Discovery And Selectivity Mechanism Study Of DNA Methyltransferase Inhibitor

Epigenetics is defined as the study of stably heritable phenotype resulting from changes in a chromosome without altering the primary DNA sequence.DNA methylation is one of the most important epigenetic phenomena,which is mediated by DNA methyltransferases（DNMTs）.Three active DNMTs,namely DNMT1,DNMT3A,and DNMT3B,have been identified in mammals.Studies have shown that aberrant expression of DNMT lead to hypermethylation of tumor suppressor genes promoter.Thus,DNMT have attracted considerable interest as promising therapeutic targets for the treatment of tumor.DNMT inhibitors has a good prospect of clinical application for the modulation of the aberrant DNA methylation pattern in a reversible way.In general,DNMT inhibitors can be divided into two groups:the nucleoside analogs and non-nucleoside compounds To date,only two nucleoside analogues azacitidine and decitabine were approved by the US Food and Drug Administration（FDA）for the treatment of myelodysplastic syndromes（MDS）,acute myelocytic leukemia（AML）,but they show poor bioavailability and chemical instability,low specificity and significant toxic side effects,which limited their clinical application.In order to overcome these limitations,a lot of efforts have been made to discover non-nucleoside DNMTi using different strategies,most of them are not quite promising for lack of potency and selectivity.Therefore,developing DNMT inhibitors with high potency,low cytotoxicity and good selectivity not only contribute to the functional study of DNMT,but also important for the development of anti-tumor drugs.In the first part of this thesis,to get insight into the molecular mechanism of selectivity inhibition of DNMTs,we performed molecular dynamics simulations and umbrella sampling to reveal the molecular basis of the binding selectivity of three representative DNMT inhibitors（SFG,DC₀5 and GSKex1）.The simulation results shown the differences of unbinding pathways of three inhibitors dissociating from DNMT1 and DNMT3A.This study provided important information for the design of DNMT1 or DNMT3A selective inhibitors,and also provided theoretical guidance for the screening of DNMT3A inhibitors in the second part of the paper.In the second part of this thesis,by combining structure-based virtual screening and in vitro bioassays,compound DY-46 was identified as a novel and potent DNMT3A inhibitor with low micromolar potency.Then through similarity searching,compound DY-46-2(IC₅₀=0.15μM)was found to be more potent than DY-46.Further selectivity assays showed that DY-46-2 was a selective inhibitor of DNMT3A,which showed good selectivity for DNMT1,DNMT3B and histone methyltransferase G9a.In addition,DY-46-2 significantly inhibited cancer cell proliferation,including leukemia and colon cancer,and showed almost no cytotoxicity in normal cells.This study provides a promising scaffold of DNMT3A inhibitors with high potency and selectivity,which provides new idea for the research of DNMT inhibitors.In summary,in this study,the selective binding mechanism of DNMT inhibitors was studied through molecular dynamics simulation,and identified a novel DNMT3A inhibitor with high potency and selectivity through virtual screening combined with bioassays.The discovery of DNMT3A inhibitors not only contribute to the functional study of DNMT,but also important for the development of anti-tumor drugs.