The costs of publication of this article were defrayed in part by the payment of page charges.CaMKII activity was measured antibody.The reaction product was measured as described for min.The reaction product was measured as described. The reaction was initiated by adding ATP and allowed to progress at C for min.The reaction product was measured as described above for the filterbased assay of eEF kinase.Cell monolayers were washed twice in PBS, scraped into ml conical tubes, and centrifuged at gat C for min.Fifty g of protein or l of thein vitro eEF 2-Hexanone phosphorylation reaction were resolved by SDSPAGE and transferred to nitrocellulose membranes.The detection was performed using horseradish peroxidaselabeled secondary antibodies and enhanced chemiluminescence detection reagent.Briefly, cells were plated in well plates and exposed to various concentrations of drug for h.The preparation of TG eEF kinase transfectants was described previously. Briefly, TG human glioblastoma cells were transfected with a pSTAR vector expressing a fulllength eEF kinase cDNA.Transfectants were selected in gml G, and resistant colonies were expanded and maintained in gml G.Briefly, the cells were treated for h, harvested by trypsinization, washed with PBS, and fixed by dropwise addition of cold ethanol for min on ice.Each point represents the mean of duplicate determinations from a representative of three experiments.To determine the effect of eEF kinase inhibition on the viability of cancer cells, a panel of cell lines was incubated with various concentrations of NH for hand assayed for viability with MTT.To further analyze whether the effects of NH on cell viability were attributable to effects on eEF kinase activity, we tested if the effect of NH on cell viability could be altered by overexpression of the enzyme.In these experiments, we compared the sensitivity to NH in two isogenic cell lines that differed in expression of eEF kinase by virtue of stable transfection of TG cells with an expression vector containing a fulllength eEF kinase cDNA. Results are representative of four similar experiments.Rottlerin, a less potent and less specific inhibitor, decreased the number of cells entering S phase. Therefore, we determined the effect of NH on the cell cycle distribution of C glioma cells.Protein kinases are attractive targets for drug discovery because of their critical role in signal transduction pathways that are critical to normal cell growth and differentiation.In addition, these enzymes provide a rich platform for medicinal chemists because they often require unique coactivators or have distinct catalytic domains as defined by both biochemical and structural analyses.The rationale for targeting eEF kinase was based on data from our laboratory and others linking aberrations in protein synthesis, including the activation of this enzyme, to the growth and viability of transformed cells. Nonetheless, our studies of the benzyl imdiazolium series uncovered a potent and relatively specific inhibitor of eEF kinase.The length of the alkyl chain attached to the N imidazolium nitrogen did not give a clear correlation with the activity of the compounds.However, it appears that an optimal chain length is required because compounds with longer than CH chain do not inhibit eEF kinase effectively.