Molecular Dynamics Study On Selective Recognition And Intracellular Delivery For Activatable Cell-penetrating Peptides

Cancer/tumor is a disease that threatens the health of human beings.Tumor imaging or treatment could be achieved by targeting the contrast agents or drugs to the tumor tissue.Cell penetrating peptides(CPPs)are no cytotoxicity,high transmembrane efficiency,and modifiable,which are widely used in gene editing,tumor imaging and therapy,cell regulation,drug delivery,intracellular delivery of nanodevices.Matrix metalloproteinase 2/9(MMP-2/9)are highly expressed in tumor tissues.The CPPs and CPPs activity-inhibiting peptides are linked by a linker peptide that can be selectively recognized and hydrolyzed by MMP-2/9.Thus,the activatable cell penetrating peptides(ACPPs)are formed,and drugs can be targeted delivery to tumor tissue by ACPPs.In this thesis,molecular dynamics(MD)simulations and umbrella sampling were employed to study the structure-activity relationship(SAR)of ACPPs,the binding and selectivity of MMP-2 to substrate,and the transmembrane process of CPPs.In this study,the maximum probability structure of ACPPs was predicted by cluster analysis,and verified by circular dichroism.The umbrella sampling method was employed to illustrate the effect of CPPs activity-inhibiting peptides on the transmembrane efficiency of CPPs.This work is to reveal the SAR of ACPPs,Molecular docking and MD were used to analyze the binding and selectivity of MMP-2 to substrate.The chemical environment of MMP-2 at PI and P1' sites was investigated,which laid a solid foundation for understanding the SAR of MMPs.In addition,this work systematically studied various factors involved in the transmembrane delivery of arginine-rich CPPs(ARCPPs).The effects of the water molecules carried in the water hole,the guanidinium-PO4 salt bridge,the length and order of hydrocarbon chain on the ARCPPs transmembrane process would be well discussed.This part is of great significance for the development of next-generation intracellular delivery strategies based on CPPs.The main conclusions of this thesis are as follows:1.The circular dichroism indicates that the a-helix ratio in ACPPs is 63%,and the random coil ratio is 32%.In the maximum probability structure of ACPPs,CPPs activity-inhibiting peptides and CPPs form multiple salt bridges,which effectively shields the positive charge of the guanidinium.2.During the adsorption and transmembrane process of ACPPs,the CPPs activity-inhibiting peptides always maintain electrostatic shielding against CPPs,which makes the transmembrane free energy barrier of CPPs in ACPPs increased by 66 kJ/mol.3.The linker peptide is as a ?-fold in the MMP,2 catalyzed groove,and there is a hydrogen bond network between the ?? chain of MMP-2 and the substrate.When the hydrogen bond network is destroyed,the binding free energy will be reduced by 76 kJ/mol.Thus,the ?? chain is critical for the stability of MMP-2/substrate complex.4.The side chain at the P1 site is exposed to the solution such that residues of the aromatic or long alkyl side chain are inhibited.In the Sl' hydrophobic pore,the chemical environment is determined by the side chain of Val112,His115 and Tyrl37,and the spatial size is limited by Val112,His115,Prol35,Ile136 and Tyr137.A locally geometric mismatch at the P1' site would significantly reduce the binding free energy of MMP-2 to the substrate.Therefore,the geometric properties should be considered when designing MMPs-speeific substrates or inhibitors by computer modeling.5.During(Arg)9 transmembrane process,the multiple salt bridge between guani-dinium and PO4 is formed,and it accounts for 65%of the overall interaction energy between(Arg)9 and phospholipid bilayers.Thus,the multiple salt bridge between guanidinium and PO4 is the driving force for(Arg)9 transmembrane process.And the number of H-bonds between guanidinium and PO4 is found to be 1.78 ± 0.3,whereas the number of H-bonds between ammonium and PO4 is 0.34±0.1.Thus,the primary interaction between the ammonium and PO4 is an ion pair rather than a salt bridge.6.The adsorption free energy(?GA)of(Arg),from solution to the phospholipid bilayers surface is found to be linearly related to the interaction energy change(?EA):?Ga=0.0426*?EA+36.7,R2=0.92.In the CPPs transmembrane process,the transmembrane free energy barrier(?Gb)is roughly correlated with the corresponding interaction energy change(?).Also,the ?GB is found to be related to the length of pore(L):(?),which makes the transmembrane efficiency estimable.The effective length(L)of water hole shows a good linear relationship with the hydrophobic thickness(2ZDc)of the phospholipid bilayer:(?).In summary,this thesis illustrates the structure-activity relationship of ACPPs,and help to design targeted drug delivery systems based on MMP-2.Also,this work provides suggestions for the next generation of intracellular delivery strategies based on CPPs,as well as for evaluating CPPs intracellular delivery efficiency by above-mentioned quantitative relationships.