title: A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing.
abstract: 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.
authors: Max-Planck-Institute for Biophysical Chemistry, Research Group Enzyme Biochemistry, 37077 Göttingen, Germany; Institute for Geophysics, Georg-August University, 37077 Göttingen, Germany. Electronic address: tmcsorl@gwdg.de.
authors: Theresa
authors: T
authors: McSorley
authors: Max-Planck-Institute for Biophysical Chemistry, Research Group Enzyme Biochemistry, 37077 Göttingen, Germany.
authors: Stephan
authors: S
authors: Ort
authors: Max-Planck-Institute for Biophysical Chemistry, Research Group Enzyme Biochemistry, 37077 Göttingen, Germany.
authors: Christian
authors: C
authors: Monnerjahn
authors: Max-Planck-Institute for Biophysical Chemistry, Research Group Enzyme Biochemistry, 37077 Göttingen, Germany. Electronic address: mkonrad@gwdg.de.
authors: Manfred
authors: M
authors: Konrad
mesh_terms: D000595:Amino Acid Sequence; D049109:Cell Proliferation; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015774:Ganciclovir / Q000494:pharmacology*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D023141:Herpesvirus 4, Equid / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir
keywords: ACV; EHV4; Enzyme prodrug activation; Equine herpes virus-4 thymidine kinase; GCV; Ganciclovir; HSV1; Herpes simplex virus thymidine kinase; IPTG; PMSF; Suicide gene therapy; TK; acyclovir; dC; dG; dT; dU; deoxycytidine; deoxyguanosine; deoxyuridine; equine herpes virus type 4; ganciclovir; herpes simplex virus type 1; isopropyl β-d-1-thiogalactopyranoside; phenylmethylsulfonyl fluoride; thymidine; thymidine kinase