Development Of Leading Compounds Of Inhibitors Targeting EZH2 Based On Synthetic Lethality

Synthetic lethality is a phenomenon that cells are death when two non-lethal genes mutated simultaneously.Presently,tumor therapy research based on synthetic lethality targets mutated genes by using RNAi high-throughput screening and CRISPR to find new targets.It has been reported that EZH2 has a synthetic lethal relationship with PBRM1,the subunit of SWI / SNF complex.EZH2 is abnormally overexpressed in a variety of cancers and is closely related to cancer aggressiveness and poor prognosis.EZH2 is a catalytic subunit of PRC2 and has histone methyltransferase activity.It further catalyzes the silencing of target genes related to occurrence and progression by catalyzing the trimethylation of histone H3K27,and further induces the occurrence and development of tumors.The purpose of this study is to find new inhibitors of EZH2,which has important theoretical and clinical significance for therapy with PBRM1 mutated cancer or EZH2 mutated cancer.In our study,we set the parameters for virtual screening by referring to the interaction between GSK126 and EZH2.We screened a total of 47,737 compounds by a two-round scoring strategy.The top 45 compounds with lowest amber scores and successfully docked into the binding pocket were selected.Then,the 45 compounds were classified and C769-0015,E218-0093,L309-0080,L501-1669 and M022-1782 were selected.Then we respectively used GSK126 and the siRNAs targeting EZH2 to treat Caki-1 and A704 to evaluate the efficacy of the putative EZH2 inhibitors.Then,we treated Caki-1 and A704 with the five potential inhibitors respectively to verify their efficacy.The relative survival was measured using the CCK8 kit.The results showed that only after L501-1669 treatment,the relative cell viability of A704 was lower than that of Caki-1.We further validated the effectiveness of L501-1669 by constructing a stable PBRM1-knockdown cell line using shRNA targeting PBRM1.The results showed that L501-1669 almost completely inhibited the cell proliferation of the PBRM1-knockdown cell line relative to the control cell line.Western blotting and the cell apoptosis assay were used to verified that L501-1669 effectively inhibited the methyltransferase activity of EZH2,and A704 showed increased apoptosis.Our study used computer virtual screening,cell biology,and molecular biology to obtain a small molecule compound targeting EZH2,provides valuable information for understanding the synthetic lethal interaction between PBRM1 and EZH2,and provide data for tumor molecular targeted therapy and drug discovery targeting EZH2.