本文選題：N肽融合抑制劑　切入點(diǎn)：gp41　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2016年博士論文

【摘要】：HIV-1融合抑制劑主要抑制病毒與靶細(xì)胞膜的融合。它以gp41亞基為作用靶點(diǎn),通過抑制病毒內(nèi)源性六股螺旋束(6HB)的形成,抑制病毒進(jìn)入靶細(xì)胞。衍生于HIV-1gp41蛋白亞基上的C末端七重復(fù)序列(CHR)的C肽融合抑制劑主要通過與N末端七重復(fù)序列(NHR)作用,抑制內(nèi)源性6HB的形成,進(jìn)而抑制病毒的膜融合,其生物活性普遍較高,IC50在納摩爾水平。T20是目前唯一上市的HIV融合抑制劑,但隨著T20的臨床使用,逐漸暴露出日益嚴(yán)重的問題:T20耐藥毒株的出現(xiàn);用藥劑量大,治療花費(fèi)高;病人依從性差等。針對(duì)這一狀況,通過對(duì)現(xiàn)有或衍生于CHR序列的C肽的突變,修飾,綴合等途徑,探索開發(fā)出與其靶點(diǎn)NHR作用更強(qiáng),結(jié)合更緊密,或作用在NHR上不同區(qū)域的C肽融合抑制劑。此后,雖然進(jìn)一步開發(fā)了C肽融合抑制劑,如T1144,也能對(duì)T20耐藥毒株具有較好的抑制作用,但其作用靶點(diǎn)都是NHR序列,這將導(dǎo)致交叉耐藥的出現(xiàn)。因此,發(fā)現(xiàn)具有結(jié)構(gòu)新穎,針對(duì)新靶點(diǎn)和新作用機(jī)制的融合抑制劑是亟需解決的難題。衍生于NHR序列的N肽融合抑制劑作用于CHR,與T20及C肽類融合抑制劑具有完全不同的作用靶點(diǎn)和作用機(jī)制,對(duì)T20耐藥毒株有效。N肽融合抑制劑要先自聚成三螺旋體,然后再和CHR作用,N肽融合抑制劑的這些特點(diǎn)使得它有可能成為解決T20藥物所面臨問題的有效途徑。但直接衍生于NHR的N肽序列較短,相互作用力弱,不具有穩(wěn)定的三螺旋結(jié)構(gòu),而且在生理?xiàng)l件下易聚沉,導(dǎo)致其生物活性低,IC50只有微摩爾的水平。本文主要針對(duì)N肽融合抑制劑易聚集、生物活性低等缺點(diǎn),通過對(duì)NHR序列進(jìn)行修飾,氨基酸替換和異肽鍵交聯(lián)的方法,設(shè)計(jì)出生物活性高,代謝穩(wěn)定性高的N肽融合抑制劑。本文的研究主要分為兩個(gè)部分:1、嵌合N肽的設(shè)計(jì)嵌合N肽是由能夠形成三螺旋的人為設(shè)計(jì)肽片段與源自NHR序列能夠特異性識(shí)別靶標(biāo)的肽片段連接而成的肽鏈。針對(duì)N肽的三聚體結(jié)構(gòu)不穩(wěn)定、易沉淀的缺點(diǎn),我們首先從頭設(shè)計(jì)能夠形成三螺旋的人為α-螺旋肽序列,其長(zhǎng)度為三個(gè)或四個(gè)七重復(fù)序列(3HR或4HR),它們可以自組裝成為三螺旋結(jié)構(gòu),再通過側(cè)鏈形成異肽鍵將三條肽鏈交聯(lián),最終得到異肽鍵交聯(lián)的人為設(shè)計(jì)三螺旋(3HR)3,(4HR)3。然后,在3HR,4HR的C端嵌合天然NHR肽片段N23,得到了嵌合并交聯(lián)的N肽(3HRN23)3,(4HRN23)3。我們利用圓二色譜(CD),分析型超離心(sva),rp-hplc,質(zhì)譜等方法確證結(jié)構(gòu)和產(chǎn)物的正確性。經(jīng)過細(xì)胞-細(xì)胞融合實(shí)驗(yàn),病毒感染,代謝穩(wěn)定性評(píng)價(jià)等實(shí)驗(yàn),我們確證了設(shè)計(jì)的n肽(3hrn23)3,(4hrn23)3均為穩(wěn)定的三螺旋結(jié)構(gòu),α螺旋達(dá)到90%以上,并且表現(xiàn)出高生物活性(ic50達(dá)到10nm左右)和高代謝穩(wěn)定性。