Aim Recent evidence has revealed that Eukaryotic elongation factor2 kinase (eEF2K) activity may confer cancer cell adaptation to metabolic stress, and high expression of eEF2K is found in several types of cancer.Therefore, eEF2K may contribute to carcinogenesis and represent a promising therapeutic target; however, inhibition of eEF2K for cancer drug discovery still remains in its infancy.This study aimed at developing a series of eEF2K inhibitor as candidate antitumor drugs in breast cancer and illustrating the possible mechanisms of its antitumor activity in vitro and in vivo.Methods In silico screening, structure modifications, MTT assay and molecular dynamics (MD) simulations were applied for the discovery of the novel eEF2K inhibitor (BLEKI03).Observations of cell morphology were executed through several methods including ERtracker, MDC and Hoechst 33258staining and GFPLC3 transfection.Flow cytometric analyses of MDC and Annexin V/PI were used for quantification of autophagy and apoptosis ratio.Western blot and ITRAQ analysis were used to explore the detailed mechanisms of BLEKI03induced ER stress, autophagic death and apoptosis in breast cancer cells.Furthermore, an in vivo xenograft mouse model was established for validating the antitumor efficacy of BLEKI03.Results Firstly, a novel eEF2K inhibitor (BLEKI03) with a good affinity for eEF2K was eventually discovered after computational screening and synthesis of a series of candidate compounds targeting eEF2K.Subsequently, our results demonstrated that BLEKI03 has remarkable antiproliferative activities and induces endoplasmic reticulum (ER) stress, autophagy and apoptosis in MCF7 and MDAMB436 cells.More importantly, the mechanism for BLEKI03induced autophagic death involves eEF2Kmediated AMPKmTORULK complex pathways.The proteomics analyses and experimental validation revealed that the BLEKI03induced mechanism was also involved BIRC6, BNIP1, SNAP29and Bif1, which might be regulated by eEF2K.Moreover, BLEKI03 exerted its antitumor activities without remarkable toxicity, and it also induced autophagy and apoptosis by targeting eEF2K in vivo.Conclusion In this study, a novel eEF2K inhibitor (BLEKI03) was discovered with remarkable antiproliferative activities and induced endoplasmic reticulum (ER) stress, autophagy and apoptosis of breast cancer in vitro and in vivo.These findings highlight a new smallmolecule eEF2K inhibitor (BLEKI03) that has the potential to impact future breast cancer therapy.