Discovery Of Small Molecular Inhibitors Targeting Key Proteins BRD4 And Menin In Transcription Regulation And Investigation Of Mechanism

Epigenetics is a subject which aims to study the change of epigenomes’ expression and function in the process of regulation,including DNA methylation,histone modification etc.This process of epigenetics is independent of change of DNA sequence.Based on this,epigenetics can be defined as “any potentially stable and heritable change in gene expression which independent of change of DNA sequence”.The advancement of science help people explore the complexity of life,and after the achievement of Human Genome Project has made epigenetics promising research direction of life science.Epigenetics in gene regulation can be divided into two parts:(1)selective regulation in gene expression,including DNA methylation,histone methylation and histone acetylation.(2)regulation in post-transcription,including siRNA and miRNA.Notably,remodeling of chromosome,gene imprinting and inactivation of protein also belong to the scopes of epigenetics.BET protein family belongs to BRD protein family,including BRD2,BRD3,BRD4 and BRDT.BRD protein family are involved in the identification of histone lysine acetylation and this process is essential for epigenetics regulation.Among these proteins,BRD4 which known as one of BET protein family has been paid great attention to since the discovery of two inhibitors,JQ-1 and I-BET.BRD4 is consist of two domains named BRD4(1)and BRD4(2).BRD4(1)plays essential role in the leukemia model.It is reported that BRD4 can inhibit the transformation and development of leukemia cells by downregulating c-Myc gene.In this thesis,we will introduce a novel category of BRD4 inhibitors,which share a trimethoxy ring and target the first bromodomain of human BRD4 protein.The IC50 value of the most potent compound DC-BD-03 is 2.01 μM.In addition,high-resolution crystal structure of the compound DC-BD-29 which is the derivative of DC-BD-03 with the first bromodomain of BRD4 was determined,which revealed the binding mode and facilitated further structure-based optimization.These compounds exhibited anti-proliferation activity,caused cell cycle arrest,and induced apoptosis in human leukemia MV4;11 cells.Besides,the results of qPCR indicates that the compound DC-BD-03 can downregulate the expression of c-Myc,Bcl2,CDK6.Thus,the results presented indicated the potential of this series of compounds as drug candidates in the therapies of BRD4-related cancers.In addition,in the process of our research of protein-protein interactions(PPIs),the fact that BRD4 interacting with histones are mentioned in some reports.In a Nature article published in 2010,Bradner and his colleagues claimed that some small molecular inhibitors were able to block the interaction between BRD4 and histones,proving the concept of “PPI inhibitors can play essential roles in epigenetic regulations”.The interaction between menin and MLL is a typical PPIs.Translocation of MLL gene was found in acute myeloid leukemia(AML),acute lymphoid leukemia(ALL)and in the treatment of leukemia and Bone marrow hyperplasia of heterogeneous syndrome,thus it is suggested that translocation of MLL gene is the cause of leukemia.MLL can fused with a lot of cofactors such as AF4,AF9 and ENL.Among these,menin is one of these important cofactors.Menin interacting with MLL is essential for leukemogenesis and thus targeting the interface can be a novel way to design and develop anti-cancer drugs.With the research of the structure of menin-MLL complex,the mechanism of the interaction is further explained and two main binding motifs named MBM1 and MBM2 are interpreted respectively.MBM1 has been researched deeper than MBM2 and is believed as the main binding domain.It is essential to discovery and develop new inhibitors targeting the interaction of menin-MLL due to the background of these related research.Our library has screened the compound library which contains 20000 compounds by FP assay.Thermal Shift assay is used to validate the activity of these compounds.Moreover,we choose MLL-fusion cells MV4;11 and KOPN-8 to test the inhibition activity of these compounds on proliferation.We have explored the crystal structure of menin and complex of menin and compounds in order to validate these compounds targeting menin-MLL.We have constructed and obtained the sequence of menin suitable for crystallization.More attempts should be continued in the future.