【作者】 杨娇艳；

【导师】 杨劭；

【作者基本信息】 华中师范大学 ， 农药学， 2008， 博士

【摘要】 稻瘟病是限制水稻高产稳产的主要因素,广泛发生在世界各稻区,每年稻瘟病爆发对农业造成巨大经济损失。甾醇14α-去甲基化酶抑制剂（DMI inhibitors,DMIs）是目前农业生产中主要使用的一类抗真菌剂,它通过抑制靶标甾醇14α-去甲基化酶(sterol 14α-demethylase,P450_14DM,CYP51)的活性而发挥杀菌抑菌作用。甾醇14α-去甲基化酶催化甾醇的14α-去甲基化反应,是真菌麦角甾醇合成过程中的一个关键酶,而麦角甾醇是真菌细胞膜的主要组分,抑制甾醇14α-去甲基化酶活性会阻碍真菌麦角甾醇的合成,麦角甾醇缺失会损害细胞膜的结构和功能,最终导致真菌死亡。为了合成和筛选稻瘟病的高效特异性杀菌剂,本研究以其病原体稻瘟病菌（Magnaporthegrisea）为研究对象,结合分子生物学技术和计算机辅助技术,从受体和配体两方面进行了研究。一方面从靶酶研究出发,对稻瘟病菌CYP51（MGCYP51）基因进行克隆和异源高效表达,同时对MGCYP51与氮唑类抗真菌剂的结合模式进行了分析,发现了靶酶的抑制剂特异结合位点;另一方面从抗真菌药物出发,利用异源表达的MGCYP51建立体外稻瘟病菌DMIs药物筛选方法,对筛选结果进行化合物结构特点分析,总结药物结构特点并用于指导抗真菌药物结构的改造;综合这两方面的研究,利用计算机药物辅助设计技术设计MGCYP51抑制剂,并进行体外筛选,获得MGCYP51抑制剂先导化合物,为稻瘟病菌新型高效特异杀菌剂的开发提供基础。具体研究结果如下。1.从稻瘟病菌中克隆了稻瘟病菌的CYP51基因（MGCYP51）,该基因的氨基酸序列与已公布的序列（XP 362183和EDJ95970）相比仅有一个氨基酸不同,在266位插入一个谷氨酸。同源比对分析发现MGCYP51具有6个保守结构域SRS1-6和血红素辅基结合区（-FGxGxHxCxGxxFA-）,其中SRS1（-YxxLxxPxFGxxVx-）和SRS4（-GQHTS-）是CYP51亚家族的识别标记。2.在大肠杆菌中获得MGCYP51高效表达。研究发现,去除N端跨膜区对于MGCYP51的原核表达至关重要。不管采用怎样的载体和宿主菌,全长MGCYP51在大肠杆菌中不表达,去除N端36个跨膜氨基酸后,截短的MGCYP51（T-MGCYP51）利用pET-30a（+）表达系统在BL21（DE3）pLysS宿主菌中获得高效表达,能维持CO结合活性。优化MGCYP51的表达菌株、表达载体、诱导温度、诱导时间和诱导剂浓度等条件后,利用pET-T-MGCYP51和pKG-T-MGCYP51重组表达菌株,在0.4mM IPTG条件下18℃诱导4h后的菌体作为材料,利用Ni-NTA琼脂糖亲和层析和GST-tag亲和层析技术,成功纯化得到MGCYP51重组蛋白。3.以原核表达的MGCYP51重组蛋白为材料,建立了DMIs抗真菌药物的体外筛选方法——结合光谱法。研究中分析了MGCYP51与DMIs抗真菌药物标准样品烯唑醇、戊唑醇、三唑醇和三唑酮的结合能力,分析了靶酶的活性、纯度及浓度对靶酶和药物间结合光谱的影响。结果表明,靶酶的P450活性、纯度及合适的靶酶浓度是获得准确结合光谱的必要条件。以表达MGCYP51的E.coli膜碎片作为研究材料,E.coli本身并无P450,避免了干扰,并且可以获得足够量且有活性的真菌靶酶CYP51,而且本身MGCYP51就是膜结合蛋白,用膜碎片作为研究材料更接近真实状态,使二者的结合光谱更能准确直接反映杀真菌剂与靶酶的结合。四种杀真菌剂与CYP51的亲和力的大小依次是烯唑醇＞戊唑醇＞三唑醇＞三唑酮,不仅顺序与杀真菌剂对稻瘟病菌的生长抑制能力实验结果相吻合,而且结合常数K_d与lgEC₅₀具有显著相关性（P＜0.05）,这说明结合常数直接反映了杀真菌剂对靶酶功能的抑制能力,利用结合光谱法测定的结合常数反映抗真菌药物的抑菌潜力是真实和可靠的。4.应用结合光谱法从随机合成的氮唑类和非氮唑类化合物中筛选MGCYP51抑制剂。共筛选出7种与靶酶的亲和力和体外抑菌活性均较高的先导化合物,包括LHEXP-10、SYL-25、SYL-35、SYL-36、ZST-17、WJ-5、WJ-15。氮唑类衍生物包括三唑并咪唑啉酮、咪唑啉酮及三唑系列,非氮唑类化合物包括有机磷化合物、含吲哚环及含吡咯环系列。通过结构-活性比较分析,表明①咪唑啉酮类化合物咪唑啉酮苯环侧链卤素取代会增强其与MGCYP51的结合,而苯环侧链烷基取代会减弱甚至丧失与MGCYP51的结合;②ZST系列三唑手性化合物中S型比R型有利于ZST系列与MGCYP51的结合,苯环上3位卤素取代有利于ZST系列与MGCYP51的结合,且溴代比氯代、氟代的效果好,而苯环上1位卤素取代却减弱化合物与MGCYP51的亲和力;③吲哚或吡咯等杂环结构可能对于化合物与MGCYP51的结合没有作用,而噻吩杂环结构却可能有利于化合物与MGCYP51的结合。5.利用同源模建、分子对接、结合光谱法和定点突变研究了MGCYP51与三氮唑类抗真菌药物的结合特点,对高效特异稻瘟病菌DMIs杀真菌剂的研发具有重要的指导作用。研究表明化合物疏水性是决定三氮唑类化合物抑制MGCYP51活性能力的最主要因素,MGCYP51活性空腔中Y112,F120,F220,H308和F497是与三氮唑类抗真菌药物发生结合作用的关键氨基酸,而且F220和F497是特异性MGCYP51抑制剂设计的靶位点。定点突变结果还表明Y112、F220和F497与MGCYP51的三维空间结构维持有关。6.根据化合物结构-活性分析及靶酶与三氮唑类化合物结合特点的分析结果,利用计算机辅助药物设计技术,设计合成19种待筛选的化合物,从中筛选出4种与MGCYP51的亲和力和体外抑菌活性均高的先导化合物,包括QY3、QY8、QY9和QY17。QY3和QY17与MGCYP51的亲和力与烯唑醇相当,比戊唑醇强,并且QY3对稻瘟病菌生长抑制的120h-EC₅₀比三唑酮的低。这些结果表明QY3可能是稻瘟病菌高效特异的DMIs杀真菌剂,具有良好的商业应用前景。更多还原

【Abstract】 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.更多还原