Theoretical Study On The Binding Mechanism Between Cytochrome P450s And Ligands

Cytochrome P450s（CYPs）are responsible for endogenous compound and exogenous drug metabolism and synthesis.CYPs are regard as versatile biocatalysts,and they can catalyze the hydroxylation of ligands at specific sites.The interaction mechanisms between CYPs and ligands have attracted extensive attention.Therefore,it is important to elucidate the binding mechanisms of ligands to CYPs for understanding the structure-function relationship of CYPs and promoting the development of protein engineering.However,the three-dimensional structures of proteins obtained based on experimental means are generally stable and single conformation,and it is difficult to capture the highly active intermediate structures and present the dynamic structural change information.The proteins structures information obtained only by experimental means cannot meet the needs of researchers for understanding of the structure-function relationships of proteins.With the rapid progress of computer simulation technology,molecular simulation has become an important tool for researchers to explore the structure-function relationships of proteins.Molecular simulation can be used to model the three-dimensional structure of proteins,predict the functions of proteins,and explore the dynamics change process of proteins in the real environment from the stable protein conformations determined by experimental means,which provides a powerful supplement to the experimental methods.In this paper,the research strategy including molecular docking,classical molecular dynamics simulations,and adaptive steered molecular dynamics simulations is used to systematically investigate the binding mechanism between several important CYPs（CYP46A1,CYP51,CYP105P1 and CYP105D6）and ligands.The main research contents were as follows:1.Theoretical research on the recognition mechanism of CYP46A1 andcholesterolThe CYP46A1 in the human brain is a potential target for treating neurodegenerative diseases such as Alzheimer’s disease and epilepsy.However,the interaction mechanism between CYP46A1 and endogenous substrate cholesterol（CH）has not been clarified,which may hinder the development process of related drugs.In this part of the study,molecular docking,classical molecular dynamics simulations,and adaptive steered molecular dynamics simulations were used to explore the micro-mechanism of CH recognition by CYP46A1.Two key factors affecting the interaction between CH and CYP46A1 are determined:the hydrophobic cavity formed by seven hydrophobic residues F80,Y109,L112,I222,W368,F371,and T475 provides nonpolar interactions to stabilize CH;The hydrogen bond formed by H81 and CH ensures the binding direction of CH in the binding site.In addition,the tunnel analysis results show that tunnel 2a is identified as the primary pathway for CH ingress/egress CYP46A1.CH affects the protein tunnel characteristic by regulating the movement of the B’helix,and its binding induces the closing of tunnel 2e and the opening of tunnel W.This study elucidates the molecular mechanism of CH recognition by CYP46A1 at the atomic level,which may provide important theoretical information for relevant drug design.2.Theoretical research on the binding mechanism of CYP46A1 and inhibitorsIn the previous study,we further investigated the interaction of CYP46A1 with existing inhibitors Soticlestat and 4-（4-methyl-1-pyrazolyl）pyridine derivative（Compound 17）by classical molecular dynamics simulations and binding free energy calculation.The binding free energy calculation results indicate that the binding affinity of Soticlestat and CYP46A1（-19.32 kcal/mol）is slightly stronger than that of Compound 17（-15.15 kcal/mol）.The common key residues anchoring the binding of the two inhibitors to CYP46A1 are Y109,L112,T306,W368,and T475.The binding mode analysis results demonstrate that Soticlestat is superior because its phenyl and pyridine groups enhanced enhance the binding affinity between inhibitor and CYP46A1.This work explains the interaction mechanism between CYP46A1 and inhibitors at the atomic level,which could provide theoretical clues for the further design of inhibitors with novel structure,high activity,and excellent selectivity.3.Theoretical research on the binding mechanism of CYP51 and azole inhibitorsThe sterol 14-αdemethylase（CYP51）catalyze methylation of lanosterol at 14-αsite to form ergosterol in fungal cell membranes,which is a target for treating fungal infection.With the continuous emergence of drug-resistant strains,existing inhibitors are facing serious resistance problems,and the development of new inhibitors has become an urgent task.In this part of the study,we have investigated the binding mechanism between four triazole inhibitors fluconazole（Flu）,voriconazole（Vor）,itraconazole（Itc）,and posaconazole（Pos）and CYP51 based on molecular docking and classical molecular dynamics simulations.The binding mode analysis results show that the four inhibitors bind to CYP51 in a similar binding mode,with the triazole ring facing the heme prosthetic group and the side chain facing the protein surface.The tunnel analysis results determine that tunnel 2f is the predominant pathway for inhibitors ingress/egress CYP51,this result is also reflected in other works related to CYP51.We also find that nonpolar parts of key residues F58,Y64,Y118,L121,Y132,L376,S378,S506,S507,and M508 can form a hydrophobic cavity,which contributes to hydrophobic interactions for the binding of inhibitors to CYP51.The binding free energy analysis results reveal that the binding affinities of long-tailed inhibitors（Itc and Pos）to CYP51 are stronger than that of short-tailed inhibitors（Flu and Vor）.This result is the theoretical explanation for the experimental fact that the long-tailed inhibitors’IC₅₀ value is lower.The structural analysis results further demonstrate that longer side chains could establish hydrophobic interactions with more residues in CYP51,which may be the reason why long-tailed inhibitors have a higher selectivity for CYP51.The research results in this study could provide the theoretical basis for the design of more effective antifungal inhibitors,and help to better understand the structure-function relationship of CYP51.4.Theoretical research on regiospecificity molecular mechanisms of substratehydroxylation catalyzed by CYP105P1 and CYP105D6The CYP105 subfamily has attracted much attention due to its potential application in biosynthesis drugs and protein engineering.Despite CYP105P1 and CYP105D6 having high sequence similarities,they can catalyze substrate Filipin I（FLI,polyene macrolide antibiotic）hydroxylation at different positions（C26 and C1′,respectively）.However,the regiospecificity micro-mechanism of FLI hydroxylated by the two enzymes remains unclear.In this part of the study,multiple simulation approaches were employed to investigate the regiospecificity micro-mechanism of the FLI hydroxylated by CYP105P1 and CYP105D6.The simulation results determine that the B-C loop region and the K1 region are the two important structural units that affect the regiospecificity of the two enzymes by influencing the opening and closing of tunnels 2a and 2c and the FLI binding modes.We also propose the“regiospecificity molecular mechanism”:The structural differences between CYP105P1 and CYP105D6in the B-C loop region can affect the types of main tunnel for FLI ingress/egress protein;The tunnel entrance residues（CYP105P1:Q80 and E184;CYP105D6:R82,R83,E85,and R87）recognize specific FLI binding mode（C26-mode or C1′-mode）;The K1region further helps stabilize FLI binding to enzymes in specific binding mode,ultimately resulting in the two enzymes with regiospecificity（hydroxylated at C26 or C1′position）.This study could give a novel viewpoint about the regiospecificity of the substrate hydroxylated by the CYP105 subfamily and provide important insights into the biosynthesis of polyene macrolide antibiotics and protein engineering.