| MiR-21 was the miRNA which was first detected by human genome.As well as most of miRNA,the tertiary structure of mi R-21 has a single "hairpin shaped".Because the human genome contains more than 1000 encoding miRNA,which is thought to be involved in the regulation of nearly 1/3 genes of human related processes,and miR-21 has been confirmed there is over expression phenomenon in human glioblastoma cells.So the research on new cancer treatment adjustment mechanism based on mi R-21 is on the rise in recent years.According to the miRNA biological structure prediction has been a problem,researchers proposed nucleotide cycle sequence diagram theory(NCM),and using MC-Fold and MC-Sym method can predict the three-dimensional structure of RNA.Besides,there are some researchers were using computational chemistry and biological experiment to study small molecule inhibitors of miR-21,and great progress has been made.This is confirmed that the computer aided drug design(CADD)plays an important role in drug discovering.At present,molecular docking,molecular dynamics simulation method and energy minimization were successfully applied to miR-21 inhibitor design.In the biotic experiment,predicting the median inhibitory concentration(IC50)of the leading compound by the MTT method can quickly screen a small molecule inhibitor activity.Because the MTT method has high sensitivity,good repeatability and easy operation,it is widely used in small molecule inhibitor activity test.In the first chapter of this paper,the origin,function mechanism and related research of miRNA,and the basic content of miR-21 were summarized.Then the miR-21 secondary structure prediction and tertiary structure prediction research progress were introduced,and the research progress of miR-21 small molecule inhibitors was introduced.Finally,the computational tools used in this paper were introduced,and the whole research idea of this thesis was briefly introduced.In the second chapter,we mainly introduced the prediction,construction and optimization process of miR-21.Firstly,we determined the 20 secondary structure coding by MC-Fold method.Then the 5 tertiary structure were determined through the MC-Sym calculation method.Finally,using the molecular dynamics simulation method to construct miR-21 structure optimization and energy analysis.In addition,cluster analysis is used to analyze the most likely conformation,ultimately determine the three level structure of mi R-21.Through the analysis of conformational changes of system energy and RMSD value stability changes further system,and combined with the RMSF fluctuation,found that the RC20 and RC56 residues of miR-21 which just in a ditch area.Combined with the molecular dynamics simulation method and energy minimization using this two RNA structure prediction method can well predict the tertiary structure of miR-21.It also provides a new idea for predicting miR-21 tertiary structure.The third chapter was mainly based on the tertiary conformation of mi R-21 predicted in the previous chapter.The Glide module in Schr?dinger10.2 was used for virtual screening to determine the compounds used for the activity test,and finally the lead compounds were identified by MTT experimental method.In order to accurately identify the binding sites of small molecules,a pocket recognition and docking phase separation method were used to determine the binding pocket of small molecules located at the median cavity site.The molecular docking between SP accuracy and XP accuracy was performed on the SPECS database,and 50 ligands small molecules were screened out.The activity of this 50 ligands were tested by MTT experiment.The research show that AN-465/43384157,AN-465/43384083,AN-465/43384085 and AN-465/43384166 of their IC50 were 8.91,9.31,12.65 and 10.8 M respectively.Because their biological activities were relatively high,so they were identified as the leading compounds used in the final research.By comparison of structures,it was found that the four small molecules have three ring structures,and they all have a structure of substitution of amino para substituted piperidine.Finally,the binding modes of these four lead compounds on miR-21 were analyzed.Founding that at the end of the four lead compounds have bonding interaction with miR-21 of two amino residues RG54,RU55 and RC56.Combined with the second chapter analysis results of RMSF,we can conclude that the RC56 residue of mi R-21 may be an important role in drug site on the miR-21,this also providing a new idea for this the discovery of small molecule inhibitors of miR-21.The results of this experiment show that the molecular docking method greatly saves the time of discovery of lead compounds,and lays the foundation for the next experimental verification. |
