Design,synthesis And Biological Characterization Of BRD4 Fluorescent Ligands

Bromodomain-containing protein 4(BRD4)is an important member of bromodomain and extra-terminal domain(BET)protein family in the BRDs protein family.As an important transcription and epigenetic factor,it extensively regulates gene expression and plays an important role in many physiological activities.BRD4 is over-expressed in many malignancies by extending the transcription of some oncogenes(such as c-Myc and c-Jun)to regulate the growth of tumor cells and it has been considered as an attractive drug target for cancer treatment.Considering that BRD4 can specifically recognize acetylated lysine(KAc),a variety of BRD4 inhibitors have been developed and they can effectively inhibit tumor growth and metastasis.Therefore,the development of BRD4 fluorescent ligand(probe)is expected to achieve the simultaneous imaging and inhibition to BRD4.Moreover,the development of BRD4 imaging agents may provide an important tool for the biological evaluation of inhibitors,the detection and identification of tumor target,and the visualization of tumor treatment.Herein,we describe the design,synthesis,and biological evaluation of two types of novel BRD4-targeted fluorescent ligands based on a potent BRD4 inhibitor 1.The content of the research work is as followed:(1)Design,synthesis and pharmacological evaluation of dye fluorescent ligands targeting BRD4.In this section,four new 1,8-naphthalimide-based fluorescent ligands were designed by using computational simulation analysis.They were synthesized,characterized by NMR and HRMS,and were pharmacologically characterized by evaluating their inhibitory to BRD4.Although they displayed decreased inhibitory effect for BRD4 compared to compound 1(IC50 value of 43.13 n M),it is basically at the same level.Among them,probe II-1 showed an acceptable decreases in inhibition to BRD4 with an IC50 value of 93.21 n M.Moreover,these probes could display excellent fluorescent properties for visualization of tumor cells,and probe II-2presented a pronounced fluorescent signal with high fluorescent labeling efficiency at1 μM and it could perfectly distinguish tumor MGC-803 cells and normal GES-1cells with low toxicity.The excitation wavelength is 468 nm,and the emission wavelength is 540 nm.In addition,compound 1 and these probes all exhibited excellent inhibitory activity against tumor cell lines,and among them probe II-1showed an excellent inhibitory activity with an IC50 value of 8.53 μM and 8.64 μM,respectively.Therefore,these results may lay a promising foundation for the visualization of tumor treatment process.(2)Design,synthesis and pharmacological evaluation of GSH-responsive fluorescent ligands targeting BRD4.Based on the high expression of GSH in tumor cells,we designed and synthesized a type of GSH-triggered BRD4 fluorescent ligands probe III 1-3 by using computational simulation analysis.They were synthesized,characterized by NMR and HRMS,and were pharmacologically characterized by evaluating their inhibitory to BRD4.Among them,Probe III-3 exhibited excellent GSH responsiveness,compared to III-1 and III-2,with a fluorescence emission wavelength of up to 495 nm.Moreover,probe III-3 also showed higher inhibitory activity than III-1 and III-2,with an IC50 value of 29.69 n M.In addition,the fluorescence signal could be turned on by intracellular GSH and probe III-3 could be successfully used for tumor cell imaging.In addition,these probes all showed a good inhibitory effect on tumor cells.Among them,probe III-3 showed higher inhibitory activity than compound 1,with an IC50 values of 5.48 μM and 1.82 μM for MGC-803 and THP-1,respectively.Compared to the dye fluorescent ligands,GSH-triggered fluorescent ligands can realize the visualization of the tumor treatment process or the integration of diagnosis based on BRD4-targeting capability and tumor microenvironment response.In summary,we herein describe the design,synthesis,and pharmacologically characterization of two types of BRD4 fluorescent ligands.Some potent BRD4 fluorescent ligands at the protein and cell levels were obtained and they could also distinguish tumor cells from normal cells with excellent membrane permeability.Our researches realized the integration of diagnosis and treatment,and laid a solid foundation for the visualization of tumor treatment.