Drug Discovery And Mechanism Studies Of Lead Compounds Targeting The Polycomb Repression Complex 2

Polycomb repressive complex 2(PRC2)is one of two main Polycomb group(PcG)protein complexs,which catalyzes the mono-,di-and tri-methylation of histone H3 at lysine 27(H3K27me1/2/3),altering the chromatin structure and maintaining the silence of specific target genes.PRC2 comprises three core protein subunits,enhancer of zeste homologue 1/2(EZH1/2),embryonic ectoderm development(EED),and suppressor of zeste 12(SUZ12),which are the basic unit of enzyme catalytic activity.EZH1 or EZH2 is the catalytic subunit of PRC2.Acting as a major proto-oncogene,EZH2 usually occurs abnormal over-expression or gain-of-functional mutations in many malignant cancers,such as breast cancer,prostatic cancer,diffuse large B-cell lymphomas(DLBCL),and follicular lymphoma(FL),leading to high levels of H3K27me3.Therefore,EZH2 and other core subunits of PRC2 are regarded as critical drug targets.Over the past decade,an increasing number of PRC2 inhibitors have been widely researched and developed,which are classed into four groups:EZH2 inhibitors,EZH2-EED protein-protein interaction(PPI)inhibitors,EED-H3K27me3 PPI inhibitors and PRC2 degraders.To date,only one EZH2 inhibitor,EPZ-6438(Tazemetostat),has passed the US FDA application for the therapy of advanced epithelioid sarcoma.Other PRC2 inhibitors are currently still in different preclinical and clinical trials stages.Although PRC2 inhibitors are promising in cancer therapy,there is an urgent demand for more potent inhibitor development.We aim to develop novel PRC2 inhibitors and elucidate their mechanism of actions,paving the way for the research and development of domestic innovative anticancer drugs.For the discovery of PRC2 inhibitors targeting the enzymatic site or PPI interface,three kinds of high-throughput screening(HTS)assays are established.The first HTS assay is a PRC2 enzymatic activity assay based on HTRF method,with a Z'-factor of 0.57,which could be used for the evaluation of inhibition activities of compounds on PRC2 catalytic reaction.The second HTS assay is developed based on AlphaScreen method to target the EED-H3K27me3 PPI.The HTS parameter Z'-factor is 0.84 representing robust for HTS campaign.This assay can also be used to test the inhibition activities of EED-H3K27me3 PPI inhibitors.The last developed HTS assay is a cell lysate-based AlphaLISA assay which is used for the discovery of EZH2-EED PPI inhibitors.Its Z'-factor is 0.56.Then a compound library composed of clinical or FDA drugs and natural products was used for HTS campaign,and several natural products were discovered as hits targeting the EZH2-EED PPI.Taken together,the above three HTS platforms lay the foundations for the discovery and development of novel inhibitors against PRC2-dependent cancers.A fast-follow study based on EED-H3K27me3 PPI inhibitor EED226 is done in this paper.About 241 of EED226 analogs are synthesized and evaluated by in vitro and in vivo assays,such as HTRF-based PRC2 activity assay,AlphaScreen-based EED-H3K27me3 PPI assay,EED protein thermal shift assay,DLBCL cells proliferation assay,live microsome metabolism assay,animal pharmacokinetics and efficacy studies.In the end,SL-ZYE-07 is selected as a candidate for preclinical study based on its excellent more potent inhibitory activities than EED226 and other analogs.The inhibitory activities of SL-ZYE-07 on PRC2 containing EZH2 wide type and EED-H3K27me3 PPI are 7.3 nM and 6.9 nM respectively.And the inhibitory activities on DLBCL proliferation are in the range of 4.7?21.1 nM.SL-ZYE-07 dose-dependently decreases the global H3K27me3 levels and upregulates the expression of PRC2 downstream target genes in DLBCL cells.SL-ZYE-07 treatment(25 mg/kg,p.o.,BID)leads to the tumor regression completely in Pfeiffer and KARPAS-422 xenograft mice models.The co-crystal structure of SL-ZYE-07 in complex with EED protein provides structural basis for development of more potent inhibitors in the future.At present,the candidate SL-ZYE-07 is in the preclinical study phase,which will be a promising domestic innovative drug for the PRC2-dependent cancer therapy.Following the first reported small molecular inhibitor astemizole against EZH2-EED PPI,we focused on the structure-guided development of novel potent EZH2-EED PPI inhibitors.Firstly,the co-crystal structure of astemizole in complex with EED protein was disclosed and the detailed binding mode between astemizole and EED protein was elucidated.Secondly,the structure of astemizole was optimized and the analogs were tested in fluorescence polarization(FP)assay,followed with structure activity relationship(SAR)study.Finally,a novel inhibitor DC-PRC2in-01 with 15-fold more potent than astemizole was discovered.The IC50 value of DC-PRC2in-01 in FP assay is 4.21 ?M,with binding affinity Kd of 4.56 ?M to EED protein.DC-PRC2in-01 disrupted the EZH2-EED complex,and induced the degradation of PRC2 core components and decrease of global H3K27me3 levels in DLBCL cells.The antiproliferation activity of DC-PRC2in-01 was determined in DLBCL cells with low micromole IC50 values and the mechanism of action(MOA)of growth inhibition is in part due to the G0/G1 arrest.Synergy effect between DC-PRC2in-01 and GSK126 was observed in DLBCL cells.In summary,our work has made progress in the development of a new potent inhibitor against EZH2-EED PPI.DC-PRC2in-01 could be an effective chemical probe for the investigation of PRC2 related physiology and pathology,and provides scaffold basis for further development of more potent inhibitors.