肺炎链球菌组氨酸激酶（VicK）的同源模建及其先导抑制剂的筛选和鉴定

发布时间：2018-06-28 17:43

本文选题：肺炎链球菌 + 组氨酸激酶　；参考：《重庆医科大学》2009年博士论文

【摘要】： 双组份系统(two-component system, TCS)是存在于细菌内的一种信号转导系统,细菌通过感受外界环境变化、调控生存、毒力因子表达来维持自身生存,是细菌适应选择压力的一种机制。在不同细菌中,该系统的结构非常类似,都由组氨酸激酶和反应调节子组成。其中,组氨酸激酶具有激酶、磷酸转移酶和磷酸酶的活性,是双组份系统通路中的核心蛋白[1]。其机制是外界信号作用于组氨酸激酶的膜外配体结合域,使组氨酸发生自身磷酸化,并将磷酸基团转移到反应调节子上,而产生一系列的调控反应[2]。由于在哺乳动物(包括人类)中未发现类似的调控系统[3],因此,组氨酸激酶是研发新型抗菌药物的潜在靶标[4]。组氨酸激酶VicK属于Pho亚家族,其最初是在枯草芽孢杆菌中被鉴定[5]。肺炎链球菌中的VicR/K[6]也被命名为MicA/B[7]或YycF/G[8],是目前肺炎链球菌中已发现的13对TCS组份之一。该通路系统具有高保守性和低G+C含量的特点,不仅影响肺炎链球菌的存活[9],还参与了其毒力、感受态的形成、以及维持菌体膜完整性等重要的作用[10]。因此,以肺炎链球菌组氨酸激酶VicK作为靶点有望筛选到有效的抗菌新药。目前,已有针对枯草芽孢杆菌及表皮葡萄球菌的组氨酸激酶YycG的筛选并得到了一些有效的抑制剂的报道[11],而对肺炎链球菌的研究中国内、外均未见相关报道。因此,本研究拟通过肺炎链球菌的组氨酸激酶VicK为研究对象,对其进行同源模建后虚拟筛选出针对该靶点的肺炎链球菌的先导抑制剂并进行实验研究。本研究内容包括以下几个部分: 1.肺炎链球菌组氨酸激酶VicK的同源模建与分析。采用同源模建的的方法构建VicK蛋白的三维结构,并用Procheck、profile_3D软件对此结构模型的合理性进行验证,采用Gromacs软件进行分子动力学优化,取平衡阶段的构象作为虚拟筛选中的受体模型。用Autodock4.0软件将结构模型与底物ADP进行自动对接,分析二者之间的相互作用。结果显示肺炎链球菌组氨酸激酶VicK与PDB编号为2c2a的Thermotoga maritima X晶体衍射结构(分辨率1.9 ?,R因子0.247)具有33%的一致序列,57%相似性;VicK模建后的结构与模板能很好的叠合;在活性口袋处的保守氨基酸残Asn145、Asn149、Lys152以及口袋内部的疏水残基在结合、水解底物的过程中发挥重要作用。结果提示:模建的肺炎链球菌组氨酸激酶VicK达到了设计标准,该模型可作为设计抗菌药的研究起点。 2.肺炎链球菌组氨酸激酶VicK先导抑制剂的虚拟筛选。将已知底物ADP叠合到模建的受体中,选择受体中距离小分子4 ?内的氨基酸残基为活性口袋,首先采用DOCK4.0对SPECS小分子数据库中的200,000个分子进行初步筛选,选择评分靠前的10,000个化合物;再用Sybyl6.8软件包中的Cscore模块进行评分,获得2,425个化合物;然后用类药性分析筛选得到308个化合物;最后采用Autodock4.0及经验进行人工挑选。结果显示通过四轮不同的筛选策略,最终从SPECS小分子数据库中得到了105个小分子化合物。 3.肺炎链球菌组氨酸激酶VicK活性鉴定。经结构数据库(http://smart.embl.de/smart/show_motif)对肺炎链球菌组氨酸激酶VicK分析,初步预测其由五个部分组成:一个跨膜信号肽,和4个已被证实的功能域:PAS、PAC、HisKA(HK)和ATPase_c。由于肺炎链球菌组氨酸激酶VicK是一个跨膜蛋白,因此需要对其胞内的HATPase_c激酶功能域片段(VicK')进行截短表达。PCR扩增肺炎链球菌(D39) VicK胞内激酶功能域序列,与pET28a载体构建重组质粒。之后,重组质粒转入大肠杆菌BL21(DE3),经IPTG诱导表达,通过SDS-PAGE和Western Blot进行鉴定,采用镍离子层析柱对表达的目的蛋白进行纯化,最后采用荧光激酶检测试剂盒检测纯化的VicK'蛋白的酶活性。结果显示原核表达的VicK'蛋白大约为35kDa,其纯度为95%,并具有水解ATP激酶的活性。 4.先导抑制剂的体外、体内的实验验证。利用表达的VicK'蛋白,初步对SPECS小分子化合物库中虚拟筛选获得的105个化合物进行筛选,以此得到具有抑制酶蛋白水解ATP作用的小分子化合物;选择对VicK'酶蛋白活性抑制率大于50%的23个化合物,进行抑菌实验,最终得到6个有抑菌作用的小分子化合物。为了进一步了解这6个化合物的抗菌特点及细胞毒性,分别采用了最小抑菌浓度(MIC)、最小杀菌浓度(MBC)以及细胞毒性实验。选取这6个化合物的不同浓度检测在不同的时间对肺炎链球菌作用的特点并绘制曲线,同时,依据此曲线来估计动物实验的体内用药量及给药时间。经腹腔感染动物摸索动物的最低肺炎链球菌致死剂量,最终选取每只实验动物的细菌浓度为5×103 CFU/ml;选用青霉素为阳性对照建立肺炎链球菌感染后治疗的动物模型;随后对此6个化合物进行动物体内实验,并设立必要的阴性及阳性对照,每组10-12只。结果显示,通过体外筛选得到6个具有抑菌作用的小分子化合物,其MIC的范围在0.28μΜ-200μΜ之间;且抑菌效果随着化合物浓度的增加而增强、抑菌的作用时间也延长,该结果表明这6个化合物抗菌作用具有明显的时间、浓度依赖关系。通过生存分析(Kaplan-Meier法)显示各组小鼠的生存时间总的比较中,化合物/药物处理因素的作用明显,具有统计学显著性。配对比较发现化合物1-6都较阴性对照组有显著的延长感染模型小鼠的存活时间(p0.01);但是化合物1、2、3和6的效果明显差于阳性对照青霉素的治疗效果(p0.05或p0.1);化合物4和5显示了比较理想的治疗效果。上述结果提示,本研究针对靶点蛋白VicK虚拟筛选出的105个先导抑制剂中,经实验验证有6个具有抑制肺炎链球菌生长的小分子化合物,为进一步研究抗菌的新型药物提供实验依据。
[Abstract]:Two - component system ( TCS ) is a kind of signal transduction system which exists in bacteria . It is a mechanism for bacteria to adapt to the selection pressure . The mechanism is that external signals act on the extracellular ligand binding domain of histidine kinase to phosphorylate histidine itself and transfer phosphoric acid groups to the reaction regulator to produce a series of regulatory responses . Since similar regulatory systems are not found in mammals , including humans , the histidine kinase is a potential target for the development of novel antibacterial drugs .

