Identification of inhibitors for a virally encoded protein kinase by 2 different screening systems: in vitro kinase assay and in-cell activity assay.

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.

Screening assay for the identification of deoxyhypusine synthase inhibitors.

The 1st step in the posttranslational hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine] modification of eukaryotic translation initiation factor 5A (eIF5A) is catalyzed by deoxyhypusine synthase (DHS). The eIF5A intermediate is subsequently hydroxylated by deoxyhypusine hydroxylase (DHH), thereby converting the eIF5A precursor into a biologically active protein. Depletion of eIF5A causes inhibition of cell growth, and the identification of eIF5A as a cofactor of the HIV Rev protein turns this host protein and therefore DHS into an interesting target for drugs against abnormal cell growth and/or HIV replication. The authors developed a 96-well format DHS assay applicable for the screening of DHS inhibitors. Using this assay, they demonstrate DHS inhibition by AXD455 (Semapimod, CNI-1493). This assay represents a powerful tool for the identification of new DHS inhibitors with potency against cancer and HIV.

New cationic lipids form channel-like pores in phospholipid bilayers.

Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5-5 micro M of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1-2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed.