Discovery And Research Of Inhibitors Targeting Protein Argine Methyltransferases PRMT1/5

Protein arginine methylation,one of the most common post-translational modifications,which catalyzed by Protein Argine Methyltransferases?PRMTs?,played an important role in regulating the physiological processes of various cells.There are three types of PRMTs protein in mammals,whose aberrant enzyme activity were associated with the development of many diseases.Therefore,PRMTs were regarded as potential drug targets in cardiovascular disease,breast cancer,and leukemia,etc.PRMT1 was the most important type I PRMT,whose catalytic activity was accounted for above 85%of total catalytic activity of mammalian PRMTs,so it is important to study its specific small molecule inhibitors.In this paper,with the aid of ligand-based CADD,we used SHAFT?SHApe FeaTure Similarity?method and selected two reported PRM1 inhibitors DB75 and MS023as templates to virtually screen the old-drug bank and got a series of candidate compounds.Then through constructed TR-FRET?Time-Resolved Fluoresence Resonance Energy Transfer?assay for initial screening,Pentamedine was founded,which was an active compound.Next,through radioisotope assay,we further confirmed that Pentamedine can inhibit PRMT1 enzyme activity,whose IC₅₀=13.2±0.3?M in vitro.Next,taking Pentamedine as the prototype,we synthesized a series of Pentamedine derivatives and studied their structure-activity relationships.As a result,we got another old drug,Hexamideine,whose IC₅₀=5.9±1.7?M in vitro.In addition,the combination of Thermal shift and NMR confirmed that the compound binds to PRMT1 protein.In order to further explore the binding mode of hexamidine and protein,we used molecular docking to predict,and with the help of NMR competitive experiment,we found that Hexamidine was a double substrate competitive inhibitor of PRMT1.Finally,the cellular level proliferation experiments showed that Hexamidine inhibited the proliferation of cancer cells where PRMT1 was overexpressed,in the time and concentration dependent manner.Western Blot experiments confirmed that the compound inhibited PRMT1cellular enzyme activity.PRMT5,the most important type?PRMT,was releated with leukemia,lymphoma and breast cancer,etc,which was regarded as a potential drug target.In this paper,with the aid of receptor-based CADD,we selected PRMT5 crystal structure to carry out molecular docking.Then through cluster analysis and manual screening,we obtained 120 candidate compounds.After initial screening of radioactive isotopes assay,we got DC_Y53,whose IC₅₀=5.7?M in vitro.In order to optimize the structure,we used similarity search and obtained DC_Y134(IC₅₀=1.7?M in vitro),which showed good selectivity among methyltransferases.Finally,cellular experiments showed that DC_Y134 inhibited the proliferation of of multiple leukemia and lymphoma cells in the time and concentration dependent manner.Among them,MV4-11 cell line showed the most sensitivity.Western Blot experiments showed that the compound could inhibit PRMT5 cellular enzyme activity,which may explain the proliferation inhibitory effect.Further flow cytometry experiments showed that DC_Y134 could block cell cycle at G0/G1 phase and promote tumor cell apoptosis.In conclusion,based on combination with CADD and biological experiments,we found Hexamideine(IC₅₀=5.9±1.7?M),the selective small molecule inhibitor targeting PRMT1,and DC_Y134(IC₅₀=1.7?M),the selective small molecule inhibitor targeting PRMT5,which will be useful molecular probes to investigate new biologcial functions of PRMT1/5 and provide clues for small molecule inhibitors.