New variants of tomato thymidine kinase 1 selected for increased sensitivity of E. Coli KY895 towards azidothymidine
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New variants of tomato thymidine kinase 1 selected for increased sensitivity of E. Coli KY895 towards azidothymidine. / Christiansen, Louise Slot; Egeblad, Louise; Munch-Petersen, Birgitte; Piškur, Jure; Knecht, Wolfgang.
I: Cancers, Bind 7, Nr. 2, 2015, s. 966-980.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - New variants of tomato thymidine kinase 1 selected for increased sensitivity of E. Coli KY895 towards azidothymidine
AU - Christiansen, Louise Slot
AU - Egeblad, Louise
AU - Munch-Petersen, Birgitte
AU - Piškur, Jure
AU - Knecht, Wolfgang
N1 - (Ekstern)
PY - 2015
Y1 - 2015
N2 - Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.
AB - Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.
KW - Azidothymidine
KW - Deoxynucleoside kinases
KW - Deoxynucleosides
KW - Deoxynucleotides
KW - Mutagenesis
KW - Nucleoside analog
KW - Nucleosides
KW - Prodrug activation
KW - Protein engineering
KW - Suicide gene therapy
UR - http://www.scopus.com/inward/record.url?scp=84934944172&partnerID=8YFLogxK
U2 - 10.3390/cancers7020819
DO - 10.3390/cancers7020819
M3 - Journal article
AN - SCOPUS:84934944172
VL - 7
SP - 966
EP - 980
JO - Cancers
JF - Cancers
SN - 2072-6694
IS - 2
ER -
ID: 255883484