本文選題：表觀遺傳學(xué) + 組蛋白去乙�；敢种苿���； 參考：《山東大學(xué)》2015年博士論文

【摘要】：本研究論文是以表觀遺傳學(xué)為基礎(chǔ),利用藥物分子雜交策略,以發(fā)現(xiàn)多靶點(diǎn)抗腫瘤藥物為目標(biāo),將時下抗腫瘤研究最熱門的領(lǐng)域組蛋白去乙�；敢种苿�(HDACI)與能夠產(chǎn)生“明星分子”一氧化氮(NO)的藥物小分子片段一氧化氮供體(NO donor)結(jié)合在一起,設(shè)計合成了25個分子結(jié)構(gòu)新穎的NO-HDACI,并對其進(jìn)行了系統(tǒng)的體內(nèi)外生物活性評價并對其結(jié)果進(jìn)行了詳細(xì)的討論。全文共分四個部分進(jìn)行分章論述。一、研究背景組蛋白去乙�；�(HDACs)是一類可以水解掉組蛋白或非組蛋白末端賴氨酸殘基上乙�；牡鞍酌讣易�,可以使染色質(zhì)形成一個轉(zhuǎn)錄抑制的狀態(tài)。HDACs活性的異常與基因的異常表達(dá)及包括癌癥在內(nèi)的多種人類疾病的發(fā)生發(fā)展有著密切的聯(lián)系。抑制HDACs可以恢復(fù)正�；虻谋磉_(dá),從而可以阻滯癌細(xì)胞的細(xì)胞周期,促進(jìn)細(xì)胞分化及細(xì)胞凋亡。因此,阻斷HDACs非正常去乙�；饔玫慕M蛋白去乙�；敢种苿�(HDACI)作為潛在的有效的抗腫瘤藥物便應(yīng)運(yùn)而生。HDACI主要包括四類：異羥肟酸類,短鏈脂肪酸類,環(huán)狀四肽類以及苯甲酰胺類。異羥肟酸類是研究最廣最深入的一類,目前該類已有三個藥物(SAHA, PXD-101和LBH-589)被FDA批準(zhǔn)上市,分別用來治療皮膚T細(xì)胞淋巴瘤、外周T細(xì)胞淋巴瘤及多發(fā)性骨髓瘤。一氧化氮(NO)作為有機(jī)體內(nèi)重要的信使小分子,參與體內(nèi)諸多生理過程,比如血管調(diào)節(jié),神經(jīng)傳遞,炎癥以及免疫反應(yīng)等過程。此外,研究表明,NO還可以抑制腫瘤細(xì)胞的增殖,轉(zhuǎn)移,血管生成以及促進(jìn)腫瘤細(xì)胞的凋亡。在體內(nèi),NO的產(chǎn)生在一氧化氮合成酶(iNOS)參與下生成以外,一氧化氮供體(NO donor)也是產(chǎn)生高濃度NO的重要方式。1,2,5-惡二唑-2-氧化物(呋咱氮氧化物)是2008年通過高通量篩選而得到的一個重要的具有開發(fā)潛力的NO供體,它可以在體外生成高濃度的NO以及在體內(nèi)具有抑制腫瘤生長的作用。因此,多年來,很多新藥特別是抗腫瘤藥物的研發(fā)過程中都會看到它的身影。近年來,越來越多的研究發(fā)現(xiàn),由NO引起的巰基亞硝酸化或蛋白質(zhì)酪氨酸硝化等共價修飾顯著影響著細(xì)胞功能。HDAC的很多家族成員可以直接或間接的以NO為靶標(biāo),而且已經(jīng)有報道證明了部分HDAC的功能也依賴于NO的調(diào)節(jié)。更重要的是,曾有研究證明了NO和HDACI在治療心肌肥厚及傷口愈合方面存在著協(xié)同作用。這就為兩者在某些疾病的聯(lián)合應(yīng)用上提供了充分的證據(jù)。分子雜交策略是新藥研發(fā)過程中發(fā)現(xiàn)多靶點(diǎn)藥物的重要手段之一,本論文的研究主要是以設(shè)計多靶點(diǎn)藥物為原則,以分子雜交策略手段將NO供體融入進(jìn)HDACI中來,合理設(shè)計、定向合成具有全新結(jié)構(gòu)的NO-HDACI,以期待可以得到體內(nèi)外抗腫瘤活性更好,靶向更明確,選擇性更高的先導(dǎo)化合物,為進(jìn)一步開發(fā)具有我國自主知識產(chǎn)權(quán)的創(chuàng)新性抗腫瘤藥物奠定基礎(chǔ)。二、苯磺酰呋咱氮氧化物一氧化氮供體型組蛋白去乙�；敢种苿┑脑O(shè)計,合成及抗腫瘤活性研究苯磺酰呋咱氮氧化物是一個經(jīng)典的1,2,5-惡二唑-2-氧化物的一氧化氮供體,在各種組織及器官中不需要酶的作用即可釋放出大量的NO。由于其對酸堿相對穩(wěn)定,該片段一直被用來與多種藥物分子進(jìn)行偶聯(lián)且大部分顯示出了比原藥更強(qiáng)的藥效。鑒于此,本課題首選該片段作為NO供體參與一氧化氮供體型HDACI的設(shè)計及合成。根據(jù)苯磺酰呋咱氮氧化物的結(jié)構(gòu)特點(diǎn)以及HDACI的結(jié)構(gòu)特征,共設(shè)計合成出了15個全新的化合物。所有的目標(biāo)化合物先后經(jīng)過了對Hela細(xì)胞提取物(主要包含HDAC1和HDAC2)和8株腫瘤細(xì)胞增殖抑制活性的評價,結(jié)果顯示大部分化合物都具有明顯的抑制活性,特別是目標(biāo)化合物5c與陽性藥SAHA相比,抑制效果尤為突出,其敏感的腫瘤細(xì)胞株為HEL細(xì)胞。而且經(jīng)對NO釋放含量的測定發(fā)現(xiàn),目標(biāo)化合物5c可以釋放大量的NO,對5c經(jīng)過NO清除劑Hemoglobin (Hb)的預(yù)處理后,發(fā)現(xiàn)其對HEL細(xì)胞增殖的抑制活性與Hb的濃度增加而呈梯度遞減,說明其對腫瘤細(xì)胞增殖的抑制是由釋放NO和抑制HDACs酶兩方面的共同作用。進(jìn)一步的機(jī)制研究表明相對于陽性藥SAHA,目標(biāo)化合物5c對HEL細(xì)胞能引起比較強(qiáng)的細(xì)胞凋亡,而且主要作用在細(xì)胞周期的G1期。裸鼠體內(nèi)實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),5c相較于陽性藥SAHA在同等劑量,相同給藥方式及給藥時間的情況下,具有明顯優(yōu)于SAHA的抑制腫瘤生長的作用,而且?guī)缀醪淮嬖隗w內(nèi)毒性。此外,目標(biāo)化合物5c在分子水平上顯示出對HDACs的亞型HDAC6明顯的選擇性,該發(fā)現(xiàn)可以作為進(jìn)一步設(shè)計探索HDAC6選擇性抑制劑的重要依據(jù)。三、苯并氧化呋咱氮氧化物一氧化氮供體型組蛋白去乙�；敢种苿┑脑O(shè)計,合成及抗腫瘤活性研究苯并氧化呋咱是1,2,5-惡二唑-2-氧化物的另一個應(yīng)用廣泛的NO供體片段,因?yàn)榭梢葬尫糯罅康腘O而應(yīng)用在心血管疾病及抗腫瘤藥物的研發(fā)上。因此,本論文的另一部分研究內(nèi)容就是將該片段作為NO供體融入目標(biāo)化合物的設(shè)計,共合成出10個全新的NO-HDACI。生物活性評價結(jié)果表明,大部分化合物都表現(xiàn)出明顯的抑制酶活的作用,而且根據(jù)初步的構(gòu)效關(guān)系可以看出其連接臂長度與苯并氧化呋咱的位置對其抑制酶活性強(qiáng)弱具有重要影響。通過MTT實(shí)驗(yàn)可以看出,目標(biāo)化合物10d和15d在抑制HCT116腫瘤細(xì)胞株的增殖上明顯的優(yōu)于陽性藥SAHA。此外,作為有NO供體存在的HDACI,在NO釋放量的評價上,目標(biāo)產(chǎn)物10d和15d在細(xì)胞內(nèi)能產(chǎn)生較大量的NO,而且隨著NO清除劑Hemoglobin濃度的增加其抑制腫瘤細(xì)胞增殖的作用減弱,充分說明了我們設(shè)計合成的目標(biāo)化合物在體外同時具有抑制HDACs酶活的作用和釋放NO的作用,這對多靶點(diǎn)抗腫瘤藥物設(shè)計合成具有積極的指導(dǎo)意義。