基于天然NHR序列的异肽键交联HIV-1融合抑制剂的发现

发布时间：2018-03-24 19:23

本文选题：N肽融合抑制剂　切入点：gp41　出处：《中国人民解放军军事医学科学院》2016年博士论文

【摘要】：HIV-1融合抑制剂主要抑制病毒与靶细胞膜的融合。它以gp41亚基为作用靶点,通过抑制病毒内源性六股螺旋束(6HB)的形成,抑制病毒进入靶细胞。衍生于HIV-1gp41蛋白亚基上的C末端七重复序列(CHR)的C肽融合抑制剂主要通过与N末端七重复序列(NHR)作用,抑制内源性6HB的形成,进而抑制病毒的膜融合,其生物活性普遍较高,IC50在纳摩尔水平。T20是目前唯一上市的HIV融合抑制剂,但随着T20的临床使用,逐渐暴露出日益严重的问题:T20耐药毒株的出现;用药剂量大,治疗花费高;病人依从性差等。针对这一状况,通过对现有或衍生于CHR序列的C肽的突变,修饰,缀合等途径,探索开发出与其靶点NHR作用更强,结合更紧密,或作用在NHR上不同区域的C肽融合抑制剂。此后,虽然进一步开发了C肽融合抑制剂,如T1144,也能对T20耐药毒株具有较好的抑制作用,但其作用靶点都是NHR序列,这将导致交叉耐药的出现。因此,发现具有结构新颖,针对新靶点和新作用机制的融合抑制剂是亟需解决的难题。衍生于NHR序列的N肽融合抑制剂作用于CHR,与T20及C肽类融合抑制剂具有完全不同的作用靶点和作用机制,对T20耐药毒株有效。N肽融合抑制剂要先自聚成三螺旋体,然后再和CHR作用,N肽融合抑制剂的这些特点使得它有可能成为解决T20药物所面临问题的有效途径。但直接衍生于NHR的N肽序列较短,相互作用力弱,不具有稳定的三螺旋结构,而且在生理条件下易聚沉,导致其生物活性低,IC50只有微摩尔的水平。本文主要针对N肽融合抑制剂易聚集、生物活性低等缺点,通过对NHR序列进行修饰,氨基酸替换和异肽键交联的方法,设计出生物活性高,代谢稳定性高的N肽融合抑制剂。本文的研究主要分为两个部分:1、嵌合N肽的设计嵌合N肽是由能够形成三螺旋的人为设计肽片段与源自NHR序列能够特异性识别靶标的肽片段连接而成的肽链。针对N肽的三聚体结构不稳定、易沉淀的缺点,我们首先从头设计能够形成三螺旋的人为α-螺旋肽序列,其长度为三个或四个七重复序列(3HR或4HR),它们可以自组装成为三螺旋结构,再通过侧链形成异肽键将三条肽链交联,最终得到异肽键交联的人为设计三螺旋(3HR)3,(4HR)3。然后,在3HR,4HR的C端嵌合天然NHR肽片段N23,得到了嵌合并交联的N肽(3HRN23)3,(4HRN23)3。我们利用圆二色谱(CD),分析型超离心(sva),rp-hplc,质谱等方法确证结构和产物的正确性。经过细胞-细胞融合实验,病毒感染,代谢稳定性评价等实验,我们确证了设计的n肽(3hrn23)3,(4hrn23)3均为稳定的三螺旋结构,α螺旋达到90%以上,并且表现出高生物活性(ic50达到10nm左右)和高代谢稳定性。该部分证明异肽键交联的方法可以有效的提高螺旋结构的稳定性和n肽的生物活性。2、以天然nhr序列为模板n三螺旋肽设计上述人为三螺旋肽与n肽嵌合的设计中,人为三螺旋部分只是为了维系平行n肽三螺旋结构的形成,会造成与n肽生物活性无关的序列过长,该部分直接以天然nhr序列为模板,并通过异肽键交联的方法设计出具有稳定三螺旋结构的n肽融合抑制剂。该n肽融合抑制剂是在天然nhr序列进行尽可能少的修饰,最终得到接近天然序列的交联n三螺旋结构,以此模拟天然nhr的生物功能和作用机制,提高n肽融合抑制剂的生物活性和稳定性。本研究中,通过对天然nhr序列的部分氨基酸替换,设计出能形成三螺旋结构的n肽n36mek2,n36mek1和n36m。并在该n肽的三螺旋结构基础上,利用异肽键交联的方法,得到异肽键交联的n肽融合抑制剂(n36mek2)3,(n36mek1)3和(n36m)3。对异肽键交联前和交联后的n肽进行了结构表征和性能测定,异肽键交联的n肽(n36mek2)3,(n36mek1)3,(n36m)3具有较好的α螺旋(螺旋率80%以上)和热稳定性(tm90℃)。它们也表现出高生物活性和良好的代谢稳定性。尤其是(n36m)3不仅序列与天然n36序列非常接近,在细胞-细胞融合实验和真病毒实验中的抑制活性达到与t20相当或高于t20活性,而且还对hiv-1流行毒株和t20耐药毒株都具有很高的生物活性。本论文共设计合成25个异肽键交联的n肽,对其中的5个n肽进行了详细的理化性质和生物性评价,确证了异肽键交联的n肽具有很高的生物活性,代谢稳定性和较低的细胞毒性,还发现异肽键的形成显著提高了n肽的α螺旋和三螺旋结构的稳定性。本论文的创新性主要表现在:1、通过对n肽的修饰和异肽键交联,设计合成出全新结构的共价交联n三螺旋肽类hiv融合抑制剂,经多种方法进行结构表征和理化性能测定,表明结果正确且达到设计目标;2、生物活性研究表明:我们设计的n肽融合抑制剂具有高活性,尤其是对t20耐药毒株同样有效。同时,具有良好的代谢稳定性,远远超过c肽类融合抑制剂。综上所述,此类抑制剂具有c肽类抑制剂不具备的特点,展现出潜在的开发前景;3、共价交联n三螺旋与天然nhr序列极为接近,可以作为c肽或小分子的分子靶标,研究其与小分子的作用机制和结构,进而为设计小分子抑制剂提供可能的结构基础。
[Abstract]:The fusion of HIV-1 fusion inhibitors mainly inhibit the viral and target cell membranes. It uses gp41 subunit as a target, through the inhibition of endogenous virus (6HB) six helix bundle formation, inhibition of viral entry into target cells. The seven C terminal repeat sequences derived from HIV-1gp41 protein subunit (CHR) of the peptide C fusion inhibitors mainly through the end of the seven N repeat sequence (NHR), inhibiting the formation of endogenous 6HB, and inhibit the virus membrane fusion, its biological activity is generally high, IC50 in the nanomolar.T20 is currently the only listed HIV fusion inhibitors, but with the clinical use of T20, gradually exposed serious problems: the emergence of drug resistance of T20 strain; large dosage, high cost of treatment; poor compliance of patients. In view of this situation, the mutation of C peptide on existing or derived from CHR sequence modification, conjugation and other ways