Establishment And Application Of A High-throughput Screening Assay For EEF2K Inhibitors

Eukaryotic elongation factor 2 kinase(eEF2K) is a kinase encoded by human eEF2 K gene. It is a highly conservative kinase, and it has no homology with the typical protein kinase in sequence. It belongs to an atypical protein kinase family named ‘?-kinase'. Previous studies have shown that eEF2 K, under the condition of certain metabolic stresses can phosphorylate eukaryotic elongation factor 2(eEF2) to restrain the activity of eEF2, thereby inhibiting the elongation of a polypeptide chain in the process of protein synthesis, reducing the consumption of energy and amino acids in cells, and helping cells live through the deprivation environment. The action of eEF2 K is dependent upon Ca2+ and calmodulin and its protective role in cells makes it a potential target for cancer treatment. The purpose of this paper is to use a high-throughput screening method to discover small molecule inhibitors of eEF2 K and their therapeutic properties.In this paper we established and optimized a high-throughput screening assay for eEF2 K small molecule inhibitors. We chose DMSO to use as a negative control, compound A484954 as a positive control, then detect the luminescence of the reaction to reflect the compounds' inhibition ratio. If the compounds can inhibit eEF2 K activity, compared with the positive control, the luminescence of the reaction will reduced. Then we can calculate the inhibition ratio of the compounds. Finally, we conducted a large scale of random screening on 56000 small molecular compounds. We get 9 compounds which have dose dependent reaction, then we chosen five compounds which IC50 values are less than 50 micromoles to test their activities in cancer cells, and the further research is underway.Our study found a good foundation for cancer or other diseases therapy which targeted eEF2 K.