Efflux Mechanism Of Substrate Pumping By Human P-Glycoprotein And Fusion Peptide Design For Organophosphate Hydrolase

Multidrug resistance（MDR）caused by overexpressed permeability-glycoprotein（P-gp）in cancer cells is one of the main barriers for the cure of cancers.The investigation of molecular mechanism of substrate pumping by human P-gp is beneficial in the design of potent P-gp inhibitors against MDR.Improving the catalytic performance of organophosphate hydrolase（OPH）is beneficial in solving the environmental and safety problems caused by organophosphorus compounds.Substrate enrichment realized by microenvironmental engineering can improve the catalytic performance of enzymes.These researches mentioned above can be realized with the help of molecular simulation.Hence,efflux mechanism of substrate pumping by human P-gp has been studied based on molecular dynamics（MD）simulation and Molecular Mechanics Poisson-Boltzmann Surface Area（MM-PBSA）method in this thesis.Then fusion peptide design for substrate enrichment was proposed and realized using MD simulation and MM-PBSA method.For the research of molecular mechanism of substrate pumping by P-gp,the transport of an antitumor drug,doxorubicin,by P-gp was firstly investigated.The conformational transition of P-gp from the inward-to outward-facing was realized using targeted MD simulations accompanied by the transport of doxorubicin.The MM-PBSA analysis identified the driving forces for the transport of doxorubicin towards the extracellular space as long-range electrostatic repulsions in the initial stage and hydrophobic interactions followed to assist further transport of doxorubicin.The driving forces are separately contributed by the positively charged residues located far away from doxorubicin and the hydrophobic residues located on the pathway of doxorubicin.The driving forces,important residues,and pathway for transporting a P-gp inhibitor,verapamil,were then investigated.The similarities and differences of molecular details between the transports of doxorubicin and verapamil were investigated.The results showed a consistent scenario in the driving forces for the transports of doxorubicin and verapamil.However,there were significant differences in the compositions and energy contributions of the residues providing the identical driving force for transporting different substrates.Only two residues（F336 and M986）are shared by the pathways for the transports of verapamil and doxorubicin.This may imply the weak competitiveness of verapamil with doxorubicin in the substrate efflux.Then,the strong binding interaction between isoleucine（I）and the substrate of OPH,parathion-methyl,was confirmed using MD simulation and MM-PBSA analysis,which are used in the above research.Fusion peptide was designed to increase the local concertation of substrate.Isoleucine was inserted into the alternate glutamic acid and lysine（EK）polypeptide to construct 30 k Da EKI polypeptide.The OPH-（EKI）_30Kfusion protein was obtained by fusing the 30 k Da EKI polypeptide and OPH.Compared with the wild-type OPH,the catalytic efficiency(k_cat/K_m)for hydrolyzing parathion-methyl of OPH-（EKI）_30K was increased by 53%due to the decrease of K_m,and the thermal stability of OPH-（EKI）_30K at 60^oC was significantly improved.In conclusion,this study explored the molecular details of P-gp for pumping different substrates using molecular simulation and provided information for the design of potent P-gp inhibitors.Then,this study demonstrated that the rational design of fusion peptide for microenvironmental engineering on the surface of enzymes was an effective strategy to improve the catalytic performance of enzyme.