to explore a and NHR targets more Strong, close combination or action on NHR in different regions of the peptide C fusion inhibitors. Since then, although further development of peptide C fusion inhibitors, such as T1144, can also have good inhibitory effect on T20 resistant strains, but the target is NHR sequence, which will lead to cross resistant drugs. Therefore, that has the advantages of novel structure, fusion inhibitors to new targets and new mechanism is a difficult problem to be solved. N peptide derived from the NHR sequence of fusion inhibitors on CHR, has the function of target and mechanism of action is quite different from the T20 and C peptide fusion inhibitors, T20 resistant strains effective.N peptide fusion inhibitors the first self assemble into three helix, and then the role of CHR, the features of N peptide fusion inhibitors makes it possible to become the effective way to solve the problems faced by T20 drugs. N peptide sequences derived from NHR directly but shorter, interaction Weak, does not have the three stable helical structure, and easy coagulation under physiological conditions, leading to its biological activity is low, IC50 only micromolar level. In this paper, N peptide fusion inhibitors and easy aggregation, low biological activity, by modifying the method of NHR sequence, amino acid substitutions and isopeptide bond cross-linking. The design of a high biological activity, high metabolic stability of N peptide fusion inhibitors. This research is mainly divided into two parts: 1. Design of chimeric N peptide chimeric peptide N is formed by three spiral can artificially designed peptide fragments derived from NHR with sequence specific recognition of target peptide fragments can be connected by chains. The trimeric structure of N peptide is not stable, easy to precipitate the shortcomings of human alpha helical peptide sequences we first de novo design can form three helix, its length is three or four seven repeats (3HR or 4HR), they can self assemble into Three spiral structure, and through the side chain isopeptide bond formation three peptide cross-linking, finally obtained the isopeptide bond crosslinking artificially designed three helix (3HR) 3 (4HR) 3., then, in 3HR, 4HR C NHR N23 natural end chimeric peptide fragments, obtained N peptide with crosslinked block (3HRN23) 3 (4HRN23), we use 3. round two chromatography (CD), analytical ultracentrifugation (SVA, RP-HPLC), mass spectrometry and other methods confirmed the correctness of the structure and the product. After cell fusion experiments, viral infections, metabolic stability evaluation experiments, we confirmed that n peptide design (3hrn23) 3. (4hrn23) 3 were three stable helical structure, alpha helix reached more than 90%, and showed high biological activity (IC50 to 10nm) stability and high metabolism. This part proved isopeptide bond crosslinking can effectively improve the stability and bioactivity of.2 n peptide helix structure, with natural NHR sequences the template n three helix Design of design of the three helix peptide human chimeric peptide and N peptide in human three helical part only to maintain parallel n peptide three helix