四、全文總結(jié)與展望本課題是以發(fā)現(xiàn)多靶點(diǎn)抗腫瘤藥物為前提,綜合運(yùn)用藥物化學(xué)、化學(xué)生物學(xué)、計算機(jī)化學(xué)等前沿學(xué)科的交叉,利用分子雜交策略將NO供體融入組蛋白去乙�；敢种苿�(HDACI)的研設(shè)計中,定向合成了兩個系列共25個具有全新骨架結(jié)構(gòu)的NO-HDACI。對所有目標(biāo)化合物進(jìn)行了分子水平的抑酶活性篩選,從中選取活性較好的化合物逐層進(jìn)行細(xì)胞水平、動物水平的抗腫瘤活性測試,利用得到的構(gòu)效關(guān)系信息對表現(xiàn)優(yōu)異的化合物進(jìn)行進(jìn)一步的結(jié)構(gòu)改造、修飾和優(yōu)化。結(jié)果表明：當(dāng)把NO供體融進(jìn)HDACI分子中時,其抗腫瘤效果會明顯提高,就目前得到的化合物5c來講,無論是在體內(nèi)還是體外的抗腫瘤活性上都明顯優(yōu)于上市藥物SAHA,同時在分子水平還表現(xiàn)出比較顯著的選擇性�；衔�5c的臨床前藥理活性還是進(jìn)一步的評價中,同時,對其進(jìn)一步的結(jié)構(gòu)優(yōu)化及活性篩選也在進(jìn)行中。該論文的呈現(xiàn),無論是為多靶點(diǎn)藥物的設(shè)計發(fā)現(xiàn)還是為選擇性HDACI的研究都奠定了良好的基礎(chǔ)。
[Abstract]:This study is based on epigenetics, using the drug molecular hybridization strategy to find multi target antitumor drugs as the target, the most popular domain of antitumor research histone deacetylase inhibitor (HDACI) and a small molecule fragment of nitric oxide (NO do) that can produce "star molecule" nitrogen oxide (NO). Nor) combined together, 25 novel NO-HDACI molecular structures were designed and synthesized, and the biological activity of the system was evaluated and the results were discussed in detail. The full text was divided into four chapters. First, the research background histone deacetylase (HDACs) is a class of proteins that can hydrolyze histone or non egg group. The family of acetylated proteinases on the white terminal lysine residue can make the chromatin form a transcriptional inhibition state of the.HDACs activity, which is closely related to the abnormal expression of genes and the development of a variety of human diseases, including cancer. Inhibition of HDACs can restore the expression of normal genes, thus blocking cancer fines. The cell cycle of the cell promotes cell differentiation and apoptosis. Therefore, the histone deacetylase inhibitor (HDACI), which blocks the abnormal deacetylation of HDACs as a potential effective antitumor drug, arises at the historic moment of.HDACI mainly including four types: hydroxamic acid, short chain fatty acids, ring four peptides, and benzamides. The hydroxamic acid is the most widely studied class. At present, three drugs (SAHA, PXD-101 and LBH-589) have been approved by FDA to treat skin T cell lymphoma, peripheral T cell lymphoma and multiple myeloma. Nitric oxide (NO), as an important messenger in the body, is involved in many physiological processes in the body. Such as vascular regulation, neurotransmission, inflammation and immune response. In addition, NO can also inhibit the proliferation, metastasis, angiogenesis, and apoptosis of tumor cells. In vivo, the production of NO is produced by nitric oxide synthase (iNOS), and the nitric oxide donor (NO donor) is also a high concentration of NO. The important way.1,2,5- oxide two azole -2- oxide (furazono NOx) is an important development potential NO donor by high throughput screening in 2008. It can produce high concentration of NO in vitro and inhibit tumor growth in vivo. Therefore, many new drugs, especially antitumor drugs, have been developed for many years. In recent years, more