Drug Discovery And Optimization Of Protein Arginine Methyltransferase And Their Anti-Tumor Effects

Epigenetics refers to heritable factors that affect gene expression without any DNA sequence changes,and they impose complex molecular regulation on various aspects of cellular growth.Uncontrolled regulations of epigenetics are closely related to the occurrence and development of various diseases.Studies have shown that abnormal expression or mutations of epigenetic enzymes are associated with a variety of tumors,resulting in the activation of several tumor signaling pathways or inhibition of tumor suppressor pathways,thereby promoting the occurrence and development of tumors.Therefore,the development of drugs targeting epigenetic targets is considered to be an important strategy for cancer treatment.At present,there are 8 epigenetic small molecule drugs including DNA methyltransferase inhibitors,histone deacetylase inhibitors and isocitrate dehydrogenase inhibitors.The protein arginine methyltransferase?PRMT?family is a group of important epigenetic targets that catalyze the methylation of arginine in various substrates in cells.According to the different state of catalytic methylation levels,PRMT family can be divided into three types.Type? PRMTs,which includes PRMT1,PRMT2,PRMT3,PRMT4,PRMT6 and PRMT8,is responsible for catalyzing the arginine monomethylation modification and asymmetric dimethylation modification?ADMA?.Type? PRMTs,which includes PRMT5 and PRMT9,is responsible for catalyzing monomethylation modification and symmetric dimethylation modification?SDMA?.Type? PRMTs,which only contains PRMT7,catalyze monomethylation modification.Among these enzymes,PRMT1 is the predominant type ?methyltransferase,which is responsible for nearly 90% of all cellular arginine methylation,and is involved in the regulation of DNA repair,RNA processing,transcriptional regulation,signal transduction,cell differentiation and other important cellular processes.Abnormal expression of PRMT1 has been reported to be associated with a variety of diseases,including cardiovascular diseases,kidney diseases,diabetes and respiratory diseases,especially cancer.Abnormalities of PRMT1 and its catalytic substrates are closely related to tumorigenesis,tumor progression and metastasis as well as drug resistance.Recently,with the great efforts of researchers in this field,many potent PRMT inhibitors have been developed.Among them,selective inhibitors with high potency that target PRMT3,PRMT4 and PRMT5 have an enzymatic IC₅₀ value of 30 nM,6 nM and 22 nM,respectively.However,there is currently no selective inhibitor of PRMT1 with potency reaching the nanomolar level.Moreover,the lack of PRMT1 chemical probes further hinders the gene function and target discovery studies for PRMT1.Therefore,developing PRMT1 inhibitors with high potency,good selectivity and high druggbilities not only contribute to the functional study of PRMT1,but also speed up the translational medicine study that benefits cancer patients.In this study,we first apply the method of virtual screening with validations in the molecular level,to identify a PRMT1 inhibitor B3 with a single-digit micromolar potency.Then we performed the binding mode analysis and found that B3 occupied the substrate site,and the conformation of its ethylenediamine side chain is very similar to the conformation of its substrate arginine.Through more thorough investigation of the catalytic mechanism of PRMT1,we modified B3 based on the substrate mimic strategy,and obtained C22,a compound with significantly improved activity that has a singledigit nanomolar inhibitory activity against PRMT1.The activity of the compound is increased by nearly 1000 fold compared with B3.Further selectivity experiments indicated that C22 is a pan-inhibitor of type ? PRMTs,with good selectivity towards PRMT ?,PRMT ? and other methyltransferases.In leukemia cells,we knocked down the PRMT1 gene and found that the proliferation of leukemia cells decreased significantly,suggesting that PRMT1 has a profound effect on the proliferation of leukemia cells.In the cellular level,C22 exhibited a strong ability to inhibit the proliferation of leukemia cells,and had almost no killing effect on normal cells.In addition,C22 could also cause cell cycle arrest and apoptosis of leukemia cells,and could significantly downregulate the transcriptional levels of key leukemogenic genes,revealing the important role of type ?PRMT enzymes in leukemia cell proliferation.To further improve the selectivity and druggabilities of C22,we designed andsynthesized 13 derivatives,and adopted salt formulation to improve the stability and solubility of the compound.Among these modified compounds,C41 showed improved selectivity towards PRMT1 and PRMT6 while maintaining the high potency.We further knocked down PRMT1 in clear cell renal cell carcinoma?ccRCC?cells,and found that the growth of ccRCC cells were significantly inhibited.At the same time,the growth and clonality of ccRCC cells were also significantly inhibited when treated with C41.In addition,C41 could also induce G1 arrest in ccRCC cells.In the animal xenograft model of ccRCC,C41 significantly inhibited the growth of A498 xenografts,and afford enhanced anti-tumor effects when combined with the first-line drug of the advanced kidney cancer,sunitinib.These studies indicate that PRMT1 play a significant role in the proliferation of ccRCC cells and is a promising drug target for ccRCC treatment.In summary,this study identified a highly potent,selective type?PRMT paninhibitor with good druggability,which not only provides an efficient chemical tool for the study of type ? PRMT gene function,but also provides a potential intervened target as well as a drug lead for the treatment of leukemia and ccRCC.