α葡萄糖苷酶與PTP1B雙靶點抑制劑的設(shè)計、合成、活性與作用機制研究
發(fā)布時間：2018-08-26 08:03
【摘要】：目的:2型糖尿病(T2DM)是以胰島素抵抗和/或胰島細胞功能衰竭為主要特征的一系列代謝紊亂綜合征,常伴隨多種急慢性并發(fā)癥,已成為影響國人健康的最主要的慢性疾病之一。由于現(xiàn)階段臨床使用的T2DM治療藥物無法有效地阻止胰島β細胞進一步壞死,且伴隨較多的副作用,因此開發(fā)療效更好的T2DM治療藥物勢在必行。多靶點藥物治療不僅可以提高治療效果,且可以避免聯(lián)合用藥產(chǎn)生的一些不良的相互作用,對于發(fā)病機制復(fù)雜的T2DM的治療具有重要意義。α葡萄糖苷酶和蛋白酪氨酸磷酸酶1B (PTP1B)是與T2DM相關(guān)的重要靶酶,若同時抑制α葡萄糖苷酶與PTP1B這兩個靶點,一方面可以降低餐后血糖,另一方面可以增強胰島素敏感性,且有助于降低肥胖型T2DM患者的體質(zhì)量。本研究旨在設(shè)計合成新型α葡萄糖苷酶/PTP1B雙靶點抑制劑,并研究其作用機制,為多靶點降糖藥的開發(fā)提供新思路。方法:(1)在化合物設(shè)計部分,本研究借助計算機輔助藥物設(shè)計的手段,以α葡萄糖苷酶與PTP1B為靶點,運用雙靶點高通量虛擬篩選的方法搜索ZINC小分子數(shù)據(jù)庫以尋找先導(dǎo)物。又通過分析α葡萄糖苷酶與PTP1B兩個靶蛋白活性位點的結(jié)構(gòu),應(yīng)用Core-hopping的方法對先導(dǎo)化合物進行結(jié)構(gòu)修飾與改造,設(shè)計新型的具有理論活性的α葡萄糖苷酶/PTP1B雙靶點抑制劑。(2)在化學(xué)合成部分,通過數(shù)據(jù)庫檢索對以上化合物進行查新,并結(jié)合相關(guān)文獻擬定化合物的合成路線。(3)在活性測試部分,首先構(gòu)建含目的基因PTPN1的重組過表達質(zhì)粒,并將其轉(zhuǎn)入感受態(tài)細胞以表達PTP1B蛋白,然后以GST融合蛋白純化法對PTP1B蛋白進行提純。然后測試合成化合物對α糖苷酶與重組人PTP1B兩個靶酶的抑制活性,并討論構(gòu)效關(guān)系。(4)在藥物-蛋白作用機制探究部分,本研究運用抑制動力學(xué)方法及透析試驗來探索小分子對α糖苷酶的抑制機制,并通過分子對接方法探究抑制劑與α葡萄糖苷酶和PTP1B兩個靶標在分子水平的結(jié)合模式。結(jié)果:(1)化合物設(shè)計部分,運用α糖苷酶與PTP1B雙靶點高通量虛擬篩選的方法發(fā)現(xiàn)先導(dǎo)化合物ZINC62431,然后將先導(dǎo)化合物分為Catalytic site binder,Linker和Second site binder三部分并依次進行結(jié)構(gòu)改造,獲得了 32個結(jié)合模式較合理且具具有合成可行性的小分子。(2)化學(xué)合成部分,確定了以兩步親核取代反應(yīng)得到目標產(chǎn)物的合成路線,并借助核磁共振氫譜、碳譜、電噴霧離子化質(zhì)譜以及高分辨質(zhì)譜等手段確證目標化合物的結(jié)構(gòu),成功完成了全部32個目標小分子的化學(xué)合成。(3)活性測試部分,我們成功構(gòu)建了 PTPN1的重組過表達質(zhì)粒pGEX-4T-1-PTPN1,并經(jīng)轉(zhuǎn)化提純,得到人重組PTP1B蛋白。以該重組PTP1B蛋白和購買的α葡萄糖苷酶為活性測試的靶蛋白,獲得了 32個合成產(chǎn)物對這兩個蛋白的體外抑制活性�；钚越Y(jié)果顯示,化合物5j對α葡萄糖苷酶的抑制活性最高(IC50= 10.11 μM),大約為已上市α葡萄