Lead Compounds Discovery And Mechanism Studies Of The Epigenetic Factor CECR2

Histone acetylation is an important histone modification of proteins,which engaged in many biological processes such as transcription,metabolism and cell signaling.Histone acetylation in chromatin opens chromatin structure,activates transcription.Conversely,histone deacetylation leads related genes silencing.The recognition of histone acetylation is mediated by the bromodomain,an evolutionarily highly conserved protein domain that exists in a wide variety of chromatin and transcription-related proteins.CECR2 is an epigenetic regulator with bromodomain that recognizes acetylated lysine residues.CECR2 participates in chromatin remodeling with SNF2H and SNF2L,and also plays a key role in DNA damage response,neurogenesis,and spermatogenesis.The mutation and abnormal expression of CECR2 are closely related to cat’s eye syndrome,breast cancer metastasis and laryngeal squamous cell carcinoma.NVS-CECR2-1 and GNE886 are currently highly effective selective inhibitors of CECR2,but there are some problems such as high cytotoxicity and unclear functions and mechanisms related to CECR2.Therefore,small molecule inhibitors targeting CECR2have greatly significance and prospects for the study of biological functions of CECR2and drug development of related diseases.In this study,we established a high-throughput screening method for CECR2 small molecule inhibitors based on Homogeneous Time Resolved Fluorescence（HTRF）technology,and optimized the reaction conditions including protein concentration,peptide concentration and DMSO concentration.The stability and reliability of this method were verified by the determination of Z-factor and IC₅₀ of the positive compound NVS-CECR2-1,and it is suitable for high-throughput screening of CECR2inhibitors.Based on the established high-throughput screening method for CECR2small molecule inhibitors,eight candidate compounds with good inhibitory activity were obtained after two rounds of screening,with IC₅₀ less than 1μM.The binding of candidate compounds and CECR2 protein was further confirmed by Micro-Scale Thermophoresis,Carr-Purcell-Meiboom-Gill（CPMG）and Saturation Transferred Difference（STD）,and the possible false positive results of HTRF experiment were excluded.Finally,the hits compound BAY was obtained,with IC₅₀=390 n M and K_d=4.27μM.The selectivity was verified by measuring its inhibitory activity against BRD family proteins,and the binding mode between BAY and CECR2 bromodomain was analyzed by molecular docking.Since BAY showed good activity at the molecular level,we further explored its activity and mechanism at the cellular level.The anti-tumor activity of BAY was verified by proliferation inhibition experiment.The proliferation of colon cancer,breast cancer,liver cancer and other tumor is inhibited by BAY.The effect of BAY on the migration of breast cancer cells was detected by scratch test,and the inhibitory effect of BAY on the migration of breast cancer cells was verified.The determination of NF-κB protein expression via Western blot assay,and BAY inhibited the expression of NF-κB protein p65 and p50,and inhibited the binding of CECR2bromodomain to acetylated p65.In conclusion,this study established a high-throughput screening method for CECR2 small molecule inhibitors based on HTRF technology,which laid a foundation for the discovery of novel CECR2 small molecule inhibitors.The novel chemical structure of hits compounds found through high-throughput screening also provides a new direction for the design and Structural optimization of novel small-molecule inhibitors of CECR2,provides a chemical probe for the study of biological functions of CECR2,and supplies a new idea for the related diseases treatment.