Biological Activity Evaluation Bromodomain Inhibitors For Antitumor

Epigenetic regulation refers to a heritable change in gene function without a change in the DNA sequence of the gene,which ultimately leads to a change in phenotype.The main mechanism is the acetylation of histones,which can be involved in the regulation of many cellular processes,including transcription,chromatin remodeling,cell cycle,cell differentiation and DNA damage and repair.The bromodomain?BRD?is the"Reader"of acetylated lysine in histones.It is the only protein domain that recognizes and binds to histone acetylated lysine.It is known that there are 61 bromodomain codes in the human genome,which are present in 46 different proteins.According to their structural similarities,they are classified into eight families.BRD4 and CREBBP belong to the group II and III of BRD.They are closely related to the occurrence and development of various diseases.Importantly,they are become new targets for the treatment of various diseases.So far,several compounds targeting these two targets have been entered the clinical trial stage.The biological activity of BRD4 and CREBBP small molecule inhibitors is evaluated.Therefore,this research will provides ideas for the subsequent development of inhibitors and drugs.Firstly,a series of small molecule inhibitors of BRD4 and CREBBP synthesized in our lab were screened by AlphaScreen and TSA experiments.Among the BRD4 inhibitors,compounds W4 and W5 have strong binding affinity for BRD4?1?.In the TSA experiment,the?Tm of two inhibitors were 9.2oC and 9.9oC.Respectively,compound W5 selectivity was superior,and there was no activity on other group II bromodomain proteins.Among the CREBBP small molecule compounds,the protein activity of compound Q4 was the best,and the?Tm was 11.5°C on the CREBBP and EP300 proteins.Next,the three compounds were tested for cell viability assay on cancer cells.IC₅₀values on various cancer cells were tested using the CellTtier-Glo luminescence reagent.The results showed that compound W4,W5 and Q4 can inhibit the proliferation of a variety of cancer cells.Among them,W5 and Q4 significantly inhibited the proliferation of AR positive prostate cancer(VCaP,IC₅₀=0.29?M and 0.43?M)and leukemia cells(MV4;11,IC₅₀=0.40?M and 0.24?M);while W5 and Q4 also significantly inhibited prostate cancer cells,such as colony formation and cell migration of 22Rv1.Especially,the inhibition rate of cell migration of compound W5 at 1?M was 77%.In the qRT-PCR experiment,compound Q4 significantly inhibited the transcription of ERG,C-MYC and PSA genes in prostate cancer cell lines?VCaP?at a concentration of 5?M.The effect of compound Q4 on the expression of AR,AR-V7 and C-MYC in the 22Rv1 were analyzed by Western blot.The experimental results showed that the expression of C-MYC was significantly inhibited when the concentration was 16?M after treatment of 22Rv1 cells with compound Q4.In summary,this research filters effective small molecule inhibitors of BRD protein,and evaluates the biological activity in hematological tumor,leukemia and solid tumor prostate cancer,breast cancer.All of them explore the mechanism of BRD protein and discover small molecule inhibitors.This study also provides a theoretical basis of drug designing.