Virtual Screening,activity Evaluation And Molecular Simulation Study Of LRRK2 Inhibitors

Leucine-rich repeat kinase 2?LRRK2?can regulate microtubule's stability,participate in immune cell maturation and inflammatory response,and play an important role in synaptic morphology and cytoskeleton formation.LRRK2 is a large protein with six domains.Its multiple amino acid mutations are the most common cause of autosomal dominant Parkinson's disease?PD?,especially the G2019S mutation located in the kinase domain.This leads to an abnormal increase in its kinase activity and eventually leads to Parkinson's disease.Studies have shown that the enhanced kinase activity caused by G2019S mutation is an important mechanism leading to dopaminergic neuron damage,which will significantly increase the risk of PD.And LRRK2 kinase inhibitors can protect against G2019S LRRK2 mutation-induced neurodegeneration,block the accumulation of increased alpha-synuclein in G2019S-LRRK2 neurons,inhibit the formation of inclusion bodies,and slow down the disease progression.Therefore,LRRK2 is considered as a new and potential target for the treatment of PD.The effect of the G2019S mutation on the structure of the LRRK2 kinase and the molecular mechanism of continuous activation caused by G2019S mutation are not yet clear.In addition,the LRRK2 inhibitors which is under studied generally have side effects such as high kidney toxicity and poor selectivity.Currently,there are no LRRK2 inhibitors on the market.Therefore,there is an urgent need to find LRRK2inhibitors with novel skeletons that inhibit both wild-type and G2019S mutation-type.The first part of this paper summarizes the biological functions,structure,common mutations of LRRK2 and the research progress of LRRK2 inhibitors,and then briefly introduces the homology modeling,virtual screening,molecular docking and molecular dynamics simulation methods used in this paper.In the second chapter,to explore the possible reasons for the increased activity of LRRK2 kinase G2019S mutant,the accelerated molecular dynamics simulation method?AMD?is used to study the effect of G2019S mutation on the LRRK2activation loop and its DYG motif and to explore the structural changes caused by mutations.Because the crystal structure of the LRRK2 kinase domain has not been resolved,a three-dimensional structure of the LRRK2 kinase domain was constructed using homology modeling method.Then,through molecular dynamics simulations,we found that the wild-type DYG motif had greater conformational flexibility.The G2019S mutation makes the DYG motif maintain in the DYG-in state through more stable hydrogen bonding interactions.In the third part,structure-based virtual screening method combined with biological activity evaluation experiments was applied to find new LRRK2 inhibitors.After three steps virtual screening based on different precision molecular docking methods,28 compounds were finally selected from the Chemdiv and Specs databases for further experimental verification.The kinase inhibitory activity experiment is divided into two steps:Firstly,the inhibitory effect of 28 compounds on the mutant G2019S kinase was initially evaluated at a compound concentration of 100?M,and 6active compounds with an inhibition rate of more than 50%were obtained.Then,the half maximal inhibitory concentration(IC₅₀)of these 6 compounds for inhibiting wild-type and G2019S mutant kinases was measured.Two small molecules with novel skeleton,compound LY2019-005 and compound LY2019-006,were indentified as potential LRRK2 kinase inhibitors.The IC₅₀s of two compounds over the wild-type kinase and G2019S mutant are 424nM,378nM and 1526nM,1165nM,respectively.In the fourth part,to analyze the action mechanism of the identified inhibitors,molecular dynamics simulations methods were performed to the complexes of LRRK2 kinase with our identified LY2019-005 and one known inhibitor LRRK2-IN-1.The structural analysis indicates that the inhibitor binds to the hydrophobic cavity formed by the p-loop,hinge region and catalytic loop region,and can form hydrogen bond interaction with the hinge region ALA1950 and?-?interaction with the p-loop region PHE1890.Among them,the hydrogen bond interaction with the kinase hinge region is the key to the compound with high inhibitory activity.This work can provide important theoretical guidance value for the further design of effective LRRK2 kinase inhibitors.In summary,firstly,this thesis revealed the structural changes caused by the LRRK2 pathogenic G2019S mutation based on accelerated molecular dynamics simulations.Then,based on the virtual screening method of molecular docking combined with biological activity evaluation methods,two LRRK2 inhibitors with higher activity were found,and the interaction mechanism between small molecule inhibitors and LRRK2 was further analyzed by molecular dynamics simulation.The obtained results can provide candidate compounds and important theoretical guidance for the future discovery of anti-PD drugs based on LRRK2 inhibitors.