Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97.
The UL97-encoded protein kinase (pUL97) of human cytomegalovirus (HCMV) plays a critical role in the control of virus replication. Deletion of the UL97 gene results in a drastic reduction in the replication efficiency. Although the exact function of pUL97 remains unclear and its sensitivity to specific inhibitors is speculative, protein kinase inhibitors of the indolocarbazole class are effective inhibitors of cytomegalovirus. Based on the phosphorylation of ganciclovir (GCV), a novel quantification system for pUL97 kinase activity was established: the phosphorylated form of GCV exerts an easily quantifiable cytotoxic effect in transfected cells. Importantly, the addition of indolocarbazole compounds, Gö6976 and NGIC-I, which were highly effective at nanomolar concentrations while other protein kinase inhibitors were not, led to a significant reduction of pUL97 kinase activity. It was also demonstrated that a catalytically inactive mutant of pUL97, K355M, and a GCV-resistant mutant, M460I, were both negative for GCV phosphorylation, although protein phosphorylation remained detectable for the latter mutant. In vitro kinase assays were used to confirm the levels of pUL97-mediated phosphorylation recorded. To generate a tool for screening large numbers of putative inhibitors that preferentially interfere with GCV as well as protein phosphorylation, pUL97-expressing cell clones with stable pUL97 kinase activity were selected. This study demonstrates that certain indolocarbazole compounds are potent pUL97 inhibitors and, therefore, represent novel candidates for antiviral drugs that target viral protein kinase functions.
Marschall
Manfred
M
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany1.
Stein-Gerlach
Matthias
M
Axxima Pharmaceuticals AG, Martinsried, Germany2.
Freitag
Martina
M
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany1.
Kupfer
Regina
R
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany1.
van den Bogaard
Miriam
M
Axxima Pharmaceuticals AG, Martinsried, Germany2.
Stamminger
Thomas
T
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany1.
D000998:Antiviral Agents / Q000494:pharmacology*; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D002999:Clone Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D003587:Cytomegalovirus / Q000187:drug effects* / Q000201:enzymology* / Q000502:physiology; D004353:Drug Evaluation, Preclinical; D004352:Drug Resistance, Microbial; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015774:Ganciclovir / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D020125:Mutation, Missense / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D047428:Protein Kinase Inhibitors; C081021:Go 6976; C075954:indolo(3,2-b)carbazole; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir