eEF-2K, also known as CaM kinase-III, is an atypical protein kinase which negatively regulates the global rate of protein synthesis through the phosphorylation and inactivation of its substrate eEF-2. Recently eEF-2K has been validated as a novel target for anti-cancer therapy. However, a detailed understanding of the role of eEF-2K in cancer biology is unavailable. Mechanistic studies can often provide an understanding of enzyme function. Therefore, we determined the kinetic mechanism of eEF-2K using a peptide substrate (Acetyl-RKKYKFNEDTERRRFL-amide). We found that eEF-2K adopts a ternary-complex, steady state ordered mechanism, with ATP binding required before the peptide substrate. A good cellular inhibitor is required for elucidating the role of eEF-2K in cancer biology. To date, NH125 is the only inhibitor used to investigate the activity of eEF-2K in cells. Although it is reported as a specific inhibitor of eEF-2K, its exact mode of action has not been reported. Through in-vitro assays and cellular studies, we found that NH125 is a non-specific inhibitor of eEF-2K that blocks eEF-2 phosphorylation in cells. There is a great demand for specific inhibitors of eEF-2K. We developed a fluorescence high throughput assay system for eEF-2K. The assay utilizes the peptide substrate labeled with a Sox moiety whose phosphorylation can be monitored at 485 nm in the presence of magnesium. We also validated the assay in a screen of 30,000 compounds in 384 well plates. We found the assay to be robust and identified a relatively specific inhibitor of eEF-2K and determined its mechanism of action. We found it behaved as a slowly reversible inhibitor of eEF-2K with a two step inhibition mechanism - fast initial binding at the enzyme active site, followed by a slower inactivation step. We propose that the nitrile group on the compound binds to the active site thiol in the enzyme covalently forming a reversible thioimidate adduct to inactivate the enzyme. / text