氨基醇类化合物对细粒棘球蚴的药效评价以及作用靶点的初步鉴定
发布时间：2018-07-31 12:46
【摘要】：棘球蚴病(Echinococcosis),又称包虫病(Hydatid diseases),是一种全球分布的重要人兽共患病。患者可通过误食入被虫卵污染的食物和水源或者接触感染的病犬等动物致病。棘球蚴在患者体内的发展较为缓慢,主要产生占位性病变,若得不到有效治疗,可导致其丧失劳动能力甚至死亡。目前,药物治疗是我国棘球蚴病患者重要的和不可或缺的治疗手段。治疗药物仅有同为苯并咪唑类的阿苯达唑和甲苯达唑两种,且治愈率均不高,亟待找到新的替代药物。氨基醇类化合物是指包括羟基(-OH)和氨基(-NH、-NHR以及-NR2)的烷烃类化合物,这类化合物表现出了不同的药理作用,如抗疟原虫、抗血吸虫、抗丝虫、抗肿瘤及抗菌活性。本研究利用建立的抗细粒棘球蚴高通量药物筛选模型对氨基醇类化合物的药效进行了评价,并对该类化合物的药物作用靶点进行了探索研究。一、建立了抗细粒棘球蚴高通量药物筛选模型通过优化和改进细粒棘球蚴原头节(羊源)和生发层细胞(鼠源)体外培养条件,分别使用美兰染色法和细胞活性测定法作为评价手段,建立了基于原头节和生发层细胞的抗细粒棘球蚴体外药物的高通量筛选方法。用甲苯达唑、甲氟喹、硝唑尼特及其代谢物进行体内和体外药效实验评估该体外药物筛选模型的效率,结果表明,该模型可真实反映药物在体外对棘球蚴的作用效果,是实现体外大规模药物筛选的基础。二、氨基醇类化合物抗细粒棘球蚴效果评价、类药性分析及毒性研究用已建立的体外药物筛选方法对131个氨基醇类化合物进行初筛,结果表明20%的待选氨基醇类化合物对原头节和生发层细胞均表现出较强的活性。随后对有效化合物分别进行了LCso(原头节)及ⅠCso(生发层细胞)测定,其中有13个化合物的LC5o及ⅠCso均低于10μg/ml。经过类药性分析和毒性预测和测定后发现,化合物16、54和124的细胞毒性低于甲氟喹,有作为先导化合物的潜能,待进一步研究后有可能发展为抗棘球蚴的新型化合物。三、 氨基醇类化合物药效团模型建立及虚拟筛选准备有活性的氨基醇类化合物建立训练集,用Discovery studio软件中的Pharmacophore模块建立药效团模型并用测试集对药效团模型进行筛选。最终选择的药效团包括一个正电荷中心、一个疏水芳香族、两个疏水中心和一个氢键供体。利用该药效团模型对ZINC数据库中的1000个化合物进行虚拟筛选,其中有101个化合物的FitValue值大于3.0,排名靠前的化合物用于进一步研究。四、 氨基醇类化合物抗棘球蚴药物靶点研究使用idTarget服务器进行药物靶点预测,筛选出的靶点通过序列比对和同源建模,模拟了细粒棘球蚴相应的药物靶点蛋白三维结构。随后应用分子对接(Autodock和Autocock vina)对上述药物靶点进一步进行筛选。最终筛选出糖原磷酸化酶作为氨基醇类化合物的候选药物靶点。体外实验结果表明,甲氟喹对细粒棘球蚴的糖原磷酸化酶仅在高浓度482.2μM有一定的抑制作用,其抑制率为57.8%。该部分的药物靶点研究方法可为此类化合物的作用机制研究以及实现基于药物靶点的药物筛选研究奠定基础。通过该课题的研究,建立抗棘球蚴药物体外高通量筛选模型,该模型基于棘球蚴囊中的原头节和生发层细胞,可真实反映药物在体外对棘球蚴的作用效果。基于上述模型,本课题还针对一类氨基醇类化合物进行了研究,从中寻找到了可作为活性候选化合物的新结构。除此之外,建立了建立了氨基醇类活性化合物的药效团模型并进行虚拟筛选。本课题还建立了基于反向分子对接技术的抗棘球蚴药物靶点筛选流程,筛选出了一个酶蛋白作为氨基醇类化合物的候选药物靶点。
[Abstract]:Echinococcosis (Echinococcosis), also known as echinococcosis (Hydatid diseases), is a global distribution of important zoonosis. Patients can be infested with food and water contaminated by eggs or infected with infected dogs and other animals. The development of echinococcosis is slow in the patient's body, mainly producing occupying lesions, if not available. Effective treatment can lead to the loss of labor and even death. At present, drug treatment is an important and indispensable treatment for the patients with echinococcosis in China. Only two kinds of drugs are treated with benzimidazole and benzidazole, and the cure rate is not high and new alternative drugs are needed. Alkanes including hydroxyl (-OH) and amino groups (-NH, -NHR and -NR2). These compounds show different pharmacological effects, such as antimalarial, anti schistosome, filariasis resistance, anti-tumor and antibacterial activity. This study evaluated the efficacy of aminoalcohols using a high flux drug screening model of Echinococcus granulosus. A high flux drug screening model for Echinococcus granulosus (Echinococcus granulosus) was established by optimizing and improving the culture conditions of echinococcosis (Yang Yuan) and germinal layer cells (Shu Yuan) in vitro, and using methylene blue staining and cell activity determination as evaluation methods. A high throughput screening method for drug resistance of Echinococcus granulosus from the primary and germinal cells. The efficiency of the drug screening model in vitro and in vitro was evaluated in vivo and in vitro by using metronidazole, methoquine, nitazolyl and their metabolites. The results showed that the model could truly reflect the effect of the drug on echinococcosis in vitro. It is the basis for large-scale drug screening in vitro. Two, the effect evaluation of amino alcohols against Echinococcus granulosus, analysis of drug resistance and toxicity study, 131 amino alcohols were screened by the established method of drug screening in vitro. The results showed that 20% of the amino alcohols were shown to both the primary and the germinal layer cells. The effective compounds