以肺炎链球菌WalK酶HATPase-c结构域为靶点的新型咪唑类化合物的抗菌效应研究

发布时间：2018-05-13 08:27

本文选题：肺炎链球菌 + 催化ATP酶结构域　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:本课题组前期采用分子对接的方法,筛选出以肺炎链球菌Wal K酶HATPase-c结构域为作用靶点的咪唑类化合物6,其在体外展示出较好的抗菌活性,但其体内保护效果不理想,且细胞毒性较高。本研究拟以肺炎链球菌Wal K酶HATPase-c结构域(CA结构域)三维结构中氨基酸功能残基为药物作用靶点,采用化合物6为先导结构模板,设计并合成一系列新型咪唑类抗菌化合物。通过化合物和三种不同的Wal K蛋白相互作用,验证新型化合物抗菌靶点位于肺炎链球菌Wal K酶CA结构域;通过体外实验考查化合物抗菌活性及毒性反应;通过建立小鼠肺炎链球菌感染模型,检测其体内抗菌效应。本研究拟最终获得毒性小、体内抗菌活性高、可能具有广谱抗革兰阳性菌的新型抗菌药物。方法:本课题拟通过分子设计—合成—生物活性测试模式,采用活性基团拼接法和取代法,合成一系列新型咪唑类抗菌药物;再对合成的咪唑类抗菌药物进行元素、1H NMR、13C NMR、MS(ESI)、熔点、远红外光谱等表征分析。利用PCR法扩增3种不同肺炎链球菌Wal K基因序列(全长、缺陷CA结构域、仅含有CA结构域),并构建3种Wal K基因序列的重组pp SUMO质粒,再将其转化到E.coli BL21中,依靠IPTG诱导表达及Ni-NTA亲和层析,得到纯度较高的可溶性目的蛋白;利用该蛋白免疫BALB/C小鼠获取多克隆抗体,经ELISA法检测其特异性Ig G抗体效价,经Western Blot鉴定Wal K全长蛋白在4种S.pn血清型菌株中的保守性。利用ATP激酶发光检测试剂盒检测以上三种蛋白的激酶活性,检测蛋白与新型咪唑类化合物作用后的激酶活性变化(IC50)。通过最小抑菌浓度(MIC)、最小杀菌浓度(MBC)及细胞毒性试验(MTT法)等实验评价化合物的体外抗菌效应以及细胞毒性作用。构建BALB/C小鼠的腹腔S.pn感染模型,通过尾静脉注射途径给药,观察小鼠的生存时间和生存率。结果:本课题组前期已经成功合成以原始化合物6为母体的15种新型咪唑类化合物,并对其中的6种化合物进行了1H NMR、13C NMR、高分辨质谱、红外光谱、熔点等结构和组成表征分析,结果提示为预期目标分子。成功构建和表达Wal K(全长、缺陷CA结构域、仅含有CA结构域)重组蛋白;以全长Wal K蛋白免疫小鼠后获得高滴度的多克隆抗体,Western Blot分析证实,CMCC(B)31207、CMCC(B)31203、CMCC(B)31693、NTCC7466(D39)等4种S.pn菌株均能稳定的表达Wal K全长蛋白。随着新型咪唑类化合物(2号、4号及5号)药物浓度的逐渐增高,Wal K全长组和仅含有CA结构域组ATP激酶活性均持续降低(均P0.05);而不同浓度的新型咪唑类化合物(2号、4号及5号)与缺陷CA结构域组作用,其ATP激酶活性变化无明显差异(P0.05)。通过最小抑菌浓度、最小杀菌浓度等体外实验证实,新合成咪唑类化合物2号、4号、5号均具有较好的抑菌及杀菌效应,其中4号化合物抑菌及杀菌效果最为明显(P0.01);细胞毒性试验证实,三种化合物细胞毒性均显著低于原始化合物(P0.01),其中以2号细胞毒性最低(1170μM)。化合物对腹腔感染D39型肺炎链球菌小鼠的治疗效果证实,与阳性对照组(PNC组)比较,原始化合物6号、新合成咪唑类化合物4号、5号的保护效果均未见明显差异(均P0.05);2号化合物与阳性对照组间比较其保护效果明显降低(P0.05)。结论:Wal K全长重组蛋白在不同血清型S.pn中保守性较好,新合成咪唑类化合物(2号、4号及5号)具有抑制Wal K酶CA结构域ATP激酶活性的效应;最小抑菌浓度和最小杀菌浓度试验均证实4号化合物在体外具有极佳的抗菌及杀菌效应;细胞毒性实验证实以2号、4号、5号化合物细胞毒性均显著低于原始化合物;体内抗菌效应试验提示新合成的咪唑类化合物4号、5号均能够明显延长腹腔感染肺炎链球菌D39小鼠的生存时间。本研究首次证实新合成的4号咪唑类化合物可能是未来治疗肺炎链球菌感染的新型药物,后续实验将着重其药物代谢学、药物动力学方面的深入研究,以期筛选出抗革兰氏阳性细菌感染的广谱新型咪唑类化合物。
[Abstract]:Objective: to select the imidazole compound 6, which is the target of the Wal K enzyme HATPase-c domain of Streptococcus pneumoniae, in the early stage of this study, it shows good antibacterial activity in vitro, but its protective effect is not ideal in vivo, and the cytotoxicity is high. This study is to use the Wal K enzyme HATPase-c domain of Streptococcus pneumoniae. A series of novel imidazole antibacterial compounds were designed and synthesized by using compound 6 as the precursor template in the three-dimensional structure of the CA domain. A series of new imidazole antibacterial compounds were designed and synthesized by using compound 6 as the leading structure template. Through the interaction of compounds and three different Wal K proteins, the anti bacteria target of the new compound was located in the Wal K enzyme domain of Streptococcus pneumoniae; Test the antibacterial activity and toxic reaction of the compound in vitro; by establishing the model of Streptococcus pneumoniae infection in mice, to detect the antibacterial effect in vivo. This study is intended to eventually obtain small toxicity, high antibacterial activity in the body, and may have a new type of antibacterial agent with broad spectrum anti gram positive bacteria. A series of new imidazole antibacterials were synthesized by active group splicing and substitution method, and then the synthetic imidazole antibacterials were characterized by elemental, 1H NMR, 13C NMR, MS (ESI), melting point and far infrared spectroscopy. The PCR method was used to amplify the Wal K sequence of 3 different Streptococcus pneumoniae (total length, defect CA structure). The domain, which only contains the CA domain), and constructs the recombinant PP SUMO plasmid of 3 Wal K gene sequences, then transforms it into E.coli BL21, and obtains the high purity soluble target protein by IPTG induced expression and Ni-NTA affinity chromatography. The polyclonal antibody is obtained by using the protein to immunize BALB/C mice and the specific Ig antibody titer of its specific Ig is detected by ELISA method. The conservatism of Wal K full-length protein in 4 S.pn serotype strains was identified by Western Blot. The kinase activity of the above three proteins was detected by the ATP kinase luminescence detection kit, and the activity of kinase activity (IC50) after the action of the protein and new imidazole compounds was detected. The minimum bacteriostasis concentration (MIC), the minimum bactericidal concentration (MBC) and cytotoxicity were obtained. The in vitro antibacterial effect and cytotoxicity of the compound were evaluated by the experiment of sex test (MTT method). The S.pn infection model of the abdominal cavity of BALB/C mice was constructed. The survival time and survival rate of the mice were observed through the injection of the tail vein. Results: the group had successfully combined the 15 new imidazole groups of the original compound 6 in the early stage. Compounds were used to analyze the structure and composition of the 6 compounds, such as 1H NMR, 13C NMR, high resolution mass spectrometry, infrared spectrum, melting point and so on. The results suggested that the target molecule was expected. The recombinant protein was successfully constructed and expressed in Wal K (full length, defective CA domain, only CA domain), and the high titer was obtained after the whole long Wal K protein was immune to mice. The Western Blot analysis showed that the 4 S.pn strains of CMCC (B) 31207, CMCC (B) 31203, CMCC (B) 31693, NTCC7466 (D39), and so on were all stable to express Wal full length protein. P0.05, while the different concentrations of the new imidazole compounds (No. 2, No. 4 and 5) and the defective CA domain group have no significant difference in the activity of ATP kinase (P0.05). Through the minimum inhibitory concentration and minimum bactericidal concentration in vitro, the new synthetic imidazole compound 2, 4 and 5 have better bacteriostasis and bactericidal effect, The bacteriostasis and bactericidal effect of compound 4 was most obvious (P0.01). Cytotoxicity test confirmed that the cytotoxicity of the three compounds was significantly lower than that of the original compound (P0.01), and the cytotoxicity of No. 2 was the lowest (1170 mu M). The therapeutic effect of the compound on the peritoneal infection of Streptococcus pneumoniae mice was confirmed, compared with the positive control group (PNC group). Compound 6, new synthetic imidazole compound No. 4, No. 5, no significant difference in protection (all P0.05), the protective effect of compound 2 was significantly lower than that between positive control groups (P0.05). Conclusion: Wal K full length recombinant protein is conserved in different serotype S.pn, and new synthetic imidazole compounds (No. 2, 4 and 5) have inhibitory effect. The effect of Wal K enzyme CA domain ATP kinase activity was made. The minimum bacteriostasis concentration and minimum bactericidal concentration test showed that compound 4 had excellent antibacterial and bactericidal effect in vitro. Cytotoxicity test showed that the cytotoxicity of compound 2, No. 4 and No. 5 was significantly lower than that of the original compound; in vivo antibacterial effect test suggested the newly synthesized mica. The number of azoles 4 and 5 can significantly prolong the survival time of the abdominal infection of Streptococcus pneumoniae D39 mice. This study is the first to confirm that the newly synthesized 4 imidazole compounds may be a new drug for the treatment of Streptococcus pneumoniae infection in the future. A broad spectrum of new imidazole compounds resistant to gram positive bacterial infections.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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