Histidine kinase VicK belongs to the Pho subfamily , which was originally identified in Bacillus subtilis . In Streptococcus pneumoniae , VicR / K - 6 mutants were also named MicA / B.7 mutants or YycF / G mutants . It is one of the 13 TCS components found in Streptococcus pneumoniae . The pathway system has the characteristics of high conservative and low G + C content , which affects not only the survival of Streptococcus pneumoniae , but also its virulence , the formation of competent state , and the maintenance of cell membrane integrity . Therefore , it is possible to screen effective antibacterial drugs by using Streptococcus pneumoniae histidine kinase ( VicK ) as a target . At present , there are some effective inhibitors for the selection of histidine kinase YycG in Bacillus subtilis and S . S . , and no relevant reports have been reported in the study of S . pneumoniae . Therefore , we have studied the pilot inhibitor of Streptococcus pneumoniae by homologous modular construction and conducted an experimental study in the study of Streptococcus pneumoniae . The contents of this study include the following parts :

1 . Homology modeling and analysis of Streptococcus pneumoniae histidine kinase VicK .

The three - dimensional structure of VicK protein was constructed by using the method of homology modeling . The rationality of the structural model was verified by using the software of Procheck and profile _ 3D . The conformation of the structure was optimized by using the software of Gromacs . The results showed that the structure and template of Streptococcus pneumoniae histidine kinase VicK and PDB No . 2c2a had an important role in the process of binding and hydrolyzing the substrate . The results showed that the model of Streptococcus pneumoniae histidine kinase VicK reached the design standard , which could be used as the starting point of the study of antimicrobial agents .

2 . Virtual screening of Streptococcus pneumoniae histidine kinase VicK leader inhibitor .

The known substrate ADP is laminated to the die - built receptor , and the small molecule 4 is selected in the receptor . The amino acid residue in SPECS small molecule database was first screened by DOCK4.0 , then the score was selected by using the Cscore module in Sybyl6.8 software package , then 308 compounds were obtained by using the Cscore module in Sybyl6.8 software package .

3 . Identification of Streptococcus pneumoniae histidine kinase VicK activity .

coli BL21 ( DE3 ) was amplified by SDS - PAGE and Western Blot . The results showed that the prokaryotic expression of VicK ' protein was about 35 kDa , the purity was 95 % , and the activity of ATP kinase was hydrolyzed .

4 . In vitro and in vivo experimental verification of the pilot inhibitor .

In order to further understand the antibacterial characteristics and cytotoxicity of these six compounds , the minimum inhibitory concentration ( MIC ) , minimum bactericidal concentration ( MBC ) and cytotoxicity test were used to further understand the antibacterial characteristics and cytotoxicity of these six compounds .

The results showed that the effects of compound 1 - 6 on the survival time of mice were significantly longer than those in the negative control group ( p < 0.01 ) . The results showed that the compound 1 - 6 had significant difference in the therapeutic effect of the positive control penicillin ( p < 0.01 ) .

The results suggested that 6 small molecule compounds with inhibiting S . pneumoniae growth were verified by experiments , which provide experimental basis for further study of anti - bacterial drugs .
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378

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