Structure-based Novel Inhibitors Compounds Discovered By Rational Design And Screening Research

Rational designing novel lead structures of enzyme-targeted inhibitors play a key role in the process of drug or pesticide research and development.Many inhibitors have been developed by traditional lead compound discovery methods,in which a myriad of naturally occuring compounds have been surveyed for their ability to control disease or fungi.However,the explosion in the fundamental knowledge of biological protein interactions has enabled a rational approach to inhibitor design based on theortical and structural biology.Precisely examining the interactions between atoms in protein targets and atoms in potential lead compounds that bind to the proteins can improve the speed and veracity of rational inhibitor design.This ability to work at high resolution with both proteins and ligand makes receptor-ligand interaction mechanism research one of the fundamental scienctific issues in inhibitor design.At present,the interaction mechanism between protein and iigand studied mainly by molecular simulation,gene engineering,spectroscopic analysis,(solid-phase) organic syntheses,and so on.In this thesis the inhibitor structure,the interaction mechanism between ligand(inhibitor)and active site of 3-Hydroxy-3-methylglutaryl-coenzyme A reductase in human(Class-ⅠHMGR), 3-Hydroxy-3-methylglutaryl-coenzyme A roductase of procaryotic organism(Class-ⅡHMGR)and sterol 14 alpha-demethylase(CYP51)in fungus were systemic researched by molecular simulation, gene engineering,spectroscopic analysis,(solid-phase)organic syntheses,and so on.Based on above information structure-based rational design and screen for novel and effective lead structure of inhibitors were performed.The present thesis is organized as follows:In chapter one,computer-aided drug design in common used methods and theories have been reviewed,and the character,function,advances in Class-ⅠHMGR,Class-ⅡHMGR,CYP51,and respective inhibitors were introduced.In chapter two,three-dimensional quantitative structure-activity relationship(3D QSAR)with comparative molecular field analysis(CoMFA)was performed on known statins inhibitors of Class-ⅠHMGR.Predictive models were established by using two different ways:(1)Models-fit,obtained by SYBYL conventional fit-atom molecular alignment rule,(2)Models-dock,obtained by docking compounds into the HMGR active site.Integrated with CoMFA 3D QSAR predictive models, molecular surface property(electrostatic and steric)mapping and structure-based(both iigand and receptor)virtual screening have been employed to explore potential novel hits for the Class-ⅠHMGR inhibitors.A representative set of thirty new compounds of non-statin-like structures but with high pIC50 values were sorted out in the present study.These compounds will be used for biological test.In chapter three,the Class-ⅡHMGR 3-Hydroxy-3-methylglutaryl-coenzyme A reductase in Streptococcus pneumoniae(SP-HMGR)was studied.In computer theory term,the theory 3D-structure of SP-HMGR was obtained by homology modeling based on crystal structures of the Class-ⅡHMGR pseudomonas mevaloniil and molecular dynamic simulation.Subsequently, molecular docking and fragment molecular orbital(FMO)were performed to evaluate the suitably docking process and key residues in active site.Based on above research structured-based virtual screening was performed,and a set of thirty hits compounds were screening out for biological test.In experiment term,the full-length SP-HMGR gene were cloned and expressed in E.coli BL21.The activity of SP-HMGR was analyzed with an UV-Vis spectrophotometer based on the oxidation or reduction of NADPH monitored at 340nm.With the above computer modeling and experiment method we obtained a potential lead inhibitor of SP-HMGR.The last chapter,in members of the cytochrome P450 superfamily,the degree of sequence identity is low(＜28%).Fortunately,the topology of the cytochrome P450 superfamily protein is quite similar,most of the secondary and supersecondary structural motifs,characteristically hydrophobic and hydrophilic segments,and the regions of the sequence containing the beme binding site,the oxygen binding site,and the site of interactions with redox partners are highly conserved.Based on these information the accuracy theory of 3D-structure of Sterol 14α-demethylase of Penicillium digitatum(PD-CYP51)and Magnaporthe grisea(MG-CYP51)were constructed by taking into account further structural information during homology model.According to the hydrophobic character in active site of CYP51 and the inhibitor molecular mechanism,Flex Pharm/PMF/GOLD and FlexX/PMF/GOLD methods were used to virtual screening for novel inhibitors of PD-CYP51 and MG-CYP51,and a representative set of nineteen and twenty-seven hits compounds were screening out respectively.The results of biological test shown that several hits compounds exhibited good potential inhibit function on enzyme and thalli level in the two research system.