title: Identification of inhibitors for a virally encoded protein kinase by 2 different screening systems: in vitro kinase assay and in-cell activity assay.
abstract: 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 microM. 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.
authors.affiliation: Axxima Pharmaceuticals AG, Munich, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Mett
authors.forename: Kerstin
authors.initials: K
authors.lastname: Hölscher
authors.forename: Heidrun
authors.initials: H
authors.lastname: Degen
authors.forename: Christina
authors.initials: C
authors.lastname: Esdar
authors.forename: Birgit Felden
authors.initials: BF
authors.lastname: De Neumann
authors.forename: Birgit
authors.initials: B
authors.lastname: Flicke
authors.forename: Tatjana
authors.initials: T
authors.lastname: Freudenreich
authors.forename: Gaby
authors.initials: G
authors.lastname: Holzer
authors.forename: Sieglinde
authors.initials: S
authors.lastname: Schinzel
authors.forename: Thomas
authors.initials: T
authors.lastname: Stamminger
authors.forename: Matthias
authors.initials: M
authors.lastname: Stein-Gerlach
authors.forename: Manfred
authors.initials: M
authors.lastname: Marschall
authors.forename: Thomas
authors.initials: T
authors.lastname: Herget
mesh_terms: D000818:Animals; D002227:Carbazoles / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D007211:Indoles / Q000737:chemistry; D015394:Molecular Structure; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
chemical_list: D002227:Carbazoles; D007211:Indoles; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase