脂肪酶SMG1小分子抑制剂的筛选及其激活机制的研究

发布时间：2018-01-11 22:25

本文关键词：脂肪酶SMG1小分子抑制剂的筛选及其激活机制的研究　出处：《华南理工大学》2016年博士论文　论文类型：学位论文

【摘要】：马拉色球菌CBS 7966来源的脂肪酶SMG1是一个偏甘油酯脂肪酶。大量研究表明,马拉色球菌对头屑和脂溢性皮炎(D/SD)的致病性与脂肪酶的活性有关,主要是通过脂肪酶介导皮脂的水解,进而产生刺激性的游离脂肪酸发生。因此脂肪酶SMG1是对抗头屑和脂溢性皮炎的一个潜在靶标。同时,脂肪酶SMG1还在生物催化中有广泛的应用,包括高纯度甘油二酯的合成、磷酯的改造以及烯烃类的环氧化等,因此有重要有工业价值。SMG1的晶体结构显示,该脂肪酶的催化口袋被一段呈loop结构的“盖子”区域覆盖,因此为闭合结构。SMG1在催化过程中必然经历一定的构象变化,使得催化口袋开放,与底物分子结合。这一构象变化过程称为脂肪酶SMG1的激活机制。对SMG1激活机制的研究,不仅可以从理论角度阐述其结构的柔性对于催化功能的影响,同时还可以为理性设计、进一步改造并提高SMG1的催化活性提供指导,以扩展SMG1在生物催化领域中的应用。本论文以脂肪酶SMG1为研究对象,旨在于发现靶向SMG1的抑制剂,为抗头屑药物的开发提供基础;并从分子水平上阐述脂肪酶SMG1在催化过程中的激活机制,为其催化活性改造提供理论依据。具体研究内容如下:1.脂肪酶SMG1小分子抑制剂的发现本论文首次以脂肪酶SMG1作为抗头屑靶标,展开小分子抑制剂的筛选。筛选的策略为计算机辅助的虚拟筛选与基于脂肪酶活性的体外高通量筛选相结合。在进行虚拟筛选前,我们首先采用同源模建的方法搭建了脂肪酶SMG1的盖子打开的开放结构,用于虚拟筛选的对接。虚拟筛选采用的化合物库来自Specs公司,其容量为17万。经过筛选和验证,我们获得了靶向脂肪酶SMG1的首个先导化合物,它针对人工底物(对硝基苯酚乙酯)的IC50为20μM,而针对天然底物(单油酸甘油酯)的IC50为0.19μM。通过对该先导化合物类似物的活性评价及对接,我们解析了该先导化合物的结构-活性关系,并提供了它与SMG1的结合模式信息。本研究将为后续以SMG1为靶标的更高活性的抑制剂的理性开发提供一个起点。2.脂肪酶SMG1的激活机制研究本论文中关于脂肪酶SMG1激活机制的研究分为两部分。(1)门控机制。激活机制的研究策略主要为,通过获得脂肪酶SMG1催化口袋的开放结构,并与闭合结构相对比,以得出激活过程的构象变化。对SMG1野生型闭合晶体结构的分析可以看出,278和102位残基连接形成“桥状”结构,将催化口袋从中间截断,并覆盖了催化三联体的丝氨酸。该“桥状”结构有一定的动态。据此我们设计了278位的突变体,以较小的残基代替野生型中较大的苯丙氨酸,来模拟278位残基向102位残基的远离,期望以此迫使催化口袋变为开放。我们获得了F278N和F278D的突变体晶体结构。通过结构分析和分子对接发现,F278N与甘油的复合晶体结构代表脂肪酶SMG1催化口袋开放的构象。通过野生型SMG1的分子动力学模拟以及PMSF抑制实验的验证得出,脂肪酶SMG1的F278和N102两个残基作为门控开关,控制着SMG1催化口袋的打开和关闭。门控机制的阐明为SMG1的理性设计提供了依据。据此,仅通过单点突变(F278N或F278T),我们设计出了高于野生型SMG1活性6倍的突变体。(2)盖子区域的动态在所有的SMG1的晶体结构中,包括闭合的野生型结构和开放的突变体复合结构,均没有观察到盖子结构的构象变化,并且盖子区域的B-因子(B-factor)相对于其它区域较低,因此盖子结构起初被认为在SMG1激活过程保持不变。接下来的研究着重进一步验证,除了门控机制以外,SMG1在激活过程中盖子区域到底会不会打开。我们采用引入二硫键的策略将SMG1的盖子结构固定于主体骨架中,并通过氧化和还原处理来控制盖子区域的固定与否。通过对盖子固定和非固定状态下的脂肪酶活性测试,我们验证了脂肪酶SMG1的激活过程中,除了102和278两个残基的门控机制以外,还需要盖子结构的打开。该研究提示了利用晶体学研究酶激活过程的缺陷。尽管激活状态的晶体结构能够给出非常丰富的信息,但它毕竟是静态的结构,并不能反映酶从非活化状态到激活状态的变化过程。尤其当酶激活状态的结构能够局部地恢复到非活化状态时,单独依赖晶体学的研究方法将会产生严重的后果。本研究中二硫键的引入是通过SMG1-L106C/V233C的双突变体蛋白获得。我们发现该突变体与含发色基团的底物相结合时,可作为一个定量的巯基氧化还原电势探针,且在生物催化的级联反应中的潜在应用。
[Abstract]:Malassezs lipase SMG1 aureus CBS 7966 source is a partial glyceride lipase. Many studies showed that Malassezia aureus against dandruff and seborrheic dermatitis (D/SD) related to the pathogenicity and lipase activity, mainly by lipase mediated hydrolysis of sebum, and free fatty acids stimulate the occurrence of therefore. Lipase SMG1 is a potential target against dandruff and seborrheic dermatitis. At the same time, has been widely used in biological catalysis of lipase SMG1, including synthesis of high purity diacylglycerol, transformation of phosphate esters and olefin epoxidation, so it is important that the crystal structure of industrial value.SMG1, the lipase a catalytic pocket is loop structure of the "lid" coverage, so for the closed structure of.SMG1 must experience some conformational changes in the catalytic process, the catalytic pocket opening, and substrate molecular junctions . this conformational change process called activation mechanism of lipase SMG1. Research on activation mechanism of SMG1, can not only expounds the effects of structural flexibility on the catalytic function from a theoretical point of view, but also for rational design, further reform and improve the catalytic activity of SMG1 to provide guidance to application of extended SMG1 in biocatalysis in the field. This paper using lipase SMG1 as the research object, aims to find targeted inhibitors of SMG1, provide a basis for the development of anti dandruff agents; and from the molecular level on the mechanism of activation in the catalytic process of lipase SMG1, provide a theoretical basis for the catalytic activity of the transformation. The specific contents are as follows: 1. lipase SMG1 inhibitors for the first time with lipase SMG1 as anti dandruff target, start screening of small molecule inhibitors. The screening strategy for virtual screening and computer aided In vitro high-throughput screening of lipase activity in combination. In virtual screening, we use homology model to build an open lid structure of lipase SMG1 