氨基醇類化合物對細(xì)粒棘球蚴的藥效評價以及作用靶點(diǎn)的初步鑒定
發(fā)布時間：2018-07-31 12:46
【摘要】：棘球蚴病(Echinococcosis),又稱包蟲病(Hydatid diseases),是一種全球分布的重要人獸共患病�；颊呖赏ㄟ^誤食入被蟲卵污染的食物和水源或者接觸感染的病犬等動物致病。棘球蚴在患者體內(nèi)的發(fā)展較為緩慢,主要產(chǎn)生占位性病變,若得不到有效治療,可導(dǎo)致其喪失勞動能力甚至死亡。目前,藥物治療是我國棘球蚴病患者重要的和不可或缺的治療手段。治療藥物僅有同為苯并咪唑類的阿苯達(dá)唑和甲苯達(dá)唑兩種,且治愈率均不高,亟待找到新的替代藥物。氨基醇類化合物是指包括羥基(-OH)和氨基(-NH、-NHR以及-NR2)的烷烴類化合物,這類化合物表現(xiàn)出了不同的藥理作用,如抗瘧原蟲、抗血吸蟲、抗絲蟲、抗腫瘤及抗菌活性。本研究利用建立的抗細(xì)粒棘球蚴高通量藥物篩選模型對氨基醇類化合物的藥效進(jìn)行了評價,并對該類化合物的藥物作用靶點(diǎn)進(jìn)行了探索研究。一、建立了抗細(xì)粒棘球蚴高通量藥物篩選模型通過優(yōu)化和改進(jìn)細(xì)粒棘球蚴原頭節(jié)(羊源)和生發(fā)層細(xì)胞(鼠源)體外培養(yǎng)條件,分別使用美蘭染色法和細(xì)胞活性測定法作為評價手段,建立了基于原頭節(jié)和生發(fā)層細(xì)胞的抗細(xì)粒棘球蚴體外藥物的高通量篩選方法。用甲苯達(dá)唑、甲氟喹、硝唑尼特及其代謝物進(jìn)行體內(nèi)和體外藥效實(shí)驗(yàn)評估該體外藥物篩選模型的效率,結(jié)果表明,該模型可真實(shí)反映藥物在體外對棘球蚴的作用效果,是實(shí)現(xiàn)體外大規(guī)模藥物篩選的基礎(chǔ)。二、氨基醇類化合物抗細(xì)粒棘球蚴效果評價、類藥性分析及毒性研究用已建立的體外藥物篩選方法對131個氨基醇類化合物進(jìn)行初篩,結(jié)果表明20%的待選氨基醇類化合物對原頭節(jié)和生發(fā)層細(xì)胞均表現(xiàn)出較強(qiáng)的活性。隨后對有效化合物分別進(jìn)行了LCso(原頭節(jié))及ⅠCso(生發(fā)層細(xì)胞)測定,其中有13個化合物的LC5o及ⅠCso均低于10μg/ml。經(jīng)過類藥性分析和毒性預(yù)測和測定后發(fā)現(xiàn),化合物16、54和124的細(xì)胞毒性低于甲氟喹,有作為先導(dǎo)化合物的潛能,待進(jìn)一步研究后有可能發(fā)展為抗棘球蚴的新型化合物。三、 氨基醇類化合物藥效團(tuán)模型建立及虛擬篩選準(zhǔn)備有活性的氨基醇類化合物建立訓(xùn)練集,用Discovery studio軟件中的Pharmacophore模塊建立藥效團(tuán)模型并用測試集對藥效團(tuán)模型進(jìn)行篩選。最終選擇的藥效團(tuán)包括一個正電荷中心、一個疏水芳香族、兩個疏水中心和一個氫鍵供體。利用該藥效團(tuán)模型對ZINC數(shù)據(jù)庫中的1000個化合物進(jìn)行虛擬篩選,其中有101個化合物的FitValue值大于3.0,排名靠前的化合物用于進(jìn)一步研究。四、 氨基醇類化合物抗棘球蚴藥物靶點(diǎn)研究使用idTarget服務(wù)器進(jìn)行藥物靶點(diǎn)預(yù)測,篩選出的靶點(diǎn)通過序列比對和同源建模,模擬了細(xì)粒棘球蚴相應(yīng)的藥物靶點(diǎn)蛋白三維結(jié)構(gòu)。隨后應(yīng)用分子對接(Autodock和Autocock vina)對上述藥物靶點(diǎn)進(jìn)一步進(jìn)行篩選。最終篩選出糖原磷酸化酶作為氨基醇類化合物的候選藥物靶點(diǎn)。體外實(shí)驗(yàn)結(jié)果表明,甲氟喹對細(xì)粒棘球蚴的糖原磷酸化酶僅在高濃度482.2μM有一定的抑制作用,其抑制率為57.8%。該部分的藥物靶點(diǎn)研究方法可為此類化合物的作用機(jī)制研究以及實(shí)現(xiàn)基于藥物靶點(diǎn)的藥物篩選研究奠定基礎(chǔ)。通過該課題的研究,建立抗棘球蚴藥物體外高通量篩選模型,該模型基于棘球蚴囊中的原頭節(jié)和生發(fā)層細(xì)胞,可真實(shí)反映藥物在體外對棘球蚴的作用效果�；谏鲜瞿Ｐ�,本課題還針對一類氨基醇類化合物進(jìn)行了研究,從中尋找到了可作為活性候選化合物的新結(jié)構(gòu)。除此之外,建立了建立了氨基醇類活性化合物的藥效團(tuán)模型并進(jìn)行虛擬篩選。本課題還建立了基于反向分子對接技術(shù)的抗棘球蚴藥物靶點(diǎn)篩選流程,篩選出了一個酶蛋白作為氨基醇類化合物的候選藥物靶點(diǎn)。
[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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R965
，
本文編號：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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R965
，
本文編號：2155624
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