of transfectants show similar expression levels for all of the mutants and wild-type TK, with no detectable expression in vector control pools (data not shown). Pools of stable transfectants were assayed for their level of GCV sensitivity over a drug range of 1-500 μM. Representative results of the prodrug sensitivities displayed by the TK mutants, wild-type TK and a vector control are shown in Fig. 3. Little to no toxicity was observed with vector alone at the lower prodrug doses. Surprisingly, no increased sensitivity was observed with either A168F or A168Y in comparison to wild-type TK for GCV (Fig. 3). Both mutant 30 and SR39 display about 100-fold increases in IC50 from wild-type TK (IC50 = 30 μM) and is similar what has been previously reported [15,16].


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)

Sensitivity of TK-expressing rat C6 constructs to GCV. Pools of stable transfectants containing vector only (pUB), wild-type TK, A168F, A168Y, Mutant 30 and SR39 were constructed and transfected in rat C6 glioma cells and evaluated for prodrug sensitivity as described in Materials and Methods. After six days of prodrug treatment, the growth inhibition was determined by staining with Alamar Blue and fluorescence recorded at 530/590 nm. Each data point (mean ± SEM, n=3 performed with at least fifteen replicates) is expressed as a percentage of the value for control wells with no prodrug treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Sensitivity of TK-expressing rat C6 constructs to GCV. Pools of stable transfectants containing vector only (pUB), wild-type TK, A168F, A168Y, Mutant 30 and SR39 were constructed and transfected in rat C6 glioma cells and evaluated for prodrug sensitivity as described in Materials and Methods. After six days of prodrug treatment, the growth inhibition was determined by staining with Alamar Blue and fluorescence recorded at 530/590 nm. Each data point (mean ± SEM, n=3 performed with at least fifteen replicates) is expressed as a percentage of the value for control wells with no prodrug treatment.
Mentions: To determine the prodrug activity of these mutants in vitro, mammalian expression vectors encoding the TK variants were constructed and used to transfect rat C6 glioma cells (see Materials and Methods). Immunoblot analyses of lysates from the pools of transfectants show similar expression levels for all of the mutants and wild-type TK, with no detectable expression in vector control pools (data not shown). Pools of stable transfectants were assayed for their level of GCV sensitivity over a drug range of 1-500 μM. Representative results of the prodrug sensitivities displayed by the TK mutants, wild-type TK and a vector control are shown in Fig. 3. Little to no toxicity was observed with vector alone at the lower prodrug doses. Surprisingly, no increased sensitivity was observed with either A168F or A168Y in comparison to wild-type TK for GCV (Fig. 3). Both mutant 30 and SR39 display about 100-fold increases in IC50 from wild-type TK (IC50 = 30 μM) and is similar what has been previously reported [15,16].

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