open, for docking. Virtual screening using virtual screening of compound libraries from Specs company, its capacity is 170 thousand. After screening and verification, we get targeting the first precursor compound lipase SMG1, the artificial substrate (p-nitrophenol acetate) of IC50 was 20 M, while the natural substrates (glycerol monooleate) IC50 0.19 M. through the evaluation of the active compounds and analogs docking, we analyzed the structure the lead compound activity relationship, and provides the information model combining it with SMG1. To provide a starting point for.2. activation of lipase SMG1 in this study for the subsequent development of rational target SMG1 have higher activity of inhibitors The research on the mechanism of lipase SMG1 activation mechanism is divided into two parts. (1) gating mechanism. Research on activation mechanism is the main strategy, through the open structure of SMG1 by lipase pocket, and compared with the closed structure, with the activation process. The conformational change analysis of the wild type SMG1 closed crystal structure it can be seen that the 278 and 102 residues are connected to form a "bridge" structure, the catalytic pocket from the middle cut, and covered the three CIS catalytic serine. The "bridge" structure has certain dynamic. So we designed 278 mutants, with smaller phenylalanine residues instead of large wild type, to simulate 278 residues away from residue 102, hoping to force the catalytic pocket is open. We obtained a mutant crystal structure of F278N and F278D. Through the analysis of the structure and molecular docking, F2 The conformation of 78N and glycerol composite crystal structure represents the lipase SMG1 catalytic pocket opening. By molecular dynamics simulations of wild-type SMG1 and PMSF inhibition experiments verify that the lipase SMG1 F278 and N102 two residues as gating switch, control of SMG1 catalytic pocket opening and closing. To clarify the gating mechanism provides the basis for for the rational design of SMG1. Therefore, only through a single point mutation (F278N or F278T), we designed the SMG1 activity 6 times higher than that of the wild type mutant. (2) the crystal structure of dynamic lid region in all of the SMG1 in the mutant composite structure including wild type structure and the open closed, no the observed conformational changes of the lid structure, and B- factor covers areas (B-factor) relative to other regions is relatively low, so that the lid structure was initially thought the activation process remained unchanged in the next research SMG1. Weight in addition to further verification, gating mechanism, SMG1 in the activation process in the end will not open lid area. We introduced two disulfide bond strategy will cover the structure of SMG1 is fixed on the main frame, and the oxidation and reduction treatment to control the lid area fixed or not. The lipase activity test on the lid and non fixed the fixed condition, we verify the activation process of lipase SMG1, 102 and 278 in addition to the gating mechanism of two residues, also need to open the lid structure. The research suggests that the defects with crystallographic studies of enzyme activation process. Although the crystal structure active are very rich information, but it is after all is the static structure, and does not reflect the enzyme from inactive state to the active state change process. When the structure of enzyme activation can be partially restored to non activated state, Research methods rely solely on Crystallography will have serious consequences. In this study, the introduction of the two disulfide bonds is obtained by double mutant SMG1-L106C/V233C. We found that the mutant containing chromophore substrate combination, can be used as a quantitative thiol oxidation reduction potential of the probe, and potential applications in cascade biological catalysis in.

【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q55

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