Discovery of a small-molecule bromodomain-containing protein 4 inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer

Jin ZHANG,Jie LIU,Liang OUYANG

(Department of Laboratory Medicine,Precision Medicine Center,State Key Laboratory of Biotherapy and Precision Medicine Key Laboratory of Sichuan Province,West China Hospital,Sichuan University and Collaborative Innovation Center,Chengdu 610041,China）

T1-41

Discovery of a small-molecule bromodomain-containing protein 4 inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer

Jin ZHANG,Jie LIU,Liang OUYANG

(Department of Laboratory Medicine,Precision Medicine Center,State Key Laboratory of Biotherapy and Precision Medicine Key Laboratory of Sichuan Province,West China Hospital,Sichuan University and Collaborative Innovation Center,Chengdu 610041,China）

OBJECTIVETo discover a small-molecule bromodomain-containing protein 4(BRD4)inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer and exploreits potential mechanisms.METHODSBRD4 interactors were analyzed by PPI network prediction and The Cancer Genome Atlas(TCGA)analysis.The interaction between BRD4 and AMPK was confirmed by co-immunoprecipitation assay.Novel BRD4 inhibitors were designed and synthesized based upon pharmacophore analysis of BRD4(1),then screened by antiproliferative activity and Alpha Screen of BRD4(1).The selectivity of the best candidate compound 8f was validated by co-crystallization,FRET assay and co-immuno precipitation assay.The mechanisms of 8f were investigated by fluorescence microscopy,electron microscopy,Western blotting,immunocy?tochemistry,siRNA and GFP-mRFP-LC3 plasmid transfections,as well as immunohistochemistry and immunofluorescence.Potential mechanisms were discovered by iTRAQ-based proteomics analysis and the therapeutic effect of 8f was assessed by xenograft breast cancer mouse and zebrafish models.RESULTSWe identified that BRD4 interacted with AMPK,which was remarkably downregulated in breast cancer.We next designed and synthesized 49 candidate compounds,and eventually discovered a selective small-molecule inhibitor of BRD4(8f).Subsequently,8f was discovered to induce autophagyassociated cell death(ACD)by BRD4-AMPK interaction,and thus activating AMPK-mTOR-ULK1-modulated autophagic pathway in breast cancer cells.Interestingly,the iTRAQ-based proteomics analyses revealed that 8f induced ACD pathways,involved in HMGB1,VDAC1/2 and eEF2.Moreover,8f displayed a therapeutic potential on both xenograft breast cancer mouse and zebrafish models.CONCLUSIONWe discovered a novel small-molecule inhibitor of BRD4 that induces BRD4-AMPK-modulated ACD in breast cancer,which may provide a candidate drug for future cancer therapy.

bromodomain-containing protein 4(BRD4);BRD4-AMPK interaction;small-molecule inhibitor of BRD4;Autophagy-associated cell death(ACD);breast cancer

The project supported by National Natural Science Foundation of China(81473091,81673290 and U1603123)

Liang OUYANG,Tel:13880674611,E-mail：klivis@163.com