以PDF为靶点的新药筛选及活性组分的分离纯化、结构鉴定与活性研究

【作者】 董国霞；

【导师】 张月琴； 余利岩； 姜威；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2009， 博士

【摘要】 随着抗生素的广泛使用甚至滥用,各种耐药病原菌大量出现,目前临床耐药菌的发展势态已对人类健康构成极大的威胁。寻找新的作用靶点,研发新型抗耐药菌的药物已成为本世纪全球关注的研究热点之一。肽脱甲酰基酶（peptide deformylase,PDF）是原核生物蛋白成熟过程中的一个关键酶,而非真核生物所必需。鉴于PDF具有作为药物筛选靶点的众多优点,近年来被广泛认同为最理想的新一代广谱抗菌药物筛选分子靶点之一。本研究采用分子生物学的方法与技术对屎肠球菌（E.faecium）的PDF酶进行克隆表达,用Ni²⁺金属螯合亲和层析柱对目的蛋白进行分离纯化,对纯化所得PDF进行Western blotting鉴定与活性检测。以纯化的PDF为靶点开展了抑酶活性物质筛选和以E.faecium为检定菌的抑菌活性物质筛选。通过对20261个微生物发酵样品的初筛和对初筛阳性样品的两次复筛,获得6株发酵产物活性稳定的阳性菌株,其中游动放线菌103A-00723和游动放线菌103A-08772的发酵产物对多种G⁺耐药细菌有抑菌活性,包括MRSA、MRSE、E.faecalis HH22（含双功能修饰酶的粪肠球菌）和VRE。经分类鉴定,这两株菌为游动放线菌属的两个新种,分别被命名为Actinoplanes sichuanensis sp.nov.和Actinoplanes Xinjiangensis sp.nov.。链霉菌I06A-01113的发酵产物抗VRE和S.aureus（金黄色葡萄球菌）的敏感株,但不抗MRSA。对获得的阳性菌株I03A-00723进行放大发酵,并对发酵产物进行分离纯化,得到95-1、95-1-h1、95-2、95-3、135、205-1、205-2和205-3八个单一组分,经结构分析95-1、95-2、135、205-1分别被确定为N^b-乙酰色胺、大豆苷元、染料木素和腺苷,95-1-h1、95-3、205-2和205-3因量太少,其结构有待进一步鉴定。本研究首次从游动放线菌中分离得到N^b-乙酰色胺、大豆苷元和染料木素,并首次发现这三个活性物质对PDF酶有抑制作用,而且对E.faecium有抗菌活性,且大豆苷元和染料木素对VRE的抗菌活性优于阳性对照Actinonin。含量极少的组分95-3对VRE的抗菌活性优于Actinonin和已报道的阳性化合物BB3497和VRC3375,与已进入临床研究的阳性化合物LBM415的抗菌活性相当,是值得深入研究的组分。本研究还以PDF的晶体结构为基础,采用sybyl7.3软件中的Surflex-Dock对我室Microbial Natural Products Database（微生物天然产物库）进行了虚拟筛选,对部分虚拟筛选阳性化合物进行了抑酶和抗菌活性检测。结果发现,虚拟筛选获得的阳性化合物Polyoxin B、Spergualin和L-4-oxalysine具有一定的抑酶和抗菌活性。同时,对实物筛选获得的阳性化合物和已知阳性抑制剂（Actinonin、LBM415、BB83698、VRC4307、VRC3375和BB3497）与PDF进行分子了对接,发现阳性化合物95-2、135和已知阳性抑制剂的虚拟筛选得分和它们的抑酶和抗菌活性相一致。本研究采用实物筛选与虚拟筛选相结合的方法,得到若干活性化合物,为寻找以PDF为靶点的新药或其先导化合物奠定了基础。更多还原

【Abstract】 With the incorrect use of antibiotics or even abuse,the rapid emergence of various G⁺ resistant bacteria,such as MRSA,MRSE,VRE,presents a very serious threat to public health.Therefore,searching for new targets and developing new drugs with novel mechanisms and potent activity against G⁺ resistant bacteria have become an urgent need and an international research project.Peptide deformylase（PDF） is an essential enzyme in the protein maturation of prokaryote,but it is absent from mammalian cells.As its many advantages as a drug target,in recent years,PDF has been widely regarded as one of ideal targets for the screening of new broad-spectrum antibiotic agents.In this study,PDF of E.faecium was cloned and expressed adopting the methods and techniques of molecular biology.The protein obtained was purified by Ni²⁺ metal chelating affinity chromatography column and was identified by Western blotting and activity assay.The screening targeted on PDF includes two parts:screening for inhibiters against E. faecium and screening for inhibiters against PDF activity.As a result,6 positive strains w -ith stable fermentation broths activity were picked out through the initial screening of 20,261 samples of microbial fermentation broths and the other twice re-screening of positive samples.Among them,fermentation broths of positive strains I03A-00723 and I03A-08772 which were identified as nova species of Actinoplanes and named as Aactinoplanes sichuanensis sp.nov,and Actinoplanes xinjiangensis sp.nov,respectively have antimicrobial activity against G⁺ resistant bacteria including MRSA,MRSE,E. faecalis HH22 and VRE.The fermentation broth of positive strain I06A-01113 which belongs to streptomyces has antimicrobial activity against VRE not MRSA.The positive strain I03A-00723 was fermented largely,its fermentation broth was isolated and active components were purified.As a result,8 components were obtained which were 95-1,95-1-h1,95-2,95-3,135,205-1,205-2 and 205-3.Among them,95-1, 95-2,135 and 205-1 were identified as N^b-acetyltryptamine,daidzein,genistein and adenosine respectively and 95-1-h1,95-3,205-2 and 205-3 haven’t been identified because of a little content of their samples.It was the first time that N^b-acetyltryptamine, daidzein and genistein were discovered to be produced by strain of Actinoplanes and to have inhibitory activity not only to enzyme PDF but also to E.faecium.Furthermore,the activity of daidzein and genistein against VRE are superior to a known PDF inhibitor Actinonin,and the activity of the minimal component 95-3 against VRE is superior to positive compounds BB3497 and VRC3375 and is approximate to LBM415 which has been in a stage of clinical study,therefore 95-3 is worthy to be studied further.Virtual screening was done with Surflex-Dock in the syby17.3 software on Microbial Natural Products Database,based on the crystal structure of PDF.Some virtual screening compounds were tested the inhibitory activity to PDF and the antibacterial activity to E.faecium.As a result,PolyoxinB,Spergualin and L-4-oxalysine were discovered to have inhibitory activity to PDF and E.faecium.Meanwhile,positive components 95-1,95-2, 135 and known positive compounds such as Actinonin,LBM415,BB83698,VRC4307, VRC3375 and BB3497 were docked with PDF crystal structure,the results showed that their virtual screening scores are correspond to their activities to PDF and E.faecium.In this study,several active compounds inhibiting PDF were obtained by applying model screening combining with virtual screening and the research results would lay foundation for finding inhibitors or lead compounds targeted on PDF.更多还原