Kokoris Mark S, Black Margaret E
Department of Pharmaceutical Sciences, Washington State University, Pullman, Washington 99164-6534, USA.
Protein Sci. 2002 Sep;11(9):2267-72. doi: 10.1110/ps.2460102.
Herpes Simplex Virus type 1 (HSV-1) thymidine kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer. Tumor cells that express HSV-1 thymidine kinase are rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme. Although ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug. From a random mutagenesis library, seven thymidine kinase variants containing multiple amino acid substitutions were identified on the basis of activity towards ganciclovir and acyclovir based on negative selection in Escherichia coli. Using a novel affinity chromatography column, three mutant enzymes and the wild-type TK were purified to homogeneity and their kinetic parameters for thymidine, ganciclovir, and acyclovir determined. With ganciclovir as the substrate, one mutant (mutant SR39) demonstrated a 14-fold decrease in K(m) compared to the wild-type enzyme. The most dramatic change is displayed by mutant SR26, with a 124-fold decrease in K(m) with acyclovir as the substrate. Such new "prodrug kinases" could provide benefit to ablative gene therapy by now making it feasible to use the relatively nontoxic acyclovir at nanomolar concentrations or ganciclovir at lower, less immunosuppressive doses.
1型单纯疱疹病毒（HSV-1）胸苷激酶（TK）是目前癌症基因治疗中应用最为广泛的自杀基因。表达HSV-1胸苷激酶的肿瘤细胞由于该酶的优先磷酸化作用而对前体药物敏感。虽然更昔洛韦（GCV）是与TK联用的首选前体药物，但这种方法在一定程度上受到该前体药物毒性的限制。通过随机诱变文库，基于在大肠杆菌中的负筛选，根据对更昔洛韦和阿昔洛韦的活性鉴定出了7个含有多个氨基酸取代的胸苷激酶变体。使用新型亲和层析柱，将三种突变酶和野生型TK纯化至均一，并测定了它们对胸苷、更昔洛韦和阿昔洛韦的动力学参数。以更昔洛韦为底物时，一种突变体（突变体SR39）的米氏常数（K(m)）与野生型酶相比降低了14倍。突变体SR26表现出最显著的变化，以阿昔洛韦为底物时其K(m)降低了124倍。这些新的“前体药物激酶”现在使得以纳摩尔浓度使用相对无毒的阿昔洛韦或以更低、免疫抑制性更小的剂量使用更昔洛韦进行消融基因治疗成为可能，从而可能为消融基因治疗带来益处。