structure formation, causing long sequence has nothing to do with the N peptide biological activity, the part directly in the natural NHR sequence as a template, and by isopeptide bond cross-linked method to design N stable peptide has three helical structure. The fusion inhibitor peptide n fusion inhibitor is modified as little as possible in the natural sequence of NHR, finally get the crosslinked n close to the natural sequence of three helix structure, biological function and mechanism to simulate the natural NHR, improve the N peptide fusion inhibitors of biological activity and stability in this study. By replacing part of natural amino acids, NHR sequence, designed to form n peptide n36mek2 three helix structure, n36mek1 and n36m. and in the three helix structure of N peptide on the basis of this, using the method of isopeptide bond crosslinking, N peptide isopeptide bond crosslinking fusion inhibitor (n36mek2) 3, (n36mek1) 3 and 3. (n36m) of isopeptide bond crosslinking before and after crosslinking of N peptide were determined by characterizing the structure and properties of N peptide, isopeptide bond crosslinked (n36mek2) 3, (n36mek1) 3 (n36m) with alpha 3 good screw (spiral rate of 80% or more) and thermal stability (tm90 C). They also showed high biological activity and good metabolic stability. Especially (n36m) not only 3 sequence and N36 sequence is very close to the natural, in cell - cell fusion inhibition experiment and real virus experiment in reach the equivalent of T20 or higher than the activity of T20, but also on HIV-1 strains and T20 resistant strains have high biological activity. This thesis is the design and synthesis of N peptide 25 isopeptide bond cross-linking, physicochemical properties and biological evaluation in detail on 5 N peptide were confirmed, isopeptide bond n peptide linked has high biological activity Metabolic stability, and low toxicity, also found that isopeptide bond formation significantly improves the stability of the alpha helix n peptide and three helix structure. The innovations of this paper are as follows: 1, through the modification of N peptide and isopeptide bond cross-linking, covalent crosslinking of N was designed and synthesized a new structure of the three helix peptide HIV fusion inhibitors, by various methods of characterization and determination of physical and chemical properties. The results showed that the correct and meet the design goals; 2, biological studies show that: we designed n peptide fusion inhibitors with high activity, especially for T20 resistant strains with effective. At the same time, with good metabolic stability, far more than C peptide fusion inhibitors. In conclusion, these inhibitors have C peptide inhibitors do not have, show the potential prospects for development; 3, covalent crosslinking of n three helix with natural NHR sequence is very close, can be used as a small peptide or C Molecular molecular targets, which study the mechanism and structure of the molecules, provide a possible structural basis for the design of small molecular inhibitors.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R91

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