Lead Compounds Discovery And Mechanism Studies Of Epigenetic Methylation Modification Related Proteins

Histone methylation modification and mRNA methylation modification are both part and parcel of epigenetic modification.The former participates in transcription regulation and the latter takes part in regulation of the processing,maturation,the translation process and the stability of target mRNA.Both of them regulate the expression of proteins crucial to cell functions.Among them,researches focus on histone lysine methylation and m~6A containing mRNA gained much more interests.Under normal conditions,these modifications precisely regulated by their“writers”,“erasers”and“readers”promote multiple important cellular processes like embryonic development,cell differentiation,DNA damage response,X-chromosome inactivation and neuron development.And the aberrant expression of these regulators,overexpression in most cases,usually breaks the homeostasis and leads to a variety of diseases like diverse malignant tumors.Nowadays,DOT1L(Disruptor of Telomeric Silencing 1-Like),YTHDF1(YTH Domain-Containing family protein 1)and YTHDF2are three epigenetic targets attracting a large amount of research.DOT1L is the only histone lysine methyltransferase without the SET domain,catalyzing the dimethylation of histone 3 lysine 79(H3K79).The existing structural and functional studies mainly focus on DOT1L's N terminal catalytic domain,but the function and structure of the remaining region are still waiting to be completely uncovered.There are dozens of small molecule inhibitors of DOT1L been reported.However,though nucleoside analogs hold better activity and selectivity,they share similar skeletal structure with poor stability and pharmacokinetic parameters which may also cause drug resistance.Besides,those don't belong to nucleoside analogs are always less effective with poor selectivity.Moreover,EPZ-5676,the only candidate entered in clinical trials,failed at last.When it comes to YTHDF1 and YTHDF2,the structural study of the targets is far from enough and the available studies so far are only focus on the YTH domain.The researches relating to the functions and mechanism of these two proteins are largely insufficient either.Nevertheless,not a single small molecular inhibitor of the two targets has been discovered and reported.Thus,it would be of great significance to develop new potent inhibitors of these targets with good selectivity.In this dissertation,we synthesized series of new DOT1L inhibitors by introducing amino side chain into a previously reported lead DOT1L inhibitor or by structural modifications on it.Via some enzyme activity and binding detections on molecular level,we confirmed that the newly synthesized compounds could bond to DOT1L and possess inhibitory activities.Then,results of cell proliferation inhibition assay,real-time quantitative polymerase chain reaction(qPCR)assay,western blot assay and cell cycle detection suggest that these new inhibitors could inhibit leukemia cell proliferation by targeting DOT1L and inducing G0/G1 phase cell cycle arrest.Moreover,binding mode and structure-activity relationship(SAR)analysis may also help further optimization.As for YTHDF1,we found the compound DC-Y20 as a hit for YTHDF1 inhibitor from our in-house compound library through fluorescence polarization(FP)based high throughput screening(HTS).We then verified that DC-Y20 could bond and block the interactions between YTHDF1 and m~6A containing mRNA by performing FP and AlphaScreen assays,as well as several qualitative and quantitative binding experiments.Besides,data obtained form competition assays,HDX MS and mutation indicates that DC-Y20 is a non-competitive inhibitor of YTHDF1 and may block its interaction with m~6A by inducing significant conformational change of the protein.When it comes to YTHDF2,we got compound DC-Y12 via FP based HTS as well.After derivatives searching and verification,we discovered a better compound DC-Y13 and confirmed the skeleton of the hits.And at last,though structure modification based on DC-Y13,we found the derivative DC-Y13-27 with improved activity and water solubility for further research and modification.In conclusion,combined with molecular and cellular biochemical assays,we did some structure modifications and found several DOT1L inhibitors with better cell proliferation inhibition activity,which may provide help and reference for further developing of the non-nucleoside inhibitor.We also discovered the first inhibitor hits of YTHDF1 and YTHDF2 respectively,which may be the lead compounds used for further drug discovery research and exploration of the functions and mechanisms of the targets,as well as the relationships between them and diseases.