were followed by LCso (primary section) and I Cso (germinal cells), respectively, of which 13 compounds of LC5o and I Cso were lower than 10 mu g/ml. after drug resistance analysis and toxicity prediction and determination. The cytotoxicity of compound 16,54 and 124 was lower than that of fluoro Quine. It is possible to develop new compounds against echinococcosis after further study. Three, the establishment of a pharmacodynamic group model of amino alcohols and the establishment of a training set of active amino alcohols by virtual screening, using the Pharmacophore module in the Discovery Studio software to establish a pharmacodynamic group model and use the test set for the drug cluster model. Screening. The final selected pharmacophore consists of a positive charge center, a aromatic fragrance family, two hydrophobic centers and a hydrogen bond donor. Using the drug cluster model, 1000 compounds in the ZINC database are virtual screened, of which 101 compounds have a FitValue value of more than 3, and the top compounds are used for further study. Four, the drug target of the amino alcohols against echinococcosis was studied by using the idTarget server to predict the drug target. The target points screened by the sequence alignment and homologous modeling were used to simulate the three-dimensional structure of the corresponding drug target protein of Echinococcus granulosus. Then, molecular docking (Autodock and Autocock Vina) was applied to the drug targets. Finally, the glycogen phosphorylase was selected as the candidate drug target of the amino alcohols. In vitro experimental results showed that the glycogen phosphorylase of Echinococcus granulosus had a certain inhibitory effect on the high concentration of 482.2 micron M, and the inhibition rate of the drug target of the 57.8%. part could be the compound of this kind of compound. Based on the study of the drug targeting, the high throughput screening model for echinococcosis in vitro is established. The model is based on the primary and germinal cells in the echinococcosis capsule, which can truly reflect the effect of the drug on the echinococcosis in vitro. In this subject, we have also studied a class of amino alcohols, and found a new structure that can be used as a candidate for active compounds. In addition, a pharmacophore model for the establishment of active compounds of amino alcohols and a virtual screening were established. The screening process has identified an enzyme protein as a candidate drug target for amino alcohols.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R965
，
本文编号：2155624
[Abstract]:Echinococcosis (Echinococcosis), also known as echinococcosis (Hydatid diseases), is a global distribution of important zoonosis. Patients can be infested with food and water contaminated by eggs or infected with infected dogs and other animals. The development of echinococcosis is slow in the patient's body, mainly producing occupying lesions, if not available. Effective treatment can lead to the loss of labor and even death. At present, drug treatment is an important and indispensable treatment for the patients with echinococcosis in China. Only two kinds of drugs are treated with benzimidazole and benzidazole, and the cure rate is not high and new alternative drugs are needed. Alkanes including hydroxyl (-OH) and amino groups (-NH, -NHR and -NR2). These compounds show different pharmacological effects, such as antimalarial, anti schistosome, filariasis resistance, anti-tumor and antibacterial activity. This study evaluated the efficacy of aminoalcohols using a high flux drug screening model of Echinococcus granulosus. A high flux drug screening model for Echinococcus granulosus (Echinococcus granulosus) was established by optimizing and improving the culture conditions of echinococcosis (Yang Yuan) and germinal layer cells (Shu Yuan) in vitro, and using methylene blue staining and cell activity determination as evaluation methods. A high throughput screening method for drug resistance of Echinococcus granulosus from the primary and germinal cells. The efficiency of the drug screening model in vitro and in vitro was evaluated in vivo and in vitro by using metronidazole, methoquine, nitazolyl and their metabolites. The results showed that the model could truly reflect the effect of the drug on echinococcosis in vitro. It is the basis for large-scale drug screening in vitro. Two, the effect evaluation of amino alcohols against Echinococcus granulosus, analysis of drug resistance and toxicity study, 131 amino alcohols were screened by the established method of drug screening in vitro. The results showed that 20% of the amino alcohols were shown to both the primary and the germinal layer cells. The effective compounds were followed by LCso (primary section) and I Cso (germinal cells), respectively, of which 13 compounds of LC5o and I Cso were lower than 10 mu g/ml. after drug resistance analysis and toxicity prediction and determination. The cytotoxicity of compound 16,54 and 124 was lower than that of fluoro Quine. It is possible to develop new compounds against echinococcosis after further study. Three, the establishment of a pharmacodynamic group model of amino alcohols and the establishment of a training set of active amino alcohols by virtual screening, using the Pharmacophore module in the Discovery Studio software to establish a pharmacodynamic group model and use the test set for the drug cluster model. Screening. The final selected pharmacophore consists of a positive charge center, a aromatic fragrance family, two hydrophobic centers and a hydrogen bond donor. Using the drug cluster model, 1000 compounds in the ZINC database are virtual screened, of which 101 compounds have a FitValue value of more than 3, and the top compounds are used for further study. Four, the drug target of the amino alcohols against echinococcosis was studied by using the idTarget server to predict the drug target. The target points screened by the sequence alignment and homologous modeling were used to simulate the three-dimensional structure of the corresponding drug target protein of Echinococcus granulosus. Then, molecular docking (Autodock and Autocock Vina) was applied to the drug targets. Finally, the glycogen phosphorylase was selected as the candidate drug target of the amino alcohols. In vitro experimental results showed that the glycogen phosphorylase of Echinococcus granulosus had a certain inhibitory effect on the high concentration of 482.2 micron M, and the inhibition rate of the drug target of the 57.8%. part could be the compound of this kind of compound. Based on the study of the drug targeting, the high throughput screening model for echinococcosis in vitro is established. The model is based on the primary and germinal cells in the echinococcosis capsule, which can truly reflect the effect of the drug on the echinococcosis in vitro. In this subject, we have also studied a class of amino alcohols, and found a new structure that can be used as a candidate for active compounds. In addition, a pharmacophore model for the establishment of active compounds of amino alcohols and a virtual screening were established. The screening process has identified an enzyme protein as a candidate drug target for amino alcohols.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R965
，
本文编号：2155624
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