本文選題：流感病毒 + 神經(jīng)氨酸酶　； 參考：《山東大學(xué)》2014年博士論文

【摘要】：流感是由流感病毒引起的一種急性呼吸道傳染性疾病,嚴(yán)重影響了人類的生命健康。近年來,人感染H5N1型高致病性禽流感病例的頻繁出現(xiàn),新型H1N1病毒開始在世界范圍內(nèi)的傳播,H7N9型禽流感病毒首次引起人類的感染,都一度造成了社會(huì)的恐慌,流感已經(jīng)成為了一個(gè)全球性的敏感話題。神經(jīng)氨酸酶(NA)是甲型和乙型流感病毒的表面蛋白,在流感病毒的感染和復(fù)制過程中發(fā)揮了重要作用,主要表現(xiàn)在：①催化宿主細(xì)胞表面受體末端的唾液酸與糖蛋白之間糖苷鍵的裂解,促進(jìn)新生病毒從感染的細(xì)胞中釋出；②阻止子代病毒從宿主細(xì)胞釋出后的聚集；③裂解呼吸道黏膜中的唾液酸,阻止病毒滅活并促進(jìn)病毒在呼吸道中的傳播。雖然甲型流感病毒NA已經(jīng)發(fā)現(xiàn)了10種亞型,但構(gòu)成其活性中心的氨基酸序列是高度保守的,這使NA成為抗流感病毒藥物設(shè)計(jì)的一個(gè)重要靶點(diǎn)。自從1999年,zanamivir(商品名Relenza)和oseltamivir(商品名Tamiflu)上市以來,NA抑制劑成為抗流感的主要手段,也成為抗流感藥物領(lǐng)域中的研究熱點(diǎn)。隨后,多種結(jié)構(gòu)類型的NA抑制劑相繼被發(fā)現(xiàn),如苯甲酸類、環(huán)戊烷類、吡咯烷類、多聚體類等。另外兩種高效NA抑制劑peramivir(商品名Rapiacta)和Laninamivir(商品名INAVIR)也已經(jīng)在少數(shù)幾個(gè)國家上市。 在已獲批準(zhǔn)的NA抑制劑中,口服劑oseltamivir是抗流感的首選藥物,在很多國家及地區(qū)被廣泛使用。最近幾年,針對(duì)oseltamivir耐藥的病毒株不斷增多,如H5N1型禽流感病毒和多種季節(jié)性H1N1、H3N2型流感病毒株,這使oseltamivir的臨床應(yīng)用受到一定限制。Zanamivir和peramivir分別通過吸入和靜脈注射方式給藥,雖然使用率低,但也有耐藥病毒株的出現(xiàn)。鑒于目前的抗流感形勢(shì)及流感病毒的潛在威脅,開發(fā)新型、高效的NA抑制劑仍具有重要現(xiàn)實(shí)意義。 本研究論文應(yīng)用基于受體和配體的合理藥物設(shè)計(jì)原理與方法,共設(shè)計(jì)合成了22類,135個(gè)化合物,經(jīng)兩種抗病毒模型初步篩選,發(fā)現(xiàn)了兩類具有較好抗流感病毒活性的活性先導(dǎo)化合物。論文共分六個(gè)章節(jié),主要包括三部分內(nèi)容,分述如下： 第一部分：通過文獻(xiàn)調(diào)研,我們發(fā)現(xiàn)很多天然產(chǎn)物,如黃酮、查爾酮及其他一些多酚類化合物都能夠較好地抑制流感病毒NA的活性,而且在細(xì)胞中還表現(xiàn)出一定的抗流感病毒增殖能力。這些化合物在結(jié)構(gòu)上不符合經(jīng)典NA抑制劑的藥效團(tuán)特征,與oseltamivir、zanamivir等抑制劑也有很大的差別。為了尋找一類結(jié)構(gòu)新穎的NA抑制劑,在本研究中,我們根據(jù)黃酮類NA抑制劑藥效團(tuán)模型,以2,4-二氨基為基本結(jié)構(gòu)片段,建立了一個(gè)結(jié)構(gòu)多樣性的小分子化合物庫。另外,對(duì)具有弱NA抑制活性的咖啡酸進(jìn)行修飾,合成了3個(gè)系列衍生物。通過體外抑酶活性篩選及結(jié)構(gòu)優(yōu)化,共得到92個(gè)化合物。其中,只有咖啡酸衍生物具有較高的NA抑制能力,而且在細(xì)胞中也表現(xiàn)出一定的抗病毒能力,例如化合物(G3)、(G4)、(M1)、(M2)和(P2)等。進(jìn)一步的酶抑制動(dòng)力學(xué)研究表明,本研究發(fā)現(xiàn)的咖啡酸衍生物為非競(jìng)爭(zhēng)性NA抑制劑,這表明這些化合物與經(jīng)典NA抑制劑的作用位點(diǎn)不同,很可能是通過一種新的結(jié)合方式對(duì)NA發(fā)揮抑制作用的。 第二部分：本課題組前期發(fā)現(xiàn)了一些具有中等活性的L-羥脯氨酸衍生物類NA抑制劑,在此基礎(chǔ)上,我們根據(jù)五元環(huán)NA抑制劑的結(jié)構(gòu)特征,繼續(xù)對(duì)其進(jìn)行研究。①將L-羥脯氨酸環(huán)上C-4位的羥基變?yōu)殡一?在吡咯環(huán)氮原子上引入疏水性基團(tuán)：②在吡咯環(huán)氮原子上引入脒基,將L-羥脯氨酸環(huán)上C-4位的羥基變?yōu)榘被?