OBJECTIVE To 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 mech...OBJECTIVE To 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.METHODS BRD4 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,immunocytochemistry,si RNA and GFP-m RFP-LC3 plasmid transfections,as well as immunohistochemistry and immunofluorescence.Potential mechanisms were discovered by i TRAQ-based proteomics analysis and the therapeutic effect of 8f was assessed by xenograft breast cancer mouse and zebrafish models.RESULTS We 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-m TOR-ULK1-modulated autophagic pathway in breast cancer cells.Interestingly,the i TRAQ-based proteomics analyses revealed that 8f induced ACD pathways,involved in HMGB1,VDAC1/2 and e EF2.Moreover,8f displayed a therapeutic potential on both xenograft breast cancer mouse and zebrafish models.CONCLUSION We 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.展开更多
Aim Nicotinamide phosphoribosyltransferase （NAMPT） is a promising therapeutic target for cardio-ce- rebrovascular diseases and tumor. Novel NAMPT inhibitors with diverse chemotypes are highly desirable for devel- op...Aim Nicotinamide phosphoribosyltransferase （NAMPT） is a promising therapeutic target for cardio-ce- rebrovascular diseases and tumor. Novel NAMPT inhibitors with diverse chemotypes are highly desirable for devel- opment of therapeutic agents. Methods We carried out a high throughput screening targeting NAMPT on a chemi- cal library of 30000 small-molecules in this study. Assays of NAD levels, anti-proliferative activity, imaging study, RNA interference were conducted in HepG2 cells or primary mouse hepatocytes. Results A non-fluorescent com- pound F671-0003 and a fluorescent compound M049-0244 were found with excellent in vitro activity （IC50：85 nmol · L^-1 and 170 nmol · L^-1 respectively） and anti-proliferative activity against HepG2 cells. These two com- pounds significantly depleted cellular NAD levels. Exogenous NMN rescued their anti-proliferative activity against HepG2 cells. Structure-activity relationship study proposed a binding mode for NAMPT inhibitor F671-0003 and highlighted the importance of hydrogen bonding, hydrophobic and -rr--rr interactions in inhibitor binding. Imaging study provided the evidence that fluorescent compound M049-0244 （3 μmol · L^-1） significantly stained living HepG2 cells. Cellular fluorescence was further verified to be NAMPT dependent by using RNA interference and NAMPT over expression transgenic mice. Conclusions This study provides novel lead compounds and a ＂first-in- class＂ fluorescent probe for imaging NAMPT.展开更多
基金supported by National Natural Science Foundation of China(81473091,81673290 and U1603123)
文摘OBJECTIVE To 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.METHODS BRD4 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,immunocytochemistry,si RNA and GFP-m RFP-LC3 plasmid transfections,as well as immunohistochemistry and immunofluorescence.Potential mechanisms were discovered by i TRAQ-based proteomics analysis and the therapeutic effect of 8f was assessed by xenograft breast cancer mouse and zebrafish models.RESULTS We 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-m TOR-ULK1-modulated autophagic pathway in breast cancer cells.Interestingly,the i TRAQ-based proteomics analyses revealed that 8f induced ACD pathways,involved in HMGB1,VDAC1/2 and e EF2.Moreover,8f displayed a therapeutic potential on both xenograft breast cancer mouse and zebrafish models.CONCLUSION We 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.
文摘Aim Nicotinamide phosphoribosyltransferase （NAMPT） is a promising therapeutic target for cardio-ce- rebrovascular diseases and tumor. Novel NAMPT inhibitors with diverse chemotypes are highly desirable for devel- opment of therapeutic agents. Methods We carried out a high throughput screening targeting NAMPT on a chemi- cal library of 30000 small-molecules in this study. Assays of NAD levels, anti-proliferative activity, imaging study, RNA interference were conducted in HepG2 cells or primary mouse hepatocytes. Results A non-fluorescent com- pound F671-0003 and a fluorescent compound M049-0244 were found with excellent in vitro activity （IC50：85 nmol · L^-1 and 170 nmol · L^-1 respectively） and anti-proliferative activity against HepG2 cells. These two com- pounds significantly depleted cellular NAD levels. Exogenous NMN rescued their anti-proliferative activity against HepG2 cells. Structure-activity relationship study proposed a binding mode for NAMPT inhibitor F671-0003 and highlighted the importance of hydrogen bonding, hydrophobic and -rr--rr interactions in inhibitor binding. Imaging study provided the evidence that fluorescent compound M049-0244 （3 μmol · L^-1） significantly stained living HepG2 cells. Cellular fluorescence was further verified to be NAMPT dependent by using RNA interference and NAMPT over expression transgenic mice. Conclusions This study provides novel lead compounds and a ＂first-in- class＂ fluorescent probe for imaging NAMPT.
