Aim Recent evidence has revealed that Eukaryotic elongation factor-2 kinase （eEF2K） activity may confer cancer cell adaptation to metabolic stress, and high expression of eEF2K is found in several types of cancer. T...Aim Recent evidence has revealed that Eukaryotic elongation factor-2 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, inhibi- tion of eEF2K for cancer drug discovery still remains in its infancy. This study aimed at developing a series of eEF2K inhibitor as candidate anti-tumor drugs in breast cancer and illustrating the possible mechanisms of its anti- tumor 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 （BL-EKI03）. Observa- tions of cell morphology were executed through several methods including ER-traeker, MDC and Hoeehst 33258 staining and GFP-LC3 transfeetion. Flow eytometrie analyses of MDC and Annexin V/PI were used for quantifica- tion of autophagy and apoptosis ratio. Western blot and ITRAQ analysis were used to explore the detailed mecha- nisms of BL-EKI03-induced ER stress, autophagie death and apoptosis in breast cancer cells. Furthermore, an in vivo xenograft mouse model was established for validating the anti-tumor efficacy of BL-EKI03. Results Firstly, a novel eEF2K inhibitor （BL-EKI03） 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 demonstra- ted that BL-EKI03 has remarkable anti-proliferative activities and induces endoplasmie retieulum （ER） stress, au- tophagy and apoptosis in MCF-7 and MDA-MB-436 cells. More importantly, the mechanism for BL-EKI03-indueed autophagie death involves eEF2K-mediated AMPK-mTOR-ULK complex pathways. The proteomies analyses and ex-perimental validation revealed that the BL-EKI03-induced mechanism was also involved BIRC6, BNIP1, SNAP29 and Bif-1, which might be regulated by eEF2K. Moreover, BL-EKI03 exerted its anti-tumor activities without re- markable toxicity, and it also induced autophagy and apoptosis by targeting eEF2K in fifo. Conclusion In this study, a novel eEF2K inhibitor （BL-EKI03） was discovered with remarkable anti-proliferative activities and in- duced endoplasmic reticulum （ER） stress, autophagy and apoptosis of breast cancer in vitro and in fifo. These findings highlight a new small-molecule eEF2K inhibitor （BL-EKI03） that has the potential to impact future breast cancer therapy.展开更多
文摘Aim Recent evidence has revealed that Eukaryotic elongation factor-2 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, inhibi- tion of eEF2K for cancer drug discovery still remains in its infancy. This study aimed at developing a series of eEF2K inhibitor as candidate anti-tumor drugs in breast cancer and illustrating the possible mechanisms of its anti- tumor 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 （BL-EKI03）. Observa- tions of cell morphology were executed through several methods including ER-traeker, MDC and Hoeehst 33258 staining and GFP-LC3 transfeetion. Flow eytometrie analyses of MDC and Annexin V/PI were used for quantifica- tion of autophagy and apoptosis ratio. Western blot and ITRAQ analysis were used to explore the detailed mecha- nisms of BL-EKI03-induced ER stress, autophagie death and apoptosis in breast cancer cells. Furthermore, an in vivo xenograft mouse model was established for validating the anti-tumor efficacy of BL-EKI03. Results Firstly, a novel eEF2K inhibitor （BL-EKI03） 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 demonstra- ted that BL-EKI03 has remarkable anti-proliferative activities and induces endoplasmie retieulum （ER） stress, au- tophagy and apoptosis in MCF-7 and MDA-MB-436 cells. More importantly, the mechanism for BL-EKI03-indueed autophagie death involves eEF2K-mediated AMPK-mTOR-ULK complex pathways. The proteomies analyses and ex-perimental validation revealed that the BL-EKI03-induced mechanism was also involved BIRC6, BNIP1, SNAP29 and Bif-1, which might be regulated by eEF2K. Moreover, BL-EKI03 exerted its anti-tumor activities without re- markable toxicity, and it also induced autophagy and apoptosis by targeting eEF2K in fifo. Conclusion In this study, a novel eEF2K inhibitor （BL-EKI03） was discovered with remarkable anti-proliferative activities and in- duced endoplasmic reticulum （ER） stress, autophagy and apoptosis of breast cancer in vitro and in fifo. These findings highlight a new small-molecule eEF2K inhibitor （BL-EKI03） that has the potential to impact future breast cancer therapy.