通過對(duì)氨基的化學(xué)修飾,引入疏水基團(tuán)；③在吡咯環(huán)氮原子上引入脒基,將L-羥脯氨酸環(huán)上C-4位的羥基變疊氮,利用Click反應(yīng)引入三氮唑,得到三類新型化合物。體外抑酶試驗(yàn)顯示,這些L-羥脯氨酸衍生物都只具有較微弱的酶抑制活性,與對(duì)照藥(oseltamivir carboxylate)相比有很大的差距。分子對(duì)接分析發(fā)現(xiàn),這些化合物中引入的基團(tuán)在空間上不能與酶活性中心的相應(yīng)位點(diǎn)結(jié)合,這可能是其活性差的主要原因。 第三部分：2006年Nature報(bào)道,Group-1的NA(N1,N4,N5和N8)在其活性中心附近有一個(gè)較大的150-cavity,該口袋可以作為一個(gè)輔助結(jié)合位點(diǎn)用于開發(fā)高選擇性的NA抑制劑。根據(jù)這一結(jié)構(gòu)特點(diǎn),我們以oseltamivir為先導(dǎo)化合物,對(duì)其結(jié)構(gòu)中的氨基進(jìn)行修飾,設(shè)計(jì)合成了取代的胍基類(T系列)和仲胺類(U系列)兩個(gè)系列,共31個(gè)化合物,化學(xué)結(jié)構(gòu)經(jīng)過1H-NMR、13C-NMR和HRMS確證。經(jīng)查閱文獻(xiàn)證實(shí),所合成的目標(biāo)化合物均為新型化合物。體外酶抑制活性試驗(yàn)顯示,本研究合成的oseltamivir衍生物大部分具有N1選擇性,U系列化合物的活性明顯高于T系列。活性最好的兩個(gè)化合物(U12)和(U19)對(duì)N1的抑制活性比oseltamivir羧酸鹽提高了8倍。在雞胚試驗(yàn)中,(U12)也被發(fā)現(xiàn)具有很好的體內(nèi)抗流感病毒活性。通過計(jì)算機(jī)模擬對(duì)接發(fā)現(xiàn),(U12)中C-5位仲胺氮原子上的聯(lián)苯甲基片段能夠很好地與150-cavity結(jié)合,而其它部分則占據(jù)酶活性中心,與晶體結(jié)構(gòu)中oseltamivir羧酸鹽的空間結(jié)構(gòu)大體上重合,這證明150-cavity完全可以作為一個(gè)輔助結(jié)合區(qū)用于開發(fā)高活性、高選擇性NA抑制劑。 結(jié)論與總結(jié)：本研究利用兩種藥物設(shè)計(jì)思路分別發(fā)現(xiàn)了兩類新型NA抑制劑,咖啡酸類和oseltamivir衍生物類�？Х人岜旧砭哂卸喾N生物活性,我們基于咖啡酸設(shè)計(jì)的部分化合物對(duì)于流感病毒在酶和細(xì)胞水平上都表現(xiàn)出較好的活性,優(yōu)于大部分報(bào)道過的活性天然產(chǎn)物�？Х人嵫苌锸且活惾碌腘A抑制劑,值得進(jìn)一步進(jìn)行研究。高致病性H5N1型禽流感病毒和H1N1型流感病毒是兩種對(duì)人類威脅最大的流感病毒,自從150-cavity被發(fā)現(xiàn)以來,選擇性NA抑制劑的研究一直備受關(guān)注,然而至今未有進(jìn)展。我們通過合理藥物設(shè)計(jì)發(fā)現(xiàn)的仲胺類oseltamivir抑制劑,在NA選擇性抑制劑的研究方面取得了突破,多個(gè)化合物對(duì)N1型NA表現(xiàn)出高活性、高選擇性,并且超過陽性對(duì)照藥oseltamivir。在目前的抗流感病毒形勢(shì)下,這一發(fā)現(xiàn)具有重要意義,為N1型流感病毒的預(yù)防和治療提供了新的研究方向。
[Abstract]:Influenza is an acute respiratory infectious disease caused by influenza virus, which seriously affects human life and health. In recent years, people have been infected with H5N1 highly pathogenic avian influenza, the new H1N1 virus began to spread around the world, and the H7N9 avian influenza virus caused human infection for the first time. The influenza has become a global and sensitive topic. Neuraminidase (NA) is the surface protein of