α葡萄糖苷酶与PTP1B双靶点抑制剂的设计、合成、活性与作用机制研究
发布时间：2018-08-26 08:03
【摘要】：目的:2型糖尿病(T2DM)是以胰岛素抵抗和/或胰岛细胞功能衰竭为主要特征的一系列代谢紊乱综合征,常伴随多种急慢性并发症,已成为影响国人健康的最主要的慢性疾病之一。由于现阶段临床使用的T2DM治疗药物无法有效地阻止胰岛β细胞进一步坏死,且伴随较多的副作用,因此开发疗效更好的T2DM治疗药物势在必行。多靶点药物治疗不仅可以提高治疗效果,且可以避免联合用药产生的一些不良的相互作用,对于发病机制复杂的T2DM的治疗具有重要意义。α葡萄糖苷酶和蛋白酪氨酸磷酸酶1B (PTP1B)是与T2DM相关的重要靶酶,若同时抑制α葡萄糖苷酶与PTP1B这两个靶点,一方面可以降低餐后血糖,另一方面可以增强胰岛素敏感性,且有助于降低肥胖型T2DM患者的体质量。本研究旨在设计合成新型α葡萄糖苷酶/PTP1B双靶点抑制剂,并研究其作用机制,为多靶点降糖药的开发提供新思路。方法:(1)在化合物设计部分,本研究借助计算机辅助药物设计的手段,以α葡萄糖苷酶与PTP1B为靶点,运用双靶点高通量虚拟筛选的方法搜索ZINC小分子数据库以寻找先导物。又通过分析α葡萄糖苷酶与PTP1B两个靶蛋白活性位点的结构,应用Core-hopping的方法对先导化合物进行结构修饰与改造,设计新型的具有理论活性的α葡萄糖苷酶/PTP1B双靶点抑制剂。(2)在化学合成部分,通过数据库检索对以上化合物进行查新,并结合相关文献拟定化合物的合成路线。(3)在活性测试部分,首先构建含目的基因PTPN1的重组过表达质粒,并将其转入感受态细胞以表达PTP1B蛋白,然后以GST融合蛋白纯化法对PTP1B蛋白进行提纯。然后测试合成化合物对α糖苷酶与重组人PTP1B两个靶酶的抑制活性,并讨论构效关系。(4)在药物-蛋白作用机制探究部分,本研究运用抑制动力学方法及透析试验来探索小分子对α糖苷酶的抑制机制,并通过分子对接方法探究抑制剂与α葡萄糖苷酶和PTP1B两个靶标在分子水平的结合模式。结果:(1)化合物设计部分,运用α糖苷酶与PTP1B双靶点高通量虚拟筛选的方法发现先导化合物ZINC62431,然后将先导化合物分为Catalytic site binder,Linker和Second site binder三部分并依次进行结构改造,获得了 32个结合模式较合理且具具有合成可行性的小分子。(2)化学合成部分,确定了以两步亲核取代反应得到目标产物的合成路线,并借助核磁共振氢谱、碳谱、电喷雾离子化质谱以及高分辨质谱等手段确证目标化合物的结构,成功完成了全部32个目标小分子的化学合成。(3)活性测试部分,我们成功构建了 PTPN1的重组过表达质粒pGEX-4T-1-PTPN1,并经转化提纯,得到人重组PTP1B蛋白。以该重组PTP1B蛋白和购买的α葡萄糖苷酶为活性测试的靶蛋白,获得了 32个合成产物对这两个蛋白的体外抑制活性。活性结果显示,化合物5j对α葡萄糖苷酶的抑制活性最高(IC50= 10.11 μM),大约为已上市α葡萄糖苷酶抑制剂Acarbose(IC50=51.32 μM)的5倍,对PTP1B的抑制活性为IC50= 13.46 μM,也好于其阳性对照熊果酸(IC50= 14.50 μM)。构效关系的研究显示,含有苯并唑环结构的化合物(包括苯并恶唑衍生物和苯并噻唑衍生物)比相应的噻唑啉衍生物具有更强的α糖苷酶/PTP1B双靶点抑制活性;与脂肪族伯胺相比,芳香性伯胺对提高α葡萄糖苷酶/PTP1B双靶点抑制活性更有利;连接吸电子取代基的芳伯胺比无取代的苯胺具有更强的α糖苷酶抑制活性;适当延长linker能更有效地抑制α糖苷酶的活性。(4)在药物-蛋白作用机制探究部分,双倒数作图法Lineweaver-Burk plot与透析试验结果显示,小分子5j是α葡萄糖苷酶的可逆性混合型抑制剂。分子对接研究显示,化合物5j与α糖苷酶和PTP1B的活性口袋在空间构象上均可以很好地相匹配,并以强烈的药物-蛋白相互作用(包括氢键,范德华,电荷相互作用和Pi-cation相互作用等)相结合。结论:本文以2型糖尿病的两个治疗靶点α葡萄糖苷酶与PTP1B的抑制活性为检测指标,利用计算机辅助药物设计手段,成功设计并合成了一系列具有α葡萄糖苷酶与PTP1B双靶点抑制活性的小分子,其中化合物5j的体外活性优于分别作用于α葡萄糖苷酶和PTP1B两个靶点的阳性对照,抑制动力学显示5j是α葡萄糖苷酶的可逆性混合型抑制剂。构效关系与对接分析的研究将指导我们对该类化合物进行深入的结构修饰与改造,以进一步改善其活性并降低副作用。本研究将为多靶点治疗药物的开发提供行之有效的方法,为T2DM的治疗提供新思路。
[Abstract]:Objective: Type 2 diabetes mellitus (T2DM) is a series of metabolic disorder syndrome characterized by insulin resistance and/or islet cell failure, often accompanied by a variety of acute and chronic complications, which has become one of the most important chronic diseases affecting the health of Chinese people. It is imperative to develop better therapeutic drugs for T2DM because of further cell necrosis and more side effects. Multitarget drug therapy can not only improve the therapeutic effect, but also avoid some adverse interactions produced by combination therapy. It is important for the treatment of T2DM with complicated pathogenesis. Protein tyrosine phosphatase 1B (PTP1B) is an important target enzyme associated with T2DM. Inhibition of both alpha-glucosidase and PTP1B targets can reduce postprandial blood glucose on the one hand, enhance insulin sensitivity on the other hand, and help reduce the body mass of obese T2DM patients. This study aimed to design and synthesize novel alpha-glucoside. METHODS: (1) In the part of compound design, we searched ZINC small molecule database by computer-aided drug design with alpha-glucosidase and PTP1B as targets, using high-throughput virtual screening method of double targets. By analyzing the structure of two target protein active sites, alpha-glucosidase and PTP1B, and using Core-hopping method to modify and modify the structure of the lead compound, a novel theoretical active alpha-glucosidase/PTP1B double-target inhibitor was designed. (2) In the chemical synthesis part, the database search was used to search for the two target protein active sites. (3) In the activity test section, the recombinant overexpression plasmid containing the target gene PTPN1 was constructed and transfected into the competent cells to express PTP1B protein, and then purified PTP1B protein by GST fusion protein purification method. Inhibitory activity of alpha-glucosidase and recombinant human PTP1B target enzymes and their structure-activity relationship were discussed. (4) In the part of exploring the mechanism of drug-protein interaction, inhibition kinetics and dialysis test were used to explore the inhibition mechanism of small molecules on alpha-glucosidase, and molecular docking method was used to explore the inhibitors and alpha-glucosidase and PTP1B. Results: (1) In the design of compounds, the lead compound ZINC 62431 was identified by high throughput dummy screening of alpha-glycosidase and PTP1B. Then the lead compounds were divided into three parts: Catalytic site binder, Linker and Econd site binder, and 32 compounds were obtained by structural modification. (2) In the part of chemical synthesis, the synthetic route of the target compound was determined by two-step nucleophilic substitution reaction, and the structure of the target compound was confirmed by means of NMR, C-NMR, ESI-MS and HRMS. (3) In the activity test, we successfully constructed the recombinant over-expression plasmid pGEX-4T-1-PTPN1 of PTPN1 and purified the recombinant human PTP1B protein. The results showed that compound 5J had the highest inhibitory activity to alpha-glucosidase (IC50 = 10.11 mu M), about 5 times that of the listed alpha-glucosidase inhibitor Acarbose (IC50 = 51.32 mu M), and the inhibitory activity to PTP1B was IC50 = 13.46 mu M, which was better than that of the positive control ursolic acid (IC50 = 14.50 mu M). The results showed that compounds containing benzoxazole ring structure (including benzoxazole derivatives and benzothiazole derivatives) had stronger alpha-glucosidase/PTP1B double-target inhibitory activity than corresponding thiazoline derivatives, and aromatic primary amines were more favorable for enhancing alpha-glucosidase/PTP1B double-target inhibitory activity than aliphatic primary amines. Substituted aromatic primary amines had stronger inhibitory activity of alpha-glucosidase than non-substituted anilines, and prolonged linker could inhibit the activity of alpha-glucosidase more effectively. (4) Lineweaver-Burk plot and dialysis test showed that small molecule 5J was a reversible mixture of alpha-glucosidase in the part of exploring the mechanism of drug-protein interaction. Molecular docking studies showed that compound 5J was well matched with the active pockets of alpha-glycosidase and PTP1B in the spatial conformation and combined with strong drug-protein interactions (including hydrogen bonding, van der Waals, charge interaction and PI-cation interaction). A series of small molecules with double-target inhibitory activities of alpha-glucosidase and PTP1B were successfully designed and synthesized by means of computer-aided drug design. Compound 5J was more active in vitro than the positive pairs of alpha-glucosidase and PTP1B, respectively. The inhibition kinetics showed that 5J was a reversible mixed inhibitor of alpha-glucosidase. The study of structure-activity relationship and docking analysis will guide us to further modify and modify these compounds to further improve their activity and reduce side effects. It provides new ideas for the treatment of T2DM.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R914;R96
本文编号：2204239
[Abstract]:Objective: Type 2 diabetes mellitus (T2DM) is a series of metabolic disorder syndrome characterized by insulin resistance and/or islet cell failure, often accompanied by a variety of acute and chronic complications, which has become one of the most important chronic diseases affecting the health of Chinese people. It is imperative to develop better therapeutic drugs for T2DM because of further cell necrosis and more side effects. Multitarget drug therapy can not only improve the therapeutic effect, but also avoid some adverse interactions produced by combination therapy. It is important for the treatment of T2DM with complicated pathogenesis. Protein tyrosine phosphatase 1B (PTP1B) is an important target enzyme associated with T2DM. Inhibition of both alpha-glucosidase and PTP1B targets can reduce postprandial blood glucose on the one hand, enhance insulin sensitivity on the other hand, and help reduce the body mass of obese T2DM patients. This study aimed to design and synthesize novel alpha-glucoside. METHODS: (1) In the part of compound design, we searched ZINC small molecule database by computer-aided drug design with alpha-glucosidase and PTP1B as targets, using high-throughput virtual screening method of double targets. By analyzing the structure of two target protein active sites, alpha-glucosidase and PTP1B, and using Core-hopping method to modify and modify the structure of the lead compound, a novel theoretical active alpha-glucosidase/PTP1B double-target inhibitor was designed. (2) In the chemical synthesis part, the database search was used to search for the two target protein active sites. (3) In the activity test section, the recombinant overexpression plasmid containing the target gene PTPN1 was constructed and transfected into the competent cells to express PTP1B protein, and then purified PTP1B protein by GST fusion protein purification method. Inhibitory activity of alpha-glucosidase and recombinant human PTP1B target enzymes and their structure-activity relationship were discussed. (4) In the part of exploring the mechanism of drug-protein interaction, inhibition kinetics and dialysis test were used to explore the inhibition mechanism of small molecules on alpha-glucosidase, and molecular docking method was used to explore the inhibitors and alpha-glucosidase and PTP1B. Results: (1) In the design of compounds, the lead compound ZINC 62431 was identified by high throughput dummy screening of alpha-glycosidase and PTP1B. Then the lead compounds were divided into three parts: Catalytic site binder, Linker and Econd site binder, and 32 compounds were obtained by structural modification. (2) In the part of chemical synthesis, the synthetic route of the target compound was determined by two-step nucleophilic substitution reaction, and the structure of the target compound was confirmed by means of NMR, C-NMR, ESI-MS and HRMS. (3) In the activity test, we successfully constructed the recombinant over-expression plasmid pGEX-4T-1-PTPN1 of PTPN1 and purified the recombinant human PTP1B protein. The results showed that compound 5J had the highest inhibitory activity to alpha-glucosidase (IC50 = 10.11 mu M), about 5 times that of the listed alpha-glucosidase inhibitor Acarbose (IC50 = 51.32 mu M), and the inhibitory activity to PTP1B was IC50 = 13.46 mu M, which was better than that of the positive control ursolic acid (IC50 = 14.50 mu M). The results showed that compounds containing benzoxazole ring structure (including benzoxazole derivatives and benzothiazole derivatives) had stronger alpha-glucosidase/PTP1B double-target inhibitory activity than corresponding thiazoline derivatives, and aromatic primary amines were more favorable for enhancing alpha-glucosidase/PTP1B double-target inhibitory activity than aliphatic primary amines. Substituted aromatic primary amines had stronger inhibitory activity of alpha-glucosidase than non-substituted anilines, and prolonged linker could inhibit the activity of alpha-glucosidase more effectively. (4) Lineweaver-Burk plot and dialysis test showed that small molecule 5J was a reversible mixture of alpha-glucosidase in the part of exploring the mechanism of drug-protein interaction. Molecular docking studies showed that compound 5J was well matched with the active pockets of alpha-glycosidase and PTP1B in the spatial conformation and combined with strong drug-protein interactions (including hydrogen bonding, van der Waals, charge interaction and PI-cation interaction). A series of small molecules with double-target inhibitory activities of alpha-glucosidase and PTP1B were successfully designed and synthesized by means of computer-aided drug design. Compound 5J was more active in vitro than the positive pairs of alpha-glucosidase and PTP1B, respectively. The inhibition kinetics showed that 5J was a reversible mixed inhibitor of alpha-glucosidase. The study of structure-activity relationship and docking analysis will guide us to further modify and modify these compounds to further improve their activity and reduce side effects. It provides new ideas for the treatment of T2DM.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R914;R96
【引证文献】
相关期刊论文 前1条
1 徐昆仑;余兰;;新型天麻素衍生物的设计、合成及抗肿瘤活性研究[J];合成化学;2018年02期
，本文编号：2204239
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2204239.html
最近更新
教材专著
