Screening And The Mechanism Studies Of Activators For P53 Signaling Pathway

P53(Tumor suppressor P53,TP53,TRP53)is a transcription factor encoded by the TP53 gene on chromosome 17,which is critical in the regulation of cell growth,cell differentiation,metabolism,and immunity.Dysfunctional P53 is associated with a variety of human diseases,promoting tumor development.Thus,P53 is generally known as a tumor suppressor.Nearly in all human tumors,the P53 signaling pathway is inactivated or dysfunctional: in some tumors,mutant P53 leads to the loss-function in tumor-suppressing,and even promoting tumor growth and metastasis in rare cases;in other tumors,wild type P53 is restricted by over-warmed negative regulatory loops.It is not rare that wild type P53 is expressing in cancer cells,for example,the colon cancer,leukemia,and some tumor virus-associated cancers(HPV and Epstein-Barr virus).For these tumors,rescued P53 biological function would significantly inhibit tumor growth.In this study,we were focusing on the screening of small molecules that can restore the P53 signaling pathway in tumors,using the interdisciplinary application of advanced tools,such as high-throughput screening,computerized drug virtual screening,and the knowledge altas,and obtained two potential P53 pathway activators,Torilin and UNBS5162.Besides,the detailed molecular mechanism of UNBS5162,a potential anti-tumor drug,was systematically analyzed.For the development of P53 signaling pathway agonists,we generated a target(wildtype P53 protein)-based computerized drug virtual screening approach and a biological phenotype(P53-transcriptional-activity)-based high-throughput luciferase repoeter drug screening approach,respectively.Later,we cross-applied these two assays in the subsequent molecular mechanism studies and the pharmacokinetic validation of candidate compounds.The natural product Torilin was found to activate the P53 signaling pathway using a computerized virtual screening system in combination with the high-throughput screening.Torilin directly binds to Cys124,the key amino acid site for maintenance of a stable P53 conformation,via 2-methyl-2-butenol,which enhances the stability of wild-type P53,activating the P53 downstream pathway to inhibit tumor cells.Besides,UNBS5162 has been picked from the high-throughput assay,which inhibits tumors both in vivo and in vitro.It promotes the degradation of MDM2(an E3 ubiquitin ligase of P53)to inhibit P53 ubiquitination,leading to the stabilized P53 protein and increased P53 protein levels in tumor cells,which induces P53 downstream gene expression.It restricts tumor cells by apoptosis and cell cycle arrest.Subsequently,for detailed molecular mechanism studies,a knowledge altas were utilized for the analysis of the phenotype-related signaling pathways and nodal molecules to narrow down the research scope,and the computerized virtual screening approach was used to explore the potential targets of UNBS5162.Torilin directly activates P53 by compoundprotein interaction,while UNBS5162 indirectly activates the P53 signaling pathway by manipulating posttranslational modification of P53 and other critical enzymes in the signaling.Two compounds activate the P53 signaling pathway using distinct but classical strategies,which might be valuable in clinical applications.Due to the different molecular mechanisms of these two compounds,they might be applied in a combination way to efficiently treat cancer,which would be pursued in the future.In summary,to perform a rapid and complete drug screening for P53 signaling pathway activator,this thesis tries to establish two drug screening system(1)P53-protein-target-based computerized drug virtual screening approach(2)P53-transcriptional-activity-based luciferase repoeter high-throughput screening approach,then obtains the pertencial P53 pathway activator,and generate an efficient pharmacological analysis approach with the help of knowledge altas,by exploratory use of molecular biology,computer science,pharmacology,and other interdisciplinary emerging technical means.