title: Expressing engineered thymidylate kinase variants in human cells to improve AZT phosphorylation and human immunodeficiency virus inhibition.
abstract: The triphosphorylated form of the nucleoside analogue AZT (AZTTP) acts as a chain terminator during reverse transcription of the human immunodeficiency virus (HIV) genome. The bottleneck in the conversion of AZT to AZTTP is the phosphorylation of AZT monophosphate (AZTMP) by cellular thymidylate kinase. Human thymidylate kinase was engineered to exhibit highly improved activity for AZTMP to AZTDP conversion. It was demonstrated here that genetically modified human cells transiently expressing these enzyme variants show more than 10-fold higher intracellular concentrations of AZTDP and AZTTP. Stable clones expressing these enzymes appear to phosphorylate AZTMP less efficiently, but first experiments indicate they are still more potent in HIV inhibition than the parental cells. It was proposed that the concept of introducing into human cells a catalytically improved human enzyme, rather than an enzyme of viral, bacterial or yeast origin, may serve as a paradigm for ameliorating the metabolic activation of an established drug.
authors.affiliation: Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
authors.forename: Birgitta M
authors.initials: BM
authors.lastname: Wöhrl
authors.affiliation: Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, Université Pierre et Marie Curie, Hôpital de la Pitié-Salpêtrière, CNRS ERS 107 CERVI, 83 boulevard de l'Hôpital, F-75651 Paris Cedex 13, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Loubière
authors.affiliation: Max-Planck-Institut für Biophysikalische Chemie, Abteilung Molekulare Genetik, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Brundiers
authors.affiliation: Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
authors.forename: Roger S
authors.initials: RS
authors.lastname: Goody
authors.affiliation: Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, Université Pierre et Marie Curie, Hôpital de la Pitié-Salpêtrière, CNRS ERS 107 CERVI, 83 boulevard de l'Hôpital, F-75651 Paris Cedex 13, France.
authors.forename: David
authors.initials: D
authors.lastname: Klatzmann
authors.affiliation: Max-Planck-Institut für Biophysikalische Chemie, Abteilung Molekulare Genetik, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Konrad
mesh_terms: D005818:Genetic Engineering; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D008826:Microbial Sensitivity Tests; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D010766:Phosphorylation; D015215:Zidovudine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D015215:Zidovudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase