Characteristics Of Interaction Between Sterol 14α-demethylase Of Magnaporthe Grisea And Inhibitors And The Screening Of Novel Fungicides

Rice blast,caused by the ascomycete fungus Magnaporthe grisea,is one of the most serious diseases for cultivated rice and causes serious recurrent epidemics throughout rice growing regions in the world.Every year,the economic loss caused by rice blast is significant.Inhibitors of fungal sterol 14α-demethylase(CYP51,P450_14DM),termed demethylase inhibitors(DMI fungicides,DMIs),are the main group of fungicides used in agriculture.DMIs inhibit the biosynthesis of ergosterol.Ergosterol depletion will result in the accumulation of methylated sterol precursors,and affect both membrane integrity and function of membrane-bound proteins,eventually cause inhibition of fungal growth.In order to obtain specific and effective fungicides for M.grisea,we have investigated the characteristics of both target enzyme CYP51 and inhibitor compounds by combining the molecular biology technique with computer-aided technique.About the target enzyme,CYP51 from M.grisea(MGCYP51)was cloned and over-expressed in E. coli.Three-dimensional structure of MGCYP51 was built via homology modeling.The characteristics of the interaction between MGCYP51 and azole fungicides were analyzed via difference binding spectra and molecular docking.About inhibitor compounds,a new DMIs screening method in vitro-binding spectrum was established and used in screening MGCYP51 inhibitors.Furthermore the structure-activity relationship was discussed to guide the fungicides' modification.Based on the results of above study,MGCYP51 inhibitors was designed via computer-aided drug design,and effective lead compounds were obtained by screening with binding spectrum.The results obtained in this study, listed below,provided basis for designation of novel,specific and effective DMIs for M. grisea.1.MGCYP51 gene was cloned.The sequences were confirmed identical to the published sequence(XP₃62183 and EDJ95970),except the 266th Glutamic acid inserted.Homologous alignment analysis indicated that MGCYP51 contained six conserved domains(substrate binding region,SRS)and heme binding region (-FGxGxHxCxGxxFA-).SRS1(-YxxLxxPxFGxxVx-)and SRS4(-GQHTS-)are CYP51 signatures.2.MGCYP51 was over-expressed in E.coli.It is founded that the truncation of N-terminal amino acids of MGCYP51 were critical for its heterologous expression in E. coli.Full-length MGCYP51 was not expressed with various combinations of expression vector and strain,unless N-terminal 36aa of MGCYP51 was truncated.The truncated MGCYP51 was successfully expressed in E.coli and the P450 activity was maintained. After optimization of MGCYP51 expression in E.coli,recombinant MGCYP51 was purified by Ni-NTA or GST sepharose resin affinity chromatography under natural conditions.3.DMIs screening method in vitro-binding spectrum was established using heterologous expression MGCYP51 membrane.The affinities of four commercial azole fungicides diniconazole,tebuconazole,triadimenol and triadimefon for MGCYP51 were analyzed.The effects of enzyme activity,purity and concentration on the binding spectra were investigated.The results showed that active enzyme,elimination of interference of other P450s and proper enzyme concentration were necessary for obtaining accurate binding spectra.The K_d values of diniconazole,tebuconazole,triadimenol and triadimefon significantly correlated to their 120h-EC₅₀on the growth of M.grisea (P＜0.05),which indicated that the binding spectra can serve as a reliable and fast method for DMIs fungicides screening.4.According to binding spectra and growth inhibition assay in vitro,seven potential effective and selective lead compounds were obtained,including LHEXP-10,SYL-25, SYL-35,SYL-36,ZST-17,WJ-5 and WJ-15,in type of organophosphorus compounds, pyrroles,indoles,imidazolinone derivatives,and azole derivatives respectively.The corollaries were concluded based on structure-activity relationship analysis.Firstly, among imidazolinone derivatives phenyl with halogen substituent could enhance its affinity for MGCYP51,and alkyl substituent was on the opposite.Secondly,among ZST series azole derivatives S isomer have stronger affinity in binding to MGCYP51 compared to R isomer.Phenyl-3-halogen substituent of ZST series compounds could enhance its affinity for MGCYP51,and Br-substituent has stronger affinity than Cl-and F-substituent.But Phenyl-1-halogen substituent of ZST series compounds could decrease its affinity for MGCYP51.Thirdly,pyrrole and indole compounds do not bind to MGCYP51,but thiophene could enhance its affinity for MGCYP51.5.The characteristics of the interaction between MGCYP51 and azole fungicides were analyzed via difference binding spectra,homology modeling,molecular docking and site-directed mutation.The results indicated that the hydrophobicity of the fungicide may be the key factor in determining its binding affinity for MGCYP51.Besides,Y112, F120,F220,H308 and F497 in active cavity are the key residues interacting with azole fungicides.Among them F220 and F497 are target sites of high selective MGCYP51 inhibitors.Y112,F120 and H308 function in the maintaining of 3D structure.6.19 compounds were designed via computer-aided drug design and screened by binding spectra and growth inhibition assay in vitro.The results showed QY3,QY8,QY9 and QY17 are potentially specific and effective lead compounds of MGCYP51 inhibitors. Furthermore,the affinities of QY3 and QY17 for MGCYP51 are basically equivalent with diniconazole and stronger than tebuconazole.120-EC₅₀of QY3 on growth of M. grisea was smaller than triadimefon.The results showed the above compounds may serve as potential fungicides for M.grisea.