該部分證明異肽鍵交聯(lián)的方法可以有效的提高螺旋結(jié)構(gòu)的穩(wěn)定性和n肽的生物活性。2、以天然nhr序列為模板n三螺旋肽設(shè)計(jì)上述人為三螺旋肽與n肽嵌合的設(shè)計(jì)中,人為三螺旋部分只是為了維系平行n肽三螺旋結(jié)構(gòu)的形成,會(huì)造成與n肽生物活性無關(guān)的序列過長(zhǎng),該部分直接以天然nhr序列為模板,并通過異肽鍵交聯(lián)的方法設(shè)計(jì)出具有穩(wěn)定三螺旋結(jié)構(gòu)的n肽融合抑制劑。該n肽融合抑制劑是在天然nhr序列進(jìn)行盡可能少的修飾,最終得到接近天然序列的交聯(lián)n三螺旋結(jié)構(gòu),以此模擬天然nhr的生物功能和作用機(jī)制,提高n肽融合抑制劑的生物活性和穩(wěn)定性。本研究中,通過對(duì)天然nhr序列的部分氨基酸替換,設(shè)計(jì)出能形成三螺旋結(jié)構(gòu)的n肽n36mek2,n36mek1和n36m。并在該n肽的三螺旋結(jié)構(gòu)基礎(chǔ)上,利用異肽鍵交聯(lián)的方法,得到異肽鍵交聯(lián)的n肽融合抑制劑(n36mek2)3,(n36mek1)3和(n36m)3。對(duì)異肽鍵交聯(lián)前和交聯(lián)后的n肽進(jìn)行了結(jié)構(gòu)表征和性能測(cè)定,異肽鍵交聯(lián)的n肽(n36mek2)3,(n36mek1)3,(n36m)3具有較好的α螺旋(螺旋率80%以上)和熱穩(wěn)定性(tm90℃)。它們也表現(xiàn)出高生物活性和良好的代謝穩(wěn)定性。尤其是(n36m)3不僅序列與天然n36序列非常接近,在細(xì)胞-細(xì)胞融合實(shí)驗(yàn)和真病毒實(shí)驗(yàn)中的抑制活性達(dá)到與t20相當(dāng)或高于t20活性,而且還對(duì)hiv-1流行毒株和t20耐藥毒株都具有很高的生物活性。本論文共設(shè)計(jì)合成25個(gè)異肽鍵交聯(lián)的n肽,對(duì)其中的5個(gè)n肽進(jìn)行了詳細(xì)的理化性質(zhì)和生物性評(píng)價(jià),確證了異肽鍵交聯(lián)的n肽具有很高的生物活性,代謝穩(wěn)定性和較低的細(xì)胞毒性,還發(fā)現(xiàn)異肽鍵的形成顯著提高了n肽的α螺旋和三螺旋結(jié)構(gòu)的穩(wěn)定性。本論文的創(chuàng)新性主要表現(xiàn)在:1、通過對(duì)n肽的修飾和異肽鍵交聯(lián),設(shè)計(jì)合成出全新結(jié)構(gòu)的共價(jià)交聯(lián)n三螺旋肽類hiv融合抑制劑,經(jīng)多種方法進(jìn)行結(jié)構(gòu)表征和理化性能測(cè)定,表明結(jié)果正確且達(dá)到設(shè)計(jì)目標(biāo);2、生物活性研究表明:我們?cè)O(shè)計(jì)的n肽融合抑制劑具有高活性,尤其是對(duì)t20耐藥毒株同樣有效。同時(shí),具有良好的代謝穩(wěn)定性,遠(yuǎn)遠(yuǎn)超過c肽類融合抑制劑。綜上所述,此類抑制劑具有c肽類抑制劑不具備的特點(diǎn),展現(xiàn)出潛在的開發(fā)前景;3、共價(jià)交聯(lián)n三螺旋與天然nhr序列極為接近,可以作為c肽或小分子的分子靶標(biāo),研究其與小分子的作用機(jī)制和結(jié)構(gòu),進(jìn)而為設(shè)計(jì)小分子抑制劑提供可能的結(jié)構(gòu)基礎(chǔ)。
[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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R91

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