以神经氨酸酶为靶点的抗流感病毒先导化合物的发现

发布时间：2018-06-06 23:31

本文选题：流感病毒 + 神经氨酸酶　；参考：《山东大学》2014年博士论文

【摘要】：流感是由流感病毒引起的一种急性呼吸道传染性疾病,严重影响了人类的生命健康。近年来,人感染H5N1型高致病性禽流感病例的频繁出现,新型H1N1病毒开始在世界范围内的传播,H7N9型禽流感病毒首次引起人类的感染,都一度造成了社会的恐慌,流感已经成为了一个全球性的敏感话题。神经氨酸酶(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复制过程中发挥了重要作用,主要表现在：①催化宿主细胞表面受体末端的唾液酸与糖蛋白之间糖苷键的裂解,促进新生病毒从感染的细胞中释出；②阻止子代病毒从宿主细胞释出后的聚集；③裂解呼吸道黏膜中的唾液酸,阻止病毒灭活并促进病毒在呼吸道中的传播。虽然甲型流感病毒NA已经发现了10种亚型,但构成其活性中心的氨基酸序列是高度保守的,这使NA成为抗流感病毒药物设计的一个重要靶点。自从1999年,zanamivir(商品名Relenza)和oseltamivir(商品名Tamiflu)上市以来,NA抑制剂成为抗流感的主要手段,也成为抗流感药物领域中的研究热点。随后,多种结构类型的NA抑制剂相继被发现,如苯甲酸类、环戊烷类、吡咯烷类、多聚体类等。另外两种高效NA抑制剂peramivir(商品名Rapiacta)和Laninamivir(商品名INAVIR)也已经在少数几个国家上市。在已获批准的NA抑制剂中,口服剂oseltamivir是抗流感的首选药物,在很多国家及地区被广泛使用。最近几年,针对oseltamivir耐药的病毒株不断增多,如H5N1型禽流感病毒和多种季节性H1N1、H3N2型流感病毒株,这使oseltamivir的临床应用受到一定限制。Zanamivir和peramivir分别通过吸入和静脉注射方式给药,虽然使用率低,但也有耐药病毒株的出现。鉴于目前的抗流感形势及流感病毒的潜在威胁,开发新型、高效的NA抑制剂仍具有重要现实意义。本研究论文应用基于受体和配体的合理药物设计原理与方法,共设计合成了22类,135个化合物,经两种抗病毒模型初步筛选,发现了两类具有较好抗流感病毒活性的活性先导化合物。论文共分六个章节,主要包括三部分内容,分述如下：第一部分：通过文献调研,我们发现很多天然产物,如黄酮、查尔酮及其他一些多酚类化合物都能够较好地抑制流感病毒NA的活性,而且在细胞中还表现出一定的抗流感病毒增殖能力。这些化合物在结构上不符合经典NA抑制剂的药效团特征,与oseltamivir、zanamivir等抑制剂也有很大的差别。为了寻找一类结构新颖的NA抑制剂,在本研究中,我们根据黄酮类NA抑制剂药效团模型,以2,4-二氨基为基本结构片段,建立了一个结构多样性的小分子化合物库。另外,对具有弱NA抑制活性的咖啡酸进行修饰,合成了3个系列衍生物。通过体外抑酶活性筛选及结构优化,共得到92个化合物。其中,只有咖啡酸衍生物具有较高的NA抑制能力,而且在细胞中也表现出一定的抗病毒能力,例如化合物(G3)、(G4)、(M1)、(M2)和(P2)等。进一步的酶抑制动力学研究表明,本研究发现的咖啡酸衍生物为非竞争性NA抑制剂,这表明这些化合物与经典NA抑制剂的作用位点不同,很可能是通过一种新的结合方式对NA发挥抑制作用的。第二部分：本课题组前期发现了一些具有中等活性的L-羟脯氨酸衍生物类NA抑制剂,在此基础上,我们根据五元环NA抑制剂的结构特征,继续对其进行研究。①将L-羟脯氨酸环上C-4位的羟基变为胍基,在吡咯环氮原子上引入疏水性基团：②在吡咯环氮原子上引入脒基,将L-羟脯氨酸环上C-4位的羟基变为氨基,通过对氨基的化学修饰,引入疏水基团；③在吡咯环氮原子上引入脒基,将L-羟脯氨酸环上C-4位的羟基变叠氮,利用Click反应引入三氮唑,得到三类新型化合物。体外抑酶试验显示,这些L-羟脯氨酸衍生物都只具有较微弱的酶抑制活性,与对照药(oseltamivir carboxylate)相比有很大的差距。分子对接分析发现,这些化合物中引入的基团在空间上不能与酶活性中心的相应位点结合,这可能是其活性差的主要原因。第三部分：2006年Nature报道,Group-1的NA(N1,N4,N5和N8)在其活性中心附近有一个较大的150-cavity,该口袋可以作为一个辅助结合位点用于开发高选择性的NA抑制剂。根据这一结构特点,我们以oseltamivir为先导化合物,对其结构中的氨基进行修饰,设计合成了取代的胍基类(T系列)和仲胺类(U系列)两个系列,共31个化合物,化学结构经过1H-NMR、13C-NMR和HRMS确证。经查阅文献证实,所合成的目标化合物均为新型化合物。体外酶抑制活性试验显示,本研究合成的oseltamivir衍生物大部分具有N1选择性,U系列化合物的活性明显高于T系列。活性最好的两个化合物(U12)和(U19)对N1的抑制活性比oseltamivir羧酸盐提高了8倍。在鸡胚试验中,(U12)也被发现具有很好的体内抗流感病毒活性。通过计算机模拟对接发现,(U12)中C-5位仲胺氮原子上的联苯甲基片段能够很好地与150-cavity结合,而其它部分则占据酶活性中心,与晶体结构中oseltamivir羧酸盐的空间结构大体上重合,这证明150-cavity完全可以作为一个辅助结合区用于开发高活性、高选择性NA抑制剂。结论与总结：本研究利用两种药物设计思路分别发现了两类新型NA抑制剂,咖啡酸类和oseltamivir衍生物类。咖啡酸本身具有多种生物活性,我们基于咖啡酸设计的部分化合物对于流感病毒在酶和细胞水平上都表现出较好的活性,优于大部分报道过的活性天然产物。咖啡酸衍生物是一类全新的NA抑制剂,值得进一步进行研究。高致病性H5N1型禽流感病毒和H1N1型流感病毒是两种对人类威胁最大的流感病毒,自从150-cavity被发现以来,选择性NA抑制剂的研究一直备受关注,然而至今未有进展。我们通过合理药物设计发现的仲胺类oseltamivir抑制剂,在NA选择性抑制剂的研究方面取得了突破,多个化合物对N1型NA表现出高活性、高选择性,并且超过阳性对照药oseltamivir。在目前的抗流感病毒形势下,这一发现具有重要意义,为N1型流感病毒的预防和治疗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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R511.7

【参考文献】