and more studies have found that covalent modification of mercapto nitrification or protein tyrosine nitrification caused by NO significantly affects a number of family members of the cell function.HDAC directly or indirectly with NO as a target, and it has been reported that part of the function of HDAC is dependent on the function of the cell. More importantly, there has been a study of the synergistic effects of NO and HDACI in the treatment of myocardial hypertrophy and wound healing. This provides sufficient evidence for the combination of some diseases. The molecular hybridization strategy is one of the most important methods for the discovery of multiple target drugs in the development of new drugs. This paper has been studied in this paper. The study is mainly based on the principle of designing multi target drugs and integrating the NO donor into HDACI by molecular hybridization strategy. It is reasonable designed and directed to synthesize a new structure of NO-HDACI, so as to expect to get better antitumor activity in vivo and in vivo, more targeted and selective, and to develop our own autonomy for further development. Two, design, synthesis and antitumor activity of benzsulfonacylfurazono nitrous oxide nitric oxide donor histone deacetylase inhibitor, benzsulfonacylfurazine oxide is a classical nitric oxide donor of 1,2,5- evil two azole oxide, in various tissues and organs. The release of a large number of NO. without the action of enzymes has been used for the coupling of a variety of drug molecules and most of the efficacy of the drug as a result of its relative stability to the acid base. In view of this, this subject is preferred as a NO donor in the design and synthesis of nitric oxide donor HDACI, based on benzsulfonyl. The structural characteristics of nitrous oxide and the structural characteristics of HDACI have been designed and synthesized. 15 new compounds have been designed and synthesized. All target compounds have been evaluated for the inhibitory activity of Hela cell extracts (mainly including HDAC1 and HDAC2) and 8 tumor cells. The results show that most of the compounds have obvious inhibitory activity. The inhibitory effect, especially the target compound 5C, is particularly prominent compared with the positive drug SAHA. The sensitive tumor cell line is HEL cells. And the target compound 5C can release a large number of NO by determination of the release content of NO, and the inhibitory activity of 5C after the NO scavenger Hemoglobin (Hb) is detected and its inhibitory activity to the proliferation of HEL cells is found. The concentration of Hb increases with a gradient decreasing, indicating that the inhibition of tumor cell proliferation is a common effect of releasing NO and inhibiting HDACs enzyme. Further mechanism studies show that relative to positive drug SAHA, the target compound 5C can induce stronger apoptosis in HEL cells, and mainly in the G1 phase of the cell cycle and nude mice. The results of the internal experiment showed that 5C had the same effect as the positive drug SAHA in the same dosage, the same way and the time of administration, which was obviously superior to SAHA in inhibiting the