Establishment Of Screening Platform For Small Molecular Inhibitors Of BRD4 And ALKBH5 And Screening And Activity Evaluation Of Inhibitors

Chromatin is a polymer complex of DNA and histone,which is the cage constructin of human genome.The heritability of eukaryotes depends on the chromatin.Histone posttranslational modifications are important regulatory mechanism of chromatin structure and function.The functions caused by different modifications make the structure and function of chromatin more diverse and complex.BRD domain is the unique protein domain which can recognize the histone acetylation modification,mediating interaction of proteins.BRD is a highly conserved protein composed of about 110 amino acids.According to the similarity of sequence length,proteins containing BRD domain can be divided into nine protein families.The largest and most studied of these families is the BET family that consists of BRD2,BRD3,BRD4 and BRDT.Proteins of BET family all have two BRD active domains BD1 and BD2 and an extra-terminal(ET)region,which bind to acetylated lysines found in H3 and H4 as well as nonhistone proteins.BRD4 is a very important protein in BET family.Its substrate is widely,including acetylation of histone lysine such as H3K9ac,H3K27ac,H3K14ac,H4K5ac,H4K16ac an so on,also some nonhistone substrate,as P-TEFb,cell cycle protein T1.BRD4 can also interact with JMJD6,cell cycle protein T1/T2,CDK9 and format complex to regulate transcription.In addition,BRD4 also has certain function in oxidative stress response,influences KEAP1/NRF2 signal pathway and also participates in DNA damage response.In the development of disease,BRD4 participate in the occurrence and development of various diseases.BRD4 is abnormal expression in a variety of tumors,including almost all kinds of blood tumors as AML,MLL,lymphoma,myeloma,and solid tumors such as testicular cancer,lung adenocarcinoma,prostate cancer,malignant pancreatic cancer,melanoma,maglignant glioma,etc.BRD4 inhibitors can be used for research and treatment of many diseases.So,BRD4 can be used as a drug target and the discovery of inhibitors is a great significance for the prevention and treatment of tumors.In addition,the modification of RNA plays an important role in RNA translation,shearing and other biological functions,affecting a variety of biological processes.m~6A is the most common modification in RNA modification,affecting almost all aspect of RNA metabolism,including RNA splicing,nuclear output,translation,efficiency and stability.It also plays a key role in sperm development,stem cell differentiation,metabolism and tumor development.ALKBH5 is the second discovered m~6A demethylase following FTO,specific removing the methylation of6-N in single strand DNA and RNA.It regulates the methylation balance of RNA with RNA methyltransferase.In function,the expression of related genes in the p53apoptosis pathway in ALKBH5 knochout mice was changed.ALKBH5 is also overexpress in glioma stem cell.Slisencing ALKBH5 can inhibit the proliferation of tumor stem cells,affecting the expression of ALKBH5 target gene.ALKBH5 crystal structure has been parsed out,on the basis of this,the study of ALKBH5 small molecular inhibitors will promote further research on the biological functions of mRNA m6A modity and provide important basis for drug design of related disease.In our research,we choose two active domains of BRD4 to establish pGEX4T-1-BRD4(BD1)and pGEX4T-1-BRD4(BD2)prokaryotic expression vector,and use prokaryotic expression system and affinity chromatography furfication to get BRD4(BD1)and BRD4(BD2)recombinant proteins.The acetylated histone H4 was synthesized as substrate.Two recombinant proteins were evaluated by HTRF method and reaction conditions were optimumed.After these,we use positive compound JQ-1 evaluate the system and get better results.A high throughput screening platform for BRD4 small molecule inhibitors was successfully established.Then we evaluated the BRD4 inhibitory activity of a series of pyrazole compounds and get a better activity compound WS-722.We use dilution method to verify that the inhibitory effect of WS-722 on BRD4 was reversible in vitro.After this,the mechanism of cell level was studied.Results show that WS-722 can inhibit the proliferation of leukemia cell THP-1.Further research has been found that WS-722can inhibit BRD4 activity and down-regulate the expression of its target gene c-Myc.What's more,WS-722 can promote the differentiation of leukemia cells,and show better antitumor activity,which lays a foundation for the discovery and optimization of such new BRD4 inhibitors in the future.Besides,based on the previous work,we use constructed prokaryotic expression vector pET28b-FDH and pMCSG19-ALKBH5 and prokaryotic expression system and affinity chromatography furfication to get FDH and ALKBH5 recombinant proteins.Based on FDH enzyme-coupled fluorescence method and AlphaScreen method,ALKBH5 activity detection system was successfully constructed.Then we optimized the system conditions and evaluate the activity detection system using a reported weak inhibitor citrate.The HTS platform of ALKBH5 small molecule inhibitor was successfully established,which will lay the foundation for the subsequent research on the biological function of ALKBH5 and the drug discovery of related diseases.