Study On The Molecular Mechanism Of Matrix Metalloproteinase-2/9 Activating Activatable Cell-penetrating Peptides

Cancer is one of the leading causes of human death in the 21 st century.The main treatment approach of cancer tumors involves surgery,as well as radiotherapy and chemotherapy.However,existing anticancer drugs are generally difficult to enter tumor cells autonomously,resulting in unsatisfactory chemotherapy effects.To solve the transmembrane problem of anticancer drugs and improve the targeting effect of drug delivery systems,activatable cell-penetrating peptides(ACPPs)have emerged.ACPPs are three-domain peptides composed of cell-penetrating peptides(CPPs),linker and polyanionic peptides.In the design of ACPPs,the positivity of CPPs is initially neutralized by polyanions through electrostatic binding.And after cleavage of the matrix metalloproteinase-2/9(MMP-2/9)-sensitive linker PLGLAG,CPPs are activated for the next step of transmembrane.Although ACPPs have showed excellent tumor targeting effect in the experiments,the process of how ACPPs are activated by MMP-2/9 to release CPPs and their loaded drugs is still unclear.Recently,computational chemistry methods such as molecular dynamics simulation,have become necessary tools for elucidating chemical properties of enzymes and designing novel therapeutic agents.This study hopes to explore the recognition mechanism between MMP-2/9 and ACPPs by molecular dynamics simulation,and use QM/MM method to explore the hydrolysis process of substrate PLGLAG by MMP-2/9.Seered molecular dynamics simulation and umbrella sampling method are also applied to calculate the free energy for explaining the release mechanism of ACPPs hydrolyzates.In this research,a series of systematic studies are conducted on the specific activation process of ACPPs by MMP-2/9.It provides a deep understanding at the molecular level for ACPPs targeted drug delivery systems,and may contribute to the design of next-generation targeted drug delivery systems.First of all,the binding process of substrate PLGLAG and ACPPs to MMP-2/9was explored.The substrate PLGLAG,which is the linker of ACPPs,is binding in the catalytic domain of MMP-9,Gly4 at the P1 site and Leu5 at the P1’ site are the key residues affecting the binding stability.Through mutational studies,it is found that the position of P1 site belongings to the substrate,rather than its residue species,plays a key role in the binding configuration of the substrate and the catalytic domain of MMP-9.When exploring the recognition mechanism between ACPPs and MMP-2/9,it is found that E9-PLGLAG-R9(E9R9)could be successfully recognized and combined by MMP-2/9,and the interaction between R9 and MMP-2/9 is smaller than that of E9 and MMP-2/9.The interaction between E9R9 and MMP-2/9 meets the conditions for the smooth removal of the shielding effect.However,the binding of reversely bound ACPPs(R9E9)to MMP-9 is not as stable as that of E9R9,its binding is not conducive to the release of the shielding effect either.This study also has found that different charges and different lengths of ACPPs will influence the binding strength and stability of ACPPs-MMP-9 combination configurations.Therefore,from the perspective of recognition and combination,E9R9 is the most suitable ACPPs.Subsequently,a deep exploration of the hydrolysis mechanism of substrate by MMP-2/9 was conducted.QM/MM results show that MMP-2 proteolysis and MMP-9proteolysis are both water-mediated four-step reactions.Water molecules not only mediate the hydrolysis reaction,but also play a role in stabilizing the intermediates.In addition,the negatively charged glutamate(MMP-2: Glu116,MMP-9: Glu227)near the catalytic zinc ion plays an important role in the whole catalytic hydrolysis reaction,by helping polarize the catalytic water molecule Wat as well as assisting the transfer of protons.Comparing the hydrolysis results of substrate PLGLAG by MMP-2 and MMP-9,the hydrolysis efficiency of MMP-2 is higher.And the rate-determining step of MMP-2 hydrolysis system is the first step of the reaction,while that of MMP-9hydrolysis system is the hydrogen bond rearrangement in the second step of the reaction.The mutation results of P1 and P1’ sites indicate that different residue types can change the reaction energy barrier of the key step,thereby changing the rate-determining step of the reaction,and affecting the hydrolysis rate of the substrate.After that,the release mechanism of hydrolyzates belonging to substrate PLGLAG and ACPPs was also explored.The huge difference in the dissociation constants between N-product and C-product indicates that N-product leave the target protein MMP-9 more easily than C-product.The mutation of P1 site has a huge impact on the dissociation constant.When P1 site is mutated to negatively charged aspartic acid,it is more conducive to the release of the shielding effect.The removal of N-R9,the CPPs terminal product after ACPPs hydrolysis,mainly needs to overcome the interaction with the shielding peptides.N-R9 basically does not bind to MMP-9 after hydrolysis.After the hydrolysis of ACPPs,the shielding peptides terminal product C-E9 has strong interactions with both MMP-9 and CPPs.As a result,in the release of C-E9,the coordination between the residue located in P1 site and the catalytic zinc ion should be destroyed first,then followed by the dissociation of shielding peptide segment E9 and CPPs segment R9.Therefore,after the hydrolysis of ACPPs,N-R9 will leave MMP-9 more easily than C-E9,thus completing the activation process of ACPPs and preparing for the next step of CPPs and loaded drugs entering target cells.This study explores the recognition mechanism between MMP-2/9 and ACPPs,the catalytic mechanism of MMP-2/9 hydrolyzing the linker,and the release mechanism of the hydrolyzates.This work systematically expounds the molecular mechanism of MMP-2/9 activating ACPPs.It is hoped that our work can provide theoretical basis and guidance for the design of a new generation of targeted drug delivery system based on MMP-2/9.