influenza A and B virus. It plays an important role in the process of infection and replication of influenza virus, which is mainly manifested in the lysis of glycosidic bonds between the end of the cell surface of the host cell and the glycoprotein. To promote the release of the new virus from the infected cells; (2) preventing the aggregation of the offspring after the release of the host cells; (3) splitting the salivary acid in the mucous membrane of the respiratory tract to prevent the inactivation of the virus and promoting the spread of the virus in the respiratory tract. Although influenza A virus NA has found 10 subtypes, but constitutes the amino acid sequence of its active center. It is highly conservative, making NA an important target for anti influenza drug design. Since 1999, zanamivir (commodity name Relenza) and oseltamivir (commodity name Tamiflu) have been listed on the market, NA inhibitors have become the main means of anti influenza, and have become a hot spot in the field of anti influenza drugs. Subsequently, a variety of structural types of NA inhibitors. One after another, such as benzoate, cyclopentane, pyrroliane, polypolymer, etc.. The other two highly efficient NA inhibitors, peramivir (commodity name Rapiacta) and Laninamivir (commodity name INAVIR), have also been listed in a few countries.
Of the approved NA inhibitors, the oral agent oseltamivir is the preferred drug for anti influenza and is widely used in many countries and regions. In recent years, the number of oseltamivir resistant virus strains, such as H5N1 avian influenza virus and multiple seasonal H1N1, H3N2 influenza virus strains, has made the clinical application of oseltamivir certain. .Zanamivir and peramivir are administered by inhalation and intravenous injection respectively, although the use rate is low, but there are also the emergence of drug resistant strains. In view of the current anti influenza situation and the potential threat of influenza virus, the development of new and efficient NA inhibitors still has important practical meaning.
