Discovery And Mechanism Of Action Study Of Small Molecule Inhibitor Targeting 53BP1-TTD

Background and Objective:Tumor suppressor p53-binding protein 1（53BP1）,a tantem tudor domain（TTD）protein,takes part in DNA Damage Repair（DDR）pathways through the specific recognition of lysine methylation on histones.The dysregulation of 53BP1 is closely related to the development of many diseases including cancer.Moreover,recent studies found that deficiency of 53BP1could increase the efficiency of precise CRISPR/Cas9 genome editing.Thus,discovery of inhibitor is beneficial to the study of biological functions of 53BP1 and the application of CRISPR/Cas9 genome editing.UNC2170 and its derivatives have been reported as 53BP1targeted small molecular inhibitors with modest activities.Therefore,it is of great significance to develop novel small molecule 53BP1 inhibitors with novel structure and strong activity.Methods:This methods of this study consists of three parts.Part 1:In order to find novel and effective precursor compounds,AlphaScreen assay was established as an in vitro biochemistry high-throughput screening platform.Part 2:3447compounds with diverse structures were screened in the laboratory by the AlphaScreen assay.And the affinity between DP308 and53BP1-TTD was detected by Microscale Themophoresis（MST）and Surface Plasmon Resonance（SPR）in vitro biophysical methods.The possible binding modes of DP308 and53BP1 were investigated by analyzing the structure-activity relationship（SARs）of DP308and its derivatives through molecular docking.Part 3:The co-crystal structure of 53BP1-TTD and DP308 complex is pursued.Results:The results of AlphaScreen assay showed that a highly stable and effective screening platform was established,with Z’factor of 0.82 and S/B of 10.01.The inhibition rate of positive polypeptide and positive inhibitor UNC2170 was also consistent with the reported activity results,and a novel and effective compound,DP308,was successfully screened with IC₅₀=1.69±0.73μM.MST and SPR results showed that the affinity（K_d）between DP308 and 53BP1 was 2.69μM and 2.71μM,respectively,indicating that DP308 had a good affinity with 53BP1 in vitro.The results of SARS and molecular docking showed that the small molecule inhibitor,DP308,interacts with the aromatic cage of 53BP1-TTD protein and occupied the substrate binding pocket.Conclusions:In this study,we discovered a novel and effective chemical scaffold with the IC₅₀ value of1.69±0.73μM through an AlphaScreen-based high-throughput screening assay.The binding affinity between DP308 and the 53BP1-TTD protein was detected by MST（K_d=2.69μM）and SPR（K_d=2.71μM）,which matches the IC₅₀ value.Further molecular docking study provided comprehensive explanation for the binding mode between DP308 and the53BP1-TTD protein.According to our results,quinoline was important for compound interacting with the aromatic binding cage of the first tudor domain of 53BP1-TTD protein.Moreover,formation of a direct hydrogen bond between amide NH and Met1584 of the second tudor domain further stabilizes the interaction,which is similar to the pattern of53BP1-TTD and H4K20me2 binding.The two tudor domains of 53BP1-TTD function are in concert to recognize H4K20me2 and DP308 blocks both interactions,which could be the reasons that DP308 is more effective than UNC2170 in vitro.Collectively,DP308 is an effective small molecule inhibitor targeting 53BP1-TTD protein.DP308 could be a promising chemical probe candidate for the study of biological function for 53BP1.Innovation:In this study,a novel and effective compound,DP308,was found through the establishment of the Alphascreen high-throughput screening platform.The application of computer drug design combined with molecular docking technology helps to analyze the structure activity and structure optimization,and enriches the small molecule inhibitors of 53BP1-TTD protein.