growth of the tumor and almost no body toxicity. In addition, the target compound 5C showed a distinct selectivity to the HDACs subtype HDAC6 at the molecular level. This discovery could be found. As an important basis for further design and exploration of selective inhibitors of HDAC6. Three, design, synthesis and antitumor activity of benzooxide furazono nitric oxide nitric oxide donor deacetylase inhibitor, benzo oxide furazan, another widely used NO donor fragment of 1,2,5- oxazolide -2- oxide, because it can be used. The release of a large number of NO was applied to the development of cardiovascular diseases and antitumor drugs. So, another part of this paper was to integrate the fragment as a NO donor in the design of the target compound, and a total of 10 new NO-HDACI. bioactivity evaluations showed that most of the compounds showed significant inhibition of enzyme activity. According to the preliminary structure-activity relationship, it can be found that the length of the connecting arm and the position of benzo oxidizing furazan have an important effect on the inhibitory activity of the inhibitor. Through the MTT experiment, we can see that the target compound 10d and 15d are obviously superior to the positive drug SAHA. in inhibiting the proliferation of HCT116 tumor cell lines, as a NO donor. In the HDACI, the target products 10d and 15d can produce a large number of NO in the cell, and the effect of the NO scavenger Hemoglobin concentration increases with the increase of the NO scavenger Hemoglobin concentration. It shows that the target compounds we designed and synthesized have the inhibition of HDACs enzyme activity and the release of NO in vitro. It has a positive guiding significance for the design and synthesis of multi target antitumor drugs. Four. The full text summary and prospect is based on the discovery of multi target antitumor drugs as the premise, the integrated use of pharmaceutical chemistry, chemical biology, Computer Chemistry and other frontier disciplines, the use of molecular hybridization strategy to integrate NO donor to histone deacetylation In the design of the enzyme inhibitor (HDACI), a total of two series of 25 new NO-HDACI. skeleton structures were synthesized, and all the target compounds were screened at molecular level. The results showed that the anti tumor effect of the NO donor was obviously improved when the HDACI molecule was incorporated into the molecule, which was obviously superior to the drug SAHA in both in vivo and in vitro, both in the body and in vitro, at the same time, at the same time. The molecular level also shows significant selectivity. The preclinical pharmacological activity of compound 5C is still further evaluated, and its further structural optimization and activity screening are also in progress. The presentation of this paper has laid a good foundation for the design of multiple target drugs or for the study of selective HDACI. Foundation.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R914;R96

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 周洲;蔣麗媛;張奕華;季暉;孫易;彭司勛;;乙酰水楊酰阿魏酸與呋咱氮氧化物和硝酸酯偶聯(lián)物的合成及其抗血栓作用[J];藥學(xué)學(xué)報;2006年11期

2 項(xiàng)光亞;楊杰;徐U，

本文編號：2043000