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Identification of Inhibitors for a Virally Encoded Protein Kinase by 2 Different Screening Systems: In Vitro Kinase Assay and In-Cell Activity Assay

Axxima Pharmaceuticals AG, Munich, Germany

Kerstin Hölscher

Axxima Pharmaceuticals AG, Munich, Germany

Heidrun Degen

Axxima Pharmaceuticals AG, Munich, Germany

Christina Esdar

Merck KGaA, Darmstadt, Germany

Birgit Felden De Neumann

Axxima Pharmaceuticals AG, Munich, Germany

Birgit Flicke

Axxima Pharmaceuticals AG, Munich, Germany

Tatjana Freudenreich

Axxima Pharmaceuticals AG, Munich, Germany

Gaby Holzer

Axxima Pharmaceuticals AG, Munich, Germany

Sieglinde Schinzel

Axxima Pharmaceuticals AG, Munich, Germany

Thomas Stamminger

Institute of Clinical and Molecular Virology, University Erlangen, Erlangen, Germany

Matthias Stein-Gerlach

Axxima Pharmaceuticals AG, Munich, Germany

Manfred Marschall

Institute of Clinical and Molecular Virology, University Erlangen, Erlangen, Germany

Thomas Herget

Axxima Pharmaceuticals AG, Munich, Germany and Merck KGaA, Darmstadt, Germany

The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 µM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates. (Journal of Biomolecular Screening 2005:36-45)

Key Words: kinase • human cytomegalovirus • cellular assay • drug discovery • pUL97

Journal of Biomolecular Screening, Vol. 10, No. 1, 36-45 (2005)
DOI: 10.1177/1087057104270269


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