Expressing engineered thymidylate kinase variants in human cells to improve AZT phosphorylation and human immunodeficiency virus inhibition

• Published in: Journal of general virology 2005-03, Vol.86 (3), p.757-764
• Main Authors: Wohrl, Birgitta M, Loubiere, Laurence, Brundiers, Ralf, Goody, Roger S, Klatzmann, David, Konrad, Manfred
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Genetic Engineering; HIV-1 - drug effects; HIV-1 - physiology; Human immunodeficiency virus; Humans; Microbial Sensitivity Tests; Microbiology; Miscellaneous; Nucleoside-Phosphate Kinase - metabolism; Phosphorylation; Virology; Zidovudine - analogs & derivatives; Zidovudine - metabolism; Zidovudine - pharmacology
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/vir.0.80529-0

1 Max-Planck-Institut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany 2 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 3 Max-Planck-Institut für Biophysikalische Chemie, Abteilung Molekulare Genetik, Am Fassberg 11, D-37077 Göttingen, Germany Correspondence Manfred Konrad mkonrad{at}gwdg.de 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. Present address: Universität Bayreuth, Lehrstuhl Biopolymere, Universitätsstr. 30, D-95447 Bayreuth, Germany. Present address: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, PO Box 19024, Clinical Division D2-100, Seattle, WA 98109, USA. These authors contributed equally to this work.