Synthesis,Evaluation And Mechanism Study Of BRD4 Small Molecule Inhibitors

Epigenetics refers to changes in heritable gene expression or cell phenotype through certain mechanisms without changing the DNA sequence.These changes can be mediated by changes in DNA methylation,histone modifications(including histone acetylation,phosphorylation,methylation,ubiquitination and proteolysis,etc.)and chromatin remodeling.There is increasing research evidence that epigenetic disorders are a common mechanism of cancer.Bromodomains(BRDs)proteins are epigenetic readers for histone acetylation involved in chromatin remodeling and transcriptional regulation.Bromodomain-containing protein 4(BRD4)is a member of the second bromodomain and extraterminal domain(BET)protein family in the BRDs protein family.It is also the most deeply studied BET protein.It has two conserved bromodomains BD1 and BD2,which can specifically recognize and bind acetylated lysine on histones and non-histones,recruit and activate positive transcription elongation factor b(P-TEFb),phosphorylating RNA Pol Ⅱ,thereby extending the transcription of some oncogenes(such as c-Myc).A large number of studies have shown that BRD4 is an epigenetic regulatory factor involved in the regulation of cell cycle,cell growth and apoptosis-related gene transcription,and is highly expressed in a variety of solid tumors and hematological tumors,and is associated with multiple carcinogenic drivers in tumors.The upregulation is also closely related to the occurrence and development of various cancers,fibrosis,inflammation and cardiovascular diseases.Therefore,the development of BRD4 inhibitors to obtain selective and effective BRD4 inhibitors has become a hot field of drug research for the treatment of solid tumors,hematological tumors,fibrosis,inflammation and cardiovascular diseases.The content of this subject is mainly based on BRD4 as an anti-tumor target,designing and synthesizing new small molecule inhibitors and evaluating some anti-tumor activities.The content of the work is roughly divided into three parts:(1)Design,synthesis and activity evaluation of new dihydropyridinone-sulfonamide small molecule inhibitors;(2)Research on the mechanism of action of new BRD4 inhibitor protein levels;(3)Research on the antitumor mechanism of new BRD4 inhibitors at the cellular level.The details are as follows:(1)Design,synthesis and activity evaluation of novel dihydropyridinone-sulfonamide small molecule inhibitorsAfter reading a lot of literature and the previous research results of the research group,a series of 30 dihydropyridinone-sulfonamide compounds were designed with the positive compound BI2536 as the research object,and then through high-throughput screening,it was found that two thirds The compound has good enzyme activity inhibition on BRD4(BD1),of which compound 13 has an IC50 value of 443.66±2.65 nM for BRD4(BD1)activity inhibition;14 has an IC50 value of 45.79±1.66 nM for BRD4(BD1)activity inhibition;Compound 15 has an IC50 value of 60.33±1.78 nM for the inhibition of BRD4(BD1)activity.Through biological activity evaluation and structure-activity relationship analysis,the following studies were carried out with compounds 13,14 and 15 as the main research objects.(2)Research on the mechanism of action of new BRD4 inhibitor protein levelsIn order to further determine the mechanism of action of the three new BRD4 inhibitors 13,14 and 15 at the protein level,we confirmed through in vitro enzyme activity screening experiments that compounds 13,14 and 15 also have good enzyme activity on BRD4(BD2)inhibition.Compound 13 had an IC50 value of 235.76±2.4 nM for BRD4(BD2)activity inhibition;14 had an IC50 value of 103.21±2.01 nM for BRD4(BD2)activity inhibition;compound 15 had an IC50 value of 114.13 ± 2.06 nM for BRD4(BD2)activity inhibition.Through the study of dilution method,it was found that compounds 13,14 and 15 like the positive compounds BI2536 and(+)-JQ1(JQ1),are reversible inhibitors,which can reversibly bind to BRD4(BD1)through non-covalent bonds and inhibit activity.In addition,we also found that in THP-1,compounds 13,14 and 15 upregulate the expression of tumor suppressor p21,promote THP-1 block G1 phase and THP-1 apoptosis to play an anti-tumor effect.(3)Research on the antitumor mechanism of new BRD4 inhibitor cellsIn the leukemia cell THP-1,it was confirmed by cellular thermal shift assy that compounds 13,14 and 15 can stabilize BRD4 in THP-1 in a concentration-dependent manner.Through cytotoxicity evaluation,we found that compounds 13,14 and 15 inhibited the proliferation of gastric cancer cells(BGC-823,SGC-7901,MKN-45 and MGC-803)and leukemia cells(THP-1)in a concentration-dependent manner.In leukemia cells THP-1 and HL60,we found that after incubating compounds 13,14 and 15 with leukemia cells for 24 h,compounds 13,14 and 15 could down-regulate the expression of c-Myc and exert an anti-tumor effect.In addition,we also found that in THP-1,compounds 13,14 and 15 can up-regulate the expression of tumor suppressor p21,promote THP-1 block G0/G1 phase and apoptosis play an anti-tumor effect.In summary,a total of 30 dihydropyridinone-sulfonamide compounds were synthesized in this subject.Through BRD4 activity screening,most compounds were found to have good activity.Through activity evaluation and structure-activity relationship analysis,further studies were conducted on compounds 13,14 and 15.Compounds 13,14 and 15 reversibly inhibit the activity of BRD4(BD1)and inhibit the proliferation of gastric cancer cells BGC-823,SGC-7901,MKN-45 and MGC-803)and leukemia cells(THP-1).Through further research,it was found that in THP-1,compounds 13,14 and 15 can down-regulate c-Myc,up-regulate the expression of p21,and show better antitumor activity by promoting THP-1 blockade in G0/G1 phase and apoptosis of THP-1.It provides an important reference basis for the research and development of new anti-tumor drugs targeting BRD4.