本文選題：Mcl-1　切入點(diǎn)：二維核磁HSQC　出處：《大連理工大學(xué)》2012年碩士論文

【摘要】：Bcl-2家族蛋白包括結(jié)構(gòu)同源、功能相反的3個(gè)亞族：(1)抗凋亡的Bcl-2-like蛋白,例如Mcl-1蛋白和Bcl-2蛋白；(2)促凋亡蛋白例如Bax和Bak；(3)僅含有BH3結(jié)構(gòu)域的BH3-only蛋白。它們之間通過(guò)共同擁有的BH3結(jié)構(gòu)域發(fā)生相互作用,調(diào)控細(xì)胞凋亡。研究證明Mcl-1和Bcl-2蛋白是腫瘤細(xì)胞逃避凋亡和獲得永生的關(guān)鍵因子,因此成為靶向抗癌藥的重要靶點(diǎn)。有機(jī)小分子通過(guò)占據(jù)Bcl-2家族蛋白發(fā)生相互作用的界面(interface):BH3溝槽來(lái)干擾Bcl-2家族蛋白之間的蛋白質(zhì)-蛋白質(zhì)相互作用,從而誘導(dǎo)腫瘤細(xì)胞凋亡,具有抗癌藥成藥前景。目前已經(jīng)有幾個(gè)Bcl-2抑制劑正處于臨床試驗(yàn)階段；但是特異的Mcl-1蛋白抑制劑還為數(shù)很少。因?yàn)镸cl-1蛋白的BH3溝槽與Bcl-2的差異明顯,現(xiàn)有的Bcl-2抑制劑大多不能同時(shí)拮抗Mcl-1蛋白。因此需要通過(guò)深入研究小分子與Mcl-1蛋白的結(jié)合模式來(lái)指導(dǎo)Mcl-1蛋白抑制劑的分子設(shè)計(jì)和構(gòu)效關(guān)系分析。 本文通過(guò)二維核磁共振技術(shù)(1H-15N HSQC NMR)研究了Mcl-1蛋白與小分子配體的相互作用,揭示了作用位點(diǎn)和結(jié)合模式,指導(dǎo)設(shè)計(jì)了通過(guò)擴(kuò)大結(jié)合位點(diǎn)獲得更高親和力的小分子Mcl-1蛋白抑制劑類(lèi)抗癌先導(dǎo)化合物。 首先,我們構(gòu)建了人源Mcl-1蛋白的表達(dá)質(zhì)粒,通過(guò)同位素標(biāo)記方法,獲得15N標(biāo)記的Mcl-1蛋白。在此過(guò)程中,優(yōu)化了Mcl-1蛋白的表達(dá)和分離純化條件,將15N標(biāo)記Mcl-1蛋白的表達(dá)量從1.3mg提高到7.2mg/L菌體的產(chǎn)量；純度達(dá)到了95%。圓二色譜分析結(jié)果顯示重組、同位素標(biāo)記、和純化的Mcl-1蛋白具有以α螺旋為主的穩(wěn)定的二級(jí)結(jié)構(gòu)。二維核磁共振結(jié)果表明各氨基酸殘基的譜峰分散均勻,證明15N標(biāo)記Mcl-1具有穩(wěn)定的、折疊良好的空間結(jié)構(gòu),滿(mǎn)足小分子核磁滴定試驗(yàn)的要求。 接下來(lái),我們對(duì)本課題組發(fā)現(xiàn)的小分子Mcl-1蛋白抑制劑S1(3-硫嗎啉基-8-氧-8H-苊并[1，，2-b]吡咯-9-腈)進(jìn)行了1H-15N HSQC NMR滴定研究。結(jié)果表明,S1引起Mcl-1蛋白化學(xué)位移0.03ppm的氨基酸60%位于BH3溝槽,其中R263殘基化學(xué)位移改變最為明顯。證明S1結(jié)合在Mcl-1蛋白的BH3溝槽中。尤其是NMR的結(jié)果表明S1主要引起了BH3溝槽的亞活性口袋：p2口袋附近的氨基酸的化學(xué)位移,包括V249,M250,V253,R263等殘基。計(jì)算機(jī)輔助分子對(duì)接結(jié)果體現(xiàn)了S1分子與Mcl-1蛋白的結(jié)合模式：S1的羰基與Mcl-1蛋白的R263殘基形成氫鍵,硫嗎啉取代基插入Mcl-1蛋白的p2口袋；S1的氰基指向但并未占據(jù)Mcl-1蛋白的p4口袋。在這一結(jié)合模式的指導(dǎo)下,本課題組合成了小分子H2(3-(4-溴苯硫基-9-3-苯丙胺基-8H-苊并[1,2-b]吡咯-8-酮)和4g(E,E)-2-(苯甲基氨基羰基)-3-苯乙烯基丙烯腈),嘗試通過(guò)延長(zhǎng)CN位置的取代基占據(jù)p4口袋。最終NMR證明它們與Mcl-1蛋白的結(jié)合模式和結(jié)合位點(diǎn)：H2和4g均能占據(jù)Mcl-1蛋白的BH3溝槽,除了引起和S1相似的p2口袋的氨基酸殘基化學(xué)位移之外,還引起p4口袋的V216、G219、V220等殘基發(fā)生明顯化學(xué)位移,表明它們同時(shí)占據(jù)了Mcl-1蛋白的p2和p4口袋。
[Abstract]:The Bcl-2 family includes three subfamilies with structural homology and opposite function: 1) Bcl-2-like protein, which is antiapoptotic. For example, Mcl-1 protein and Bcl-2 protein 2) Apoptosis-promoting proteins such as Bax and Bax 3) contain only BH3-only proteins with BH3 domain. They interact with each other through the shared BH3 domain. Regulation of apoptosis. Studies have shown that Mcl-1 and Bcl-2 proteins are key factors for tumor cells to escape apoptosis and gain immortality. Therefore, organic small molecules interfere with protein-protein interaction between Bcl-2 family proteins by occupying the interface where Bcl-2 family proteins interact with each other, thus inducing apoptosis of tumor cells. Several Bcl-2 inhibitors are in the clinical trial stage, but the specific Mcl-1 protein inhibitors are still few, because the BH3 channel of Mcl-1 protein is obviously different from that of Bcl-2. Most of the existing Bcl-2 inhibitors can not antagonize Mcl-1 protein at the same time, so it is necessary to further study the binding pattern of small molecules to Mcl-1 protein to guide the molecular design and structure-activity relationship analysis of Mcl-1 protein inhibitors. In this paper, the interaction of Mcl-1 protein with small molecular ligands was studied by 1H-15N HSQC NMRs, and the interaction sites and binding patterns were revealed. Small molecular Mcl-1 protein inhibitors were designed to obtain higher affinity by expanding binding sites. First of all, we constructed the expression plasmid of human Mcl-1 protein, and obtained 15N labeled Mcl-1 protein by isotope labeling method. In the process, we optimized the expression, isolation and purification conditions of Mcl-1 protein. The expression of 15N labeled Mcl-1 protein was increased from 1.3mg to the yield of 7.2mg/L cell, the purity was 95%. The results of circular dichroism analysis showed that the recombinant was labeled by isotope, The results of 2D NMR showed that the peaks of the amino acid residues were uniform, which proved that the 15N-labeled Mcl-1 had a stable and well-folded spatial structure. It meets the requirement of nuclear magnetic titration test of small molecule. Next, we performed a 1H-15N HSQC NMR titration study on the small molecular Mcl-1 protein inhibitor S1, 3-thiomolinyl -8-oxo-8H-acenaphthene [1h2b] pyrrole-9- nitrile. The results showed that 60% of the amino acids of the Mcl-1 protein shift 0.03ppm caused by S _ S _ 1 were located in the BH3 channel. The chemical shift of R263 residues is the most obvious. It is proved that S1 binds in the BH3 grooves of Mcl-1 protein, especially the results of NMR show that S1 mainly causes the chemical shift of amino acids near the subactive pocket of BH3 groove: p2. The results of computer-assisted molecular docking show that the carbonyl group of S1 molecule binds to Mcl-1 protein, and the carbonyl group of Mcl-1 protein forms hydrogen bond with R263 residue of Mcl-1 protein. The thiomolane substituent inserted into the p2 pocket of Mcl-1 protein, the cyanyl group of S1 was directed but did not occupy the p4 pocket of Mcl-1 protein. In this paper, we combine small molecule H _ 2O _ 3-N _ 4- bromophenyl thio _ 3-9-3- phenylamphetamine _ (-8H _ (acenaphthene) [1H _ (2-b)] pyrrole -8one) and 4g ~ ((1)) (E _ (2)) ((phenylmethylamino carbonyl) -3- (styrylacrylonitrile)) to try to occupy p4 pockets by extending the substitution group of CN position. Finally, NMR certificate is obtained. The binding patterns and binding sites of Mcl-1 proteins to Mcl-1 proteins were determined that they could occupy the BH3 grooves of Mcl-1 proteins. In addition to the chemical shifts of amino acid residues in p2 pockets similar to S1, they also resulted in obvious chemical shifts of V216G219V220 and other residues in p4 pockets, indicating that they occupy both p2 and p4 pockets of Mcl-1 proteins at the same time.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R341

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