Limits...
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

Superposition of HSV TK active site residues (residue numbers 58, 128-132,168-172, 222-225 (unlabeled in figure)). All three panels have consistent coloring: wild-type: yellow; A168F: blue; and A168Y: green. Models of wild-type and mutant proteins in complex with: A. Thymidine (thy); B. GCV.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2570551&req=5

Figure 4: Superposition of HSV TK active site residues (residue numbers 58, 128-132,168-172, 222-225 (unlabeled in figure)). All three panels have consistent coloring: wild-type: yellow; A168F: blue; and A168Y: green. Models of wild-type and mutant proteins in complex with: A. Thymidine (thy); B. GCV.

Mentions: Despite the replacement of a small hydrophobic side chain at position 168 with either an aromatic phenylalanine residue or bulky and polar tyrosine, many of the original contacts between protein and ligand are conserved after molecular dynamics/simulated annealing. In fact, these dramatic mutations are mainly accommodated by rearrangements in side-chain positions while the backbone undergoes relatively minor adjustments (Fig. 4). These rearrangements allow the maintenance of hydrophobic and ionic interactions between the protein and both the nucleoside base analog and the sugar constituents of all ligands. Most notably, the hydrophobic tyrosine at position 172 (Y172) and methionine 128 (M128) residues sandwich the ring and define its placement within the binding site.


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)

Superposition of HSV TK active site residues (residue numbers 58, 128-132,168-172, 222-225 (unlabeled in figure)). All three panels have consistent coloring: wild-type: yellow; A168F: blue; and A168Y: green. Models of wild-type and mutant proteins in complex with: A. Thymidine (thy); B. GCV.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Superposition of HSV TK active site residues (residue numbers 58, 128-132,168-172, 222-225 (unlabeled in figure)). All three panels have consistent coloring: wild-type: yellow; A168F: blue; and A168Y: green. Models of wild-type and mutant proteins in complex with: A. Thymidine (thy); B. GCV.
Mentions: Despite the replacement of a small hydrophobic side chain at position 168 with either an aromatic phenylalanine residue or bulky and polar tyrosine, many of the original contacts between protein and ligand are conserved after molecular dynamics/simulated annealing. In fact, these dramatic mutations are mainly accommodated by rearrangements in side-chain positions while the backbone undergoes relatively minor adjustments (Fig. 4). These rearrangements allow the maintenance of hydrophobic and ionic interactions between the protein and both the nucleoside base analog and the sugar constituents of all ligands. Most notably, the hydrophobic tyrosine at position 172 (Y172) and methionine 128 (M128) residues sandwich the ring and define its placement within the binding site.

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