| Epigenetics usually means the heritable changes in gene expression that do not involve changes to the underlying DNA sequence — a change in phenotype without a change in genotype.Epigenetic regulation has various forms,which control cell fate and regulate the normal growth and development of individuals in an orderly manner,and the process is reversible.It also plays critical roles in the occurrence and development of various diseases,including tumor,diabetes,heart disease and intestinal diseases.Therefore,epigenetics and epigenetic-targeted drug development has been a hot topic of research.The main research contents of epigenetics include: methylation of DNA,modifications of histones,non-coding RNAs,and so on.So far,there are 8 inhibitors have been approved,belonging to three targets.They are 2 DNA methylase inhibitors,5 histone deacetylase inhibitors,and 1 isocitrate dehydrogenase(IDH)inhibitor.The polycomb repressive complexe 2(PRC2)is one of the important histone methyltransferases.It is a complex consisting of subunits as EZH2,EED,SUZ12 and Rb Ap46/48,which mainly catalyzes the methylation of H3K27,resulting in the trimethylation of H3K27 in the promoter region and transcriptional silencing of the target gene.PRC2 affects multiple biological processes of cells mainly through transcriptional silencing target genes.Abnormal expression and/or mutation of EZH2 are found in tumors such as lymphoma,pancreatic cancer,melanoma,breast cancer,bladder cancer,and gastric cancer.Therefore,inhibition of the activity of the PRC2 complex is considered to be an emerging means of treating tumors,and many scientists have devoted a lot of energy to this.However,no compounds have been approved at present.Most of the inhibitors targeting PRC2 are enzymatic inhibitors against EZH2,and some are undergoing clinical trials.Accumulating evidences indicated that beyond playing its role in a PRC2-dependent manner as a histone modifier,EZH2 also functions both as a transcriptional suppressor and a transcriptional co-activator,not depending onH3K27me3 and on the different cellular contexts,such as Actin,GATA4,AR and ER.Therefore,inhibition of the enzymatic activity of EZH2 alone does not potently inhibit the proliferation of tumor cells which express high expression of EZH2 and/or mutant EZH2.H3K27me3 recognition by EED is essential in stimulating basal PRC2 activity and propagating H3K27 methylation in repressive chromatin for gene silencing.And the binding of H3K27me3 to EED causes a conformational change of the stimulation-responsive motif in EZH2,leading to the enhanced catalytical efficiency,which can be illustrated by crystal structures of the PRC2 complex.This implicated that finding disabled the PRC2 complexes in cancer by destroying protein interactions is an alternative strategy for development of PRC2 inhibitors.In the present study,we aimed to find a pharmacologic approach to supress the PRC2 function by disruption of PRC2 complex.Therefore,firstly,we used the novel bioluminescence energy resonance transfer(Nano BRET)method to establish a high-throughput screening platform for compounds screening in living cells.In addition,we had also established evaluation method for such inhibitors based on fluorescence polarization(FP)at the molecular level.Then,an in-house compound bank more than 1000 listed drugs were screened using Nano BRET method.A potential compound was discovered and its anti-tumor effect and mechanism were further evaluated.1.Establish methods based on FP and Nano BRET,a high-throughput screening platforms for EZH2-EED protein interaction inhibitors screeningCompound screening methods based on the physical phenomenon of fluorescence polarization have become the most important screening method for PPI inhibitors at present,and can be used to select compounds for target selectivity screening at the molecular level.However,the potential compounds obtained at the molecular level screening always tend to behave in general at the cellular level evaluation.Therefore,in our screening,we hope to establish a high-throughput screening model for direct screening evaluation at the cellular level.Nano BRET is a novel bioluminescence energy resonance transfer method and can be used for compounds screening in living cells,which are mainly applied in this study.Byconstructing two protein fusion expression plasmids by combining EZH2 and EED proteins with a fluorescent donor and a fluorescent receptor respectively,these plasmids are simultaneously introduced into the tool cells for target proteins expression.The interaction of EZH2 and EED allows the fluorescent donor and the fluorescent acceptor to be sufficiently close.With the addition of the reaction substrate,the fluorescence produced by the fluorescent donor catalyzes the substrate,which diverts and activates the fluorescent donor to emit fluorescence.When incubating the tool cells with compounds together,it is possible to determine if the compound can disrupt the interaction between the EZH2-EED proteins by measuring the intensity and ratio of the two fluorescent signals.By optimizing the experimental conditions,we found the optimal expression plasmid combination EZH2 FN31N+EED FC14 H,that is,the N-labeled fluorescent donor Nano Luc in the EZH2 protein,and the fluorescent receptor Halo Tag in the C-segment of the EED protein,and the two plasmids were transfected into HEK293 T tool cells according to the ratio of 1:1,which had better signal values and sensitivity.The Z’ factor is about 1,which means the method has good stability and reliability,and can be used to EZH2-EED PPI inhibitor high-throughput screening at the living cell level.2.Discovery of AZD9291 as a lead compound targeting EZH2-EED disruption and evaluation of its antitumor effectBased on our established inhibitor screening platform,we screened more than 1,000 compounds in our in-house compound bank,and found that AZD9291 disrupted the interaction between EZH2 and EED significantly.The compound is a three-generation EGFR inhibitor that was approved by the US Food and Drug Administration(FDA)in 2015 for the treatment of non-small cell lung cancer with EGFR T790 M mutation.Our results indicated that AZD9291 can directly bind to EZH2 protein,disrupt the interaction between EZH2-EED,inhibiting tumor cell proliferation,which was independent on its inhibition of EGFR signaling pathway.In addition,we also found that this compound not only inhibited the binding of EZH2-EED,thereby decreasing the stability of PRC2,but also decreased the expression of EZH2 m RNA,which then affected the expression of EZH2-regulated genes and inhibited motility of tumor cells.The results of mic RNA Array analysis indicated that AZD9219 may through down-regulating the expression of mi R-34 a,decreased EZH2 m RNA,and inhibited EZH2 protein expression.3.In a classical way,micro RNAs(mi RNAs)regulate m RNA expression through the binding between the 5’-end seed sequence and the 3’UTR(3’-Untranslated Region)region of the m RNA.These two regions eventually integrated into the RNA silencing complexes and regulate gene expression based on their complete pairing levels and site.However,some studies have shown that some mi RNAs can regulate genes expression by targeting the 5’UTR and CDS regions of m RNA.Even some mi RNAs do not need to bind to the m RNA through their seed sequences,but regulate the expression of m RNA through other bases.Using the NCBI database to perform Blast on the 3’UTR region of mi R-34 a and EZH2,we found that mi R-34 a and mi R-3157 could directly bind to 3’UTR of EZH2 though a non-classical seed sequence,inducing EZH2 m RNA degradation,then decreased the protein level of EZH2.This new regulation model may be a complement to the existing regulation mechanisms of mi RNAs,which need to be further confirmed.In summary,we established a screening platform for EZH2-EED PPI inhibitors,used this platform and found the potential compound AZD9291,evaluated its anti-tumor effects and regulation mechanism.The discovery of this compound can provide a new structure for the development of such kind inhibitors. |
PDF Full Text Request