Combing Optimization Screening And Activity Evaluation To Discover A Novel BCR-ABL/T315I Tyrosine Kinase Inhibitor

Leukemia is a clonal disease caused by abnormal hematopoietic stem cells.Due to abnormal regulation,uncontrolled proliferation,differentiation and apoptosis of cells are blocked,and a large number of cells proliferate in hematopoietic tissues,accumulate,infiltrate into other organs without hematopoietic function in the body,and inhibit normal hematopoietic function.Chronic myeloid leukemia(CML)is one of the highest incidence types of leukemia in China,accounting for 15-20% of adults,and the prevalence is positively correlated with age,and the incidence of male is higher than that of female.CML is a malignant tumor caused by clonal proliferation of bone marrow hematopoietic stem cells.Hematological manifestations include leukocytosis,thrombocytosis and mild anemia,and common physical manifestations include fatigue,abdominal pain and hepatomegaly.BCR-ABL fusion gene is found in more than 90% of CML patients by hematological examination,so it is considered to be an important target for anti-CML drugs.The ABL proto-oncogene on chromosome9 translocate to the breakpoint BCR on chromosome 22,resulting in the formation of the BCR-ABL fusion gene,which can bind to ATP and interfere with the normal signal transduction pathway in cells,leading to malignant proliferation of cells.The emergence of tyrosine kinase inhibitors(TKI)has changed the treatment of CML.Imatinib is the first TKI approved by the FDA for the treatment of CML so far.However,the emergence of Imatinib resistance affects the quality of life of patients and greatly reduces the survival time.The resistance problem is caused by a variety of reasons,and the T135 I mutation as the BCR-ABL "gatekeeper" is the most common cause of Imatinib resistance.Currently,there are no clinically available drugs with long-term efficacy and minimal adverse effects.The first chapter introduces CML.It includes the causes and treatment status of CML.Aiming at the relationship between CML and BCR-ABL tyrosine kinase,including the pathogenesis and kinase structure,the research status of tyrosine kinase inhibitors and the main reasons for drug resistance;T315I mutation is the focus of research,including mutation types and drug research status.Subsequently,the whole research process was described.In the second chapter,the small molecule compounds were downloaded from the database by computer virtual screening technology,and the drug-like screening,rigid screening,semi-flexible screening and cluster analysis were carried out respectively to obtain the small molecule that can bind well with T315 I protein.In the second chapter,small molecular compounds were downloaded from the database by computer virtual screening technology,and screened by drug-like screening,rigid screening,semi-flexible screening and clustering analysis,and the small molecules that could bind well with T315 I protein were obtained.In the third chapter,the CML Pharmacophore screening model was established,and the small molecules with good binding to T315 I protein were screened,and the small molecules with good binding to T315 I protein and potential inhibition of CML activity were obtained.In the fourthly Chapter,biological experiments were carried out,including cell viability,cytotoxicity,cell cycle,cell apoptosis,mitochondrial membrane potential,Caspase-3 activity verification,cell reactive oxygen species(ROS),and Western blot.Compound 4 was found to have a killing effect on leukemia cells and a good inhibitory effect on Ba F3/T315 I cells.Compound 4 also induced cell cycle arrest in S phase,caused cell autophagy and apoptosis,reduced Caspase-3 enzyme activity,MMP membrane potential level,decreased ROS,and inhibited the phosphorylation of Crkl,BCR-ABL and STAT5 proteins and P62 autophagy pathway.In the fifth Chapter,Molecular Dynamics simulation(MD)is carried out to study the interaction modes of 3IK3-IM,3IK3-R,3IK3-S and 3IK3-RS systems.The interaction modes between 3IK3 protein and different conformations of the compound and the sites affecting the activity were analyzed by system equilibrium and computer binding free energy.In this study,compound 4 was found to have a good inhibitory effect on T315 I cells by combining computer screening,biological activity experiment and molecular dynamics simulation.The results show that the selected compounds can be used as lead compounds for further research and provide theoretical and material basis for the discovery of ideal therapeutic drugs for CML.