Based on the principle and method of rational drug design based on receptor and ligand, 22 classes and 135 compounds were designed and synthesized. Through the preliminary screening of two kinds of antiviral models, two kinds of active precursor compounds with good anti influenza virus activity were found. The thesis is divided into six chapters, including three parts. The following are the following:
Part one: through literature research, we have found that many natural products, such as flavonoids, chalcone and some other polyphenols, can better inhibit the activity of influenza virus NA and also exhibit a certain ability to resist influenza virus proliferation in cells. These compounds are structurally inconsistent with the classic NA inhibitors. In order to find a class of novel NA inhibitors, in order to find a class of novel NA inhibitors, in this study, we set up a small molecular compound library of structural diversity based on the model of the flavonoid NA inhibitor pharmacophore and the basic structural fragment of the 2,4- two amino group. In addition, we have a weak NA inhibitory activity. 3 series of derivatives were synthesized by modifying the caffeic acid. 92 compounds were obtained by screening and structural optimization in vitro. Only the caffeic acid derivatives had high NA inhibition ability, and they also showed a certain antiviral ability in the cells, such as compounds (G3), (G4), (M1), (M2) and (P2), and so on. The enzyme inhibition kinetics study showed that the caffeic acid derivatives found in this study were non competitive NA inhibitors, which showed that these compounds were different from the classical NA inhibitors, and were likely to inhibit NA through a new binding method.
The second part: we have discovered some medium active L- hydroxyproline derivatives as NA inhibitors. On this basis, we continue to study the structure of the five membered ring NA inhibitors. (1) the hydroxyl groups of the C-4 bit on the L- hydroxyproline ring are transformed into guanidine, and the hydrophobic groups are introduced into the pyrrole ring nitrogen atom. (2) introducing amidamido on pyrrole ring nitrogen atom, changing the hydroxyl group of C-4 bit on L- hydroxyproline ring into amino group, introducing hydrophobic group through chemical modification of amino group, introducing amidamidin on pyrrole ring nitrogen atom, converting the hydroxyl group of C-4 bit on L- hydroxyproline ring to azide, and using Click to reverse the introduction of three azolds, and get three new types of compounds. The inhibition test showed that all the L- hydroxyproline derivatives only had weak enzyme inhibition activity, and there was a large gap compared with the control drug (oseltamivir carboxylate). Molecular docking analysis found that the group introduced in these compounds could not combine with the corresponding site of the enzyme active center in space, which may be the main difference in the activity of these compounds. Reason.
The third part: in 2006, Nature reported that Group-1's NA (N1, N4, N5 and N8) has a large 150-cavity near its active center, which can be used as an auxiliary binding site for the development of highly selective NA inhibitors. According to this structure, we modify the amino group in its structure with oseltamivir as a pilot compound. Two series of substituted guanidine base class (T Series) and Zhong Anlei (U Series) were designed and synthesized. A total of 31 compounds were confirmed by 1H-NMR, 13C-NMR and HRMS. The synthesized target compounds were proved to be new compounds. In vitro enzyme inhibitory activity test showed that most of the synthesized oseltamivir derivatives were synthesized. N1 selectivity, the activity of U series compounds was significantly higher than that of the T series. The two compounds (U12) and (U19) with the best activity were 8 times higher than the oseltamivir carboxylates. In the chicken embryo test, (U12) was also found to have a good anti influenza virus activity in the body. Through the computer simulation docking, the C-5 site secondary amine nitrogen atom was found in (U12). The diphenyl methyl segments can be well combined with 150-cavity, while the others occupy the active center of the enzyme, which coincides with the spatial structure of the oseltamivir carboxylate in the crystal structure. This proves that 150-cavity can be used as an auxiliary binding zone for the development of high activity and high selective NA inhibitors.
Conclusion and summary: in this study, two new types of NA inhibitors, caffeic acid and oseltamivir derivatives have been found with two kinds of drug design ideas. Caffeic acid itself has a variety of biological activities. Some of the reported active natural products. Caffeic acid derivatives are a new class of NA inhibitors. It is worth further study. The highly pathogenic H5N1 avian influenza virus and the H1N1 influenza virus are the two most human influenza viruses. Since the discovery of 150-cavity, the study of selective NA inhibitors has attracted much attention. We have not progressed so far. We have made a breakthrough in the study of NA selective inhibitors through the oseltamivir inhibitors of secondary amines found in rational drug design. Many compounds have high activity, high selectivity to N1 type NA, and more than the positive control drug oseltamivir. in the anti influenza virus situation before the target, this discovery is heavy. Its significance provides a new research direction for the prevention and treatment of influenza N1 virus.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R511.7

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相關(guān)期刊論文 前2條

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本文編號(hào)：1988582