Max-Planck-Institute for Biophysical Chemistry, Research Group Enzyme Biochemistry, 37077 Göttingen, Germany; Institute for Geophysics, Georg-August University, 37077 Göttingen, Germany.
Max-Planck-Institute for Biophysical Chemistry, Research Group Enzyme Biochemistry, 37077 Göttingen, Germany.
Biochem Pharmacol. 2014 Feb 1;87(3):435-44. doi: 10.1016/j.bcp.2013.11.011. Epub 2013 Dec 4.
The limitations of the ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV1 TK: EC 2.7.1.21) system as a suicide gene therapy approach have been extensively studied over the years. In our study, we focused on improving the cytotoxic profile of the GCV/equine herpes virus-4 thymidine kinase (EHV4 TK: EC 2.7.1.21) system. Our approach involved the structure-guided mutagenesis of EHV4 TK in order to switch its ability to preferentially phosphorylate the natural substrate deoxythymidine (dT) to that of GCV. We performed steady-state kinetic analysis, genetic complementation in a thymidine kinase-deficient Escherichia coli strain, isothermal titration calorimetry, and analysis of GCV-induced cell killing through generation of HEK 293 stable cell-lines expressing EHV4 TK mutants and wild-type EHV4 TK. We found that the EHV4 TK S144H-GFP mutant preferentially phosphorylates GCV and confers increased GCV-induced cytotoxicity compared to wild-type EHV4 TK.
多年来，人们对更昔洛韦（GCV）/单纯疱疹病毒胸苷激酶（HSV1 TK：EC 2.7.1.21）系统作为自杀基因治疗方法的局限性进行了广泛研究。在我们的研究中，我们专注于改善 GCV/马疱疹病毒-4 胸苷激酶（EHV4 TK：EC 2.7.1.21）系统的细胞毒性特征。我们的方法涉及 EHV4 TK 的结构导向突变，以改变其优先磷酸化天然底物脱氧胸苷（dT）的能力，使其更倾向于 GCV。我们通过生成表达 EHV4 TK 突变体和野生型 EHV4 TK 的 HEK 293 稳定细胞系，进行了稳态动力学分析、在胸苷激酶缺陷型大肠杆菌菌株中的遗传互补、等温滴定量热法以及分析 GCV 诱导的细胞杀伤作用。我们发现 EHV4 TK S144H-GFP 突变体优先磷酸化 GCV，并与野生型 EHV4 TK 相比，赋予了更高的 GCV 诱导细胞毒性。
