Study On The Interaction Between KRAS Protein And Its Targeted Inhibitors Through Molecular Dynamics Simulation

KRAS is the most well-known protein in the RAS protein family（HRAS,NRAS and KRAS）.Mutations in KRAS can lead to a range of cancers,including colorectal cancer（CRC）,non-small cell lung cancer（NSCLC）and pancreatic cancer（PDAC）.Among them,pancreatic cancer caused by G12D mutation accounts for 45%,lung cancer caused by G12C mutation is as high as 46%,and colorectal cancer caused by G12S mutation also accounts for 15%.Therefore,in drug research,the importance of KRAS is beyond doubt.With the continuous breakthroughs in the analysis of the KRAS crystal protein structure,people’s understanding of the structure and function of KRAS is also getting deeper and deeper.Although these crystal structures can reveal the structural information of KRAS protein,they are static and cannot reveal the dynamic mechanism of KRAS protein.Therefore,at present,people mainly use molecular dynamics（MD）simulation method to study the dynamic mechanism of KRAS protein or other biomacromolecules.In this paper,the mutant KRAS protein and its targeted inhibitors were selected as the research objects,which were MRTX1133 inhibitor and KRAS^G12D protein,AMG510 inhibitor and KRAS^G12C protein,G12Si-5 inhibitor and KRAS^G12S protein.Based on the three resolved crystal structures,conventional unbiased molecular dynamics simulation,Markov state model（MSM）and cluster analysis were used to analyze the KRAS protein and its targeted inhibitors.The inhibition mechanism as well as the dynamic interactions were studied in detail.The first chapter of this article introduces the background and related content of the research topic.Including the gene,structure and mechanism of KRAS protein,it also introduces the research progress of KRAS inhibitors,focusing on the KRAS^G12Cand KRAS^G12D inhibitors that have made major breakthroughs.In the second chapter of the thesis,the theory of molecular dynamics simulation and the analysis methods used in the thesis are mainly outlined.Including the basic principles of molecular dynamics simulation,main steps,force fields,solvent models,energy minimization algorithms,constant temperature and constant pressure algorithms,periodic boundary conditions,commonly used simulation softwares,in addition,cluster analysis and Markov state model methods are discussed in detail.In the third chapter of the thesis,in order to explore the binding mechanism of MRTX1133 with KRAS^G12D,especially how MRTX1133 could bind the active state KRAS^G12Dwithout triggering the active function of KRAS^G12D.We used a combination of all-atom molecular dynamics simulations and Markov state model（MSM）to understand the inhibition mechanism of MRTX1133 and its analogues.The stationary probabilities derived from MSM show that MRTX1133 and its analogs can stabilize the inactive or active states of KRAS^G12D into different conformations.More remarkably,by scrutinizing the conformational differences,MRTX1133 and its analogs were hydrogen bonded to Gly60 to stabilize the switch II region and left switch I region in a dynamically inactive conformation,thus achieving an inhibitory effect.In the fourth chapter of the thesis,we used molecular dynamics to explore the dynamic interaction of three mutant proteins(KRAS^G12C,KRAS^G12Dand KRAS^G12S)and their inhibitors（AMG510,MRTX1133 and G12Si-5）at the atomic level.Through the calculation of Root Mean Square Deviation（RMSD）,it was found that the backbone conformations of the three mutant proteins were basically similar,and the three inhibitors were all stable.Comparing the Root Mean Square Fluctuation（RMSF）values revealed that although the overall residue fluctuations of the three mutant proteins were similar,there were significant differences in the switch I and switch II regions.Interactions not exhibited in the crystal structure of the mutant KRAS protein were also discovered during the simulations.In addition,the distance analysis of residues G60 and T35 revealed that AMG510,MRTX1133,and G12Si-5 inactivate KRAS^G12C,KRAS^G12D,and KRAS^G12S,respectively.The fifth chapter of the thesis summarizes the work of the full text,and puts forward the direction of the next step of this study.