The Role of Herpes Simplex Virus-1 Thymidine Kinase Alanine 168 in Substrate Specificity



Candice L Willmon1, Django Sussman2, Margaret E Black*, 1
1 Department of Pharmaceutical Sciences, Washington State University, Pullman, WA
2 Fred Hutchinson Cancer Research Center, Seattle, WA, USA


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© Willmon et al.; Licensee Bentham Open.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/), which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.

* Address correspondence to these authors at the Dept. of Pharmaceutical Sciences, P.O. Box 646534, Washington State University, Pullman, WA 99164-6534, USA; Tel: (509) 335-6265; Fax: (509) 335-5902; E-mail: blackm@mail.wsu.edu


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.

Keywords: Herpes Simplex Virus 1 Thymidine Kinase, ganciclovir, substrate specificity, gene therapy.