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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

Deduced amino acid sequence of the two HSV TK mutants, mutant 30 and SR39. The top line shows the amino acid sequence of the residues 159-174 of wild-type HSV TK. Two highly conserved tripeptide motifs are boxed and denoted as Sites 3 and 4. The amino acids in bold in the top line denote codons that were targeted for the creation for the randomized libraries and subsequent mutations are shown in the following lines [15,16].
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Figure 1: Deduced amino acid sequence of the two HSV TK mutants, mutant 30 and SR39. The top line shows the amino acid sequence of the residues 159-174 of wild-type HSV TK. Two highly conserved tripeptide motifs are boxed and denoted as Sites 3 and 4. The amino acids in bold in the top line denote codons that were targeted for the creation for the randomized libraries and subsequent mutations are shown in the following lines [15,16].

Mentions: In order to improve the poor catalytic performance of the enzyme towards GCV, we employed protein engineering to optimize HSV TK for increased sensitivity to GCV. Our previous studies used random sequence mutagenesis to target six amino acid residues that neighbor two highly conserved tripeptide motifs shown to be involved in substrate binding (Fig. 1) [15,16]. Several variants conferred enhanced prodrug sensitivity in rat C6 glioma transfected cells. In a xenograft tumor model, the most promising two mutants, mutant 30 and SR39, displayed impaired tumor growth at doses of GCV that did not impact wild-type HSV TK transfected tumors [17]. Molecular modeling of these multiple amino acid substituted variants led us to suggest that substitutions at position 168 may be crucial for the observed substrate alterations found in mutant 30 and SR39 [17]. To examine the role of A168 in substrate specificity we created individual substitutions to reflect mutations at A168 found in mutant 30 or SR39. These mutant and wild-type HSV TK enzymes were expressed and purified to near homogeneity from E. coli, and characterized for their kinetic properties. The substrate binding and catalytic efficiency information generated from theses studies were used to correlate functional enzyme data and modeled structures with three different substrates. Our results reveal that the phenylalanine (F) and tyrosine (Y) substitutions at the A168 are mainly accommodated by the side chain rearrangements that maintain interactions between HSV TK and the nucleoside analogs. Furthermore, these mutants were stably transfected in a rat C6 glioma cell line and evaluated for GCV sensitivity.


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)

Deduced amino acid sequence of the two HSV TK mutants, mutant 30 and SR39. The top line shows the amino acid sequence of the residues 159-174 of wild-type HSV TK. Two highly conserved tripeptide motifs are boxed and denoted as Sites 3 and 4. The amino acids in bold in the top line denote codons that were targeted for the creation for the randomized libraries and subsequent mutations are shown in the following lines [15,16].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Deduced amino acid sequence of the two HSV TK mutants, mutant 30 and SR39. The top line shows the amino acid sequence of the residues 159-174 of wild-type HSV TK. Two highly conserved tripeptide motifs are boxed and denoted as Sites 3 and 4. The amino acids in bold in the top line denote codons that were targeted for the creation for the randomized libraries and subsequent mutations are shown in the following lines [15,16].
Mentions: In order to improve the poor catalytic performance of the enzyme towards GCV, we employed protein engineering to optimize HSV TK for increased sensitivity to GCV. Our previous studies used random sequence mutagenesis to target six amino acid residues that neighbor two highly conserved tripeptide motifs shown to be involved in substrate binding (Fig. 1) [15,16]. Several variants conferred enhanced prodrug sensitivity in rat C6 glioma transfected cells. In a xenograft tumor model, the most promising two mutants, mutant 30 and SR39, displayed impaired tumor growth at doses of GCV that did not impact wild-type HSV TK transfected tumors [17]. Molecular modeling of these multiple amino acid substituted variants led us to suggest that substitutions at position 168 may be crucial for the observed substrate alterations found in mutant 30 and SR39 [17]. To examine the role of A168 in substrate specificity we created individual substitutions to reflect mutations at A168 found in mutant 30 or SR39. These mutant and wild-type HSV TK enzymes were expressed and purified to near homogeneity from E. coli, and characterized for their kinetic properties. The substrate binding and catalytic efficiency information generated from theses studies were used to correlate functional enzyme data and modeled structures with three different substrates. Our results reveal that the phenylalanine (F) and tyrosine (Y) substitutions at the A168 are mainly accommodated by the side chain rearrangements that maintain interactions between HSV TK and the nucleoside analogs. Furthermore, these mutants were stably transfected in a rat C6 glioma cell line and evaluated for GCV sensitivity.

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