Theoretical Study On The Molecular Mechanism Of Regioselectivity In Midazolam Metabolism Mediated By Cytochrome P450 3A4

Cytochrome P450 enzymes(CYPs)are a class of heme-containing monooxygenases that catalyze the biosynthesis and metabolism of a series of endogenous and exogenous compounds.There are 57 CYP isoforms in human body,in which cytochrome P450 3A4(CYP3A4)is the most important drug metabolic enzyme.The large and malleable active site of CYP3A4 allows for the binding of a variety of structurally diverse compounds,and even simultaneous binding of multiple ligands,leading to CYP3A4 being responsible for the metabolisms of approximately 50% of clinically used drugs and the metabolism process is complex.CYP3A4-mediated drug metabolisms can be allosteric regulated by substrate concentrations and other drugs,making the metabolic regioselectivity and resulting in unusual kinetic characteristics in the metabolic process.Understanding of the complex microscopic processes of CYP3A4-mediated drug metabolism and its regulatory mechanisms is of great significance for drug development,rational drug use and disease treatment.With the rapid development of computer science,molecular simulation technology has become a significant research tool for studying the structure and function of enzymes,and is widely used in many research fields such as three-dimensional structure prediction,catalytic mechanism and metabolic regulation of enzymes,providing a lot of basic data and information for us.In this thesis,the molecular mechanism of CYP3A4-mediated regioselective metabolism has been systematically investigated using a research strategy combining multiple computational methods and using midazolam(MDZ)as a probe.The main research elements of this thesis are as follows:1.Mechanism of homologous allostery in midazolam metabolism regulated by its concentrationIn this work,three types of MDZ binding poses(Pose A,Pose B and Pose C)in CYP3A4 active site were identified,the "sandwich" structure(L216,P218 and L482)was considered as the key factor affecting the binding mode of the substrate,the diversity of MDZ binding modes may be responsible for the diversity of its metabolites;the mechanism of substrate concentration remotely regulates its metabolic regioselectivity through homotropic allostery in MDZ metabolism was also elucidated by using homology modelling,molecular docking,umbrella sampling,classical molecular dynamics simulations and accelerated molecular dynamics simulations.This work may contribute to the understanding of the complex molecular mechanism of CYP3A4-mediated drug metabolism,thus providing important theoretical clues for drug development and related disease treatment.2.Mechanism of heterologous allostery in midazolam metabolism regulated by testosteroneIn this work,we investigated the phenomenon of CYP3A4-mediated drug-drug interactions(DDIs)taking MDZ and testosterone(TST)as research subjects and using classical molecular dynamics simulations and Well-Tempered Metadynamics.We predicted two peripheral allosteric sites of CYP3A4 Site 1 and Site 2,found the binding of TST in the allosteric site provides favorable conditions for the transition of substrate binding mode by opening the "sandwich" structure and enlarging the volume of the active site;elucidated the dynamic process of the transition of substrate binding modes,identified conformational changes of residues R105 and F108 were key factors influencing the substrate binding mode transition;pointed out that there were multiple pathways involved in the heterologous allostery of MDZ metabolism regulated by TST,and Site 2 seems to be more pronounced.The results of this study would help us to better understand the mechanism of CYP3A4-mediated DDIs,which may provide a theoretical basis for the development of rational prescribing and related disease treatment.3.Role of residue F108 in the homologous and heterologous allosteryResidue F108 was identified as playing an important role in the CYP3A4-mediated MDZ metabolism and the allosteric effects in our previous work.To further clarify the important role of F108 in the allosteric effects of MDZ metabolism,we combined residue mutation method with classical molecular dynamics simulations and WellTempered Metadynamics to clarify the effects of F108 on MDZ binding stability and the transition of MDZ binding mode in terms of both homologous and heterologous allostery,we found that the the steric effect between F108 side chain and MDZ is favorable for the stable binding of MDZ in the active site,and the π-π interaction facilitates the transition of MDZ binding mode.F108 was recognized as gating residue of CYP3A4 protein channels,and the volume of F108 side chain was closely related to the length,radius size,and other characteristics of protein channels.We also provided a theoretical explanation for the experimental phenomenon that F108 A and F108 W mutations affecting the regioselectivity in MDZ metabolism.The results would helpful for the determing of relevant experimental facts and improve the understanding of the structure-function relationship of CYP3A4,which can provide valuable theoretical guidance for the study of CYP3A4 genetic polymorphisms and the development of personalized treatment.