Mutation of herpesvirus thymidine kinase to generate ganciclovir-specific kinases for use in cancer gene therapies.
Understanding the functional and mechanistic properties of the multi-substrate herpes simplex virus type-1 thymidine kinase (HSV-1 TK) remains critical to defining its role as a major pharmacological target in herpesvirus and gene therapies for cancer. An inherent limitation of the activity of HSV-TK is the >70-fold difference in the K(m)s for phosphorylation of thymidine over the pro-drug ganciclovir (GCV). To engineer an HSV-1 TK isoform that is specific for GCV as the preferred substrate, 16 site-specific mutants were generated. The mutations were concentrated at conserved residues involved in nucleoside base binding, Gln125 and near sites 3 and 4 involved in catalysis and substrate binding. The substrate preferences of each mutant enzyme were compared with wild-type HSV-1 TK. One mutant, termed Q7530 TK, had a lower K(m) for GCV than thymidine. Expression of the Q7530 TK in tumor cells indicated comparable metabolism to and improved sensitivity to GCV over wild-type HSV-1 TK, with minimal thymidine phosphorylation activity. A molecular modeling simulation of the different HSV-1 TK active-sites was done for GCV and thymidine binding. It was concluded that mutations at Gln125 and near site 4, especially at Ala168, were responsible for loss of deoxypyrimidine substrate binding.
Mercer
Kelly E
KE
Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23507, USA.
Ahn
Christina E
CE
Coke
Amanda
A
Compadre
Cesar M
CM
Drake
Richard R
RR
D015774:Ganciclovir / Q000378:metabolism*; D015316:Genetic Therapy; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009369:Neoplasms / Q000628:therapy; D013329:Structure-Activity Relationship; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism
D013937:Thymidine Kinase; D015774:Ganciclovir