小分子先导药物与Mcl-1蛋白相互作用的二维核磁研究

发布时间：2018-03-24 05:09

本文选题：Mcl-1　切入点：二维核磁HSQC　出处：《大连理工大学》2012年硕士论文

【摘要】：Bcl-2家族蛋白包括结构同源、功能相反的3个亚族：(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)仅含有BH3结构域的BH3-only蛋白。它们之间通过共同拥有的BH3结构域发生相互作用,调控细胞凋亡。研究证明Mcl-1和Bcl-2蛋白是肿瘤细胞逃避凋亡和获得永生的关键因子,因此成为靶向抗癌药的重要靶点。有机小分子通过占据Bcl-2家族蛋白发生相互作用的界面(interface):BH3沟槽来干扰Bcl-2家族蛋白之间的蛋白质-蛋白质相互作用,从而诱导肿瘤细胞凋亡,具有抗癌药成药前景。目前已经有几个Bcl-2抑制剂正处于临床试验阶段；但是特异的Mcl-1蛋白抑制剂还为数很少。因为Mcl-1蛋白的BH3沟槽与Bcl-2的差异明显,现有的Bcl-2抑制剂大多不能同时拮抗Mcl-1蛋白。因此需要通过深入研究小分子与Mcl-1蛋白的结合模式来指导Mcl-1蛋白抑制剂的分子设计和构效关系分析。本文通过二维核磁共振技术(1H-15N HSQC NMR)研究了Mcl-1蛋白与小分子配体的相互作用,揭示了作用位点和结合模式,指导设计了通过扩大结合位点获得更高亲和力的小分子Mcl-1蛋白抑制剂类抗癌先导化合物。首先,我们构建了人源Mcl-1蛋白的表达质粒,通过同位素标记方法,获得15N标记的Mcl-1蛋白。在此过程中,优化了Mcl-1蛋白的表达和分离纯化条件,将15N标记Mcl-1蛋白的表达量从1.3mg提高到7.2mg/L菌体的产量；纯度达到了95%。圆二色谱分析结果显示重组、同位素标记、和纯化的Mcl-1蛋白具有以α螺旋为主的稳定的二级结构。二维核磁共振结果表明各氨基酸残基的谱峰分散均匀,证明15N标记Mcl-1具有稳定的、折叠良好的空间结构,满足小分子核磁滴定试验的要求。接下来,我们对本课题组发现的小分子Mcl-1蛋白抑制剂S1(3-硫吗啉基-8-氧-8H-苊并[1，，2-b]吡咯-9-腈)进行了1H-15N HSQC NMR滴定研究。结果表明,S1引起Mcl-1蛋白化学位移0.03ppm的氨基酸60%位于BH3沟槽,其中R263残基化学位移改变最为明显。证明S1结合在Mcl-1蛋白的BH3沟槽中。尤其是NMR的结果表明S1主要引起了BH3沟槽的亚活性口袋：p2口袋附近的氨基酸的化学位移,包括V249,M250,V253,R263等残基。计算机辅助分子对接结果体现了S1分子与Mcl-1蛋白的结合模式：S1的羰基与Mcl-1蛋白的R263残基形成氢键,硫吗啉取代基插入Mcl-1蛋白的p2口袋；S1的氰基指向但并未占据Mcl-1蛋白的p4口袋。在这一结合模式的指导下,本课题组合成了小分子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),尝试通过延长CN位置的取代基占据p4口袋。最终NMR证明它们与Mcl-1蛋白的结合模式和结合位点：H2和4g均能占据Mcl-1蛋白的BH3沟槽,除了引起和S1相似的p2口袋的氨基酸残基化学位移之外,还引起p4口袋的V216、G219、V220等残基发生明显化学位移,表明它们同时占据了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.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

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