Discovery Of Novel PRMT5 Small Molecule Inhibitors And Study On The Mechanism Of 11-MT On Lung Cancer Cells

Malignant tumors remain one of the main causes of death.Therefore,anti-tumor drug research and development is a hot spot in industry and academia.Epigenetic modification and autophagy,which regulate the mechanism of cell death,play an important role in tumorigenesis and development.Epigenetics refers to the regulation of gene expression and its function by influencing the chemical modification of DNA,histone,and the chromatin conformation at the transcriptional level without changing the nucleotide sequence of the gene.These changes can be inherited steadily with ontogeny and cell proliferation.The abnormalities of epigenetic modification-related gene are often associated with diseases,such as cancers,diabetes and Alzheimer's diseases.Autophagy is the key mechanism to control the fate of cells,it contains dual function in the process of tumor development:in the early stage of tumor occurrence,autophagy can clear the unfavorable factors in the cell and inhibit the development of tumor;but once the tumor forms,autophagy becomes a powerful weapon for tumor cells to resist adverse environments.Apart from cancers,the dysfunction of autophagy is also associated with heart failure,neurodegenerative diseases,inflammation etc.Protein arginine methyltransferase 5?PRMT5?is an epigenetic modification-related enzyme,which can catalyze methylation modification of histone and non-histone,its expression level is usually up-regulated in a variety of human malignant tumor cells,so it is a potential anti-tumor target.At present,two inhibitors of PRMT5 are in the clinical research stage I.In the first chapter,we discovered two new types of PRMT5 inhibitors by combining computer-aided drug design with chemical synthesis,and the mechanism was expected to studied.In the first section of this chapter,we obtained four new small molecules with5-methylbenzoxazole by virtual screening based on the pharmacophore model of positive compound EPZ015666.It was founded that compound 4 and 10 can inhibit the activity of PRMT5 with IC₅₀0 value of 8.1?M and 6.5?M,respectively.20 derivatives,based on the structure of 4,were selected and purchased from the SPECS database by similarity substructure search.It was showed that none of the derivatives was superior to 4 in the activity inhibition test of PRMT5.At the cellular level,Compound 4 was able to inhibit the proliferation of lymphoma cell line Jeko-1 and leukemia cell MV4-11 in a dose-dependent manner,and it was able to significantly down-regulate the level of H4R3me2s a substrate of PRMT5.In the second,we reconstructed the screening model based on the crystal structure of PRMT5 in the PDB database?5EML and 4GQB?and obtained a novel small molecule P16with 5-benzyl-2-phenylthiazolone.And then,the total 44 compounds including P16 were obtained by substructure similarity search and chemical synthesis methods.Radioisotope results showed that compound 5 and 19 showed significant inhibitory activity to PRMT5,with IC₅₀ of 0.77 and 6.60?M,respectively.At the cellular level,compound 5 and 19inhibited the proliferation of leukemia cell line MV4-11 in a concentration-dependent manner.The cycle and apoptosis assays revealed that compounds induced apoptosis in leukemia cells and arrested its cycle at G1 phase;the experiment on effect of dimethylation level of the SmD3,a substrate of PRMT5,revealed that both compounds could down-regulate the expression level of SmD3me2s in a concentration-dependent manner.It was concluded that the inhibitory activity of the compounds on the proliferation of tumor cells is the direct result of inhibiting the expression of PRMT5 substrate protein.These two types compounds are expected to be new small molecular probes to study the function of prmt5.Autophagy,a highly conserved manner,is a self-protective mechanism unique to eukaryotic cells.Under the influence of stress factors,the unfavorable environment may induce basal autophagy to protect cells from harsh environments.The switch that regulates autophagy in a tumor environment is critical for treating diseases such as cancer.Anticancer drugs derived from natural products are very important clinical drugs,in which alkaloids account for the proportion of natural products cannot be underestimated.In the third part of the thesis,we explored the role of autophagy in the carcinogenesis and development of cancer in the research background of the anti-lung cancer mechanism of aspidosperma alkaloids.Eleven alkaloids were isolated from the apocynaceae plants.In vitro activity screening showed that 11-methoxytabersonine?11-MT?could significantly inhibit the proliferation of lung cancer cells A549 and H157.Further study of the mechanism showed that 11-MT could induce both autophagy and programmed necrosis of tumor cells,and autophagy had protective effect on tumor cells.The combination of autophagy inhibitor and 11-MT can further promote the death of lung cancer cells.In summary,this paper investigated the discovery and mechanism of small Molecule inhibitors on the key enzyme PRMT5 related to epigenetic modification,and at the same time,it can be investigated that the mechanism of the alkaloid 11-MT derived from natural products on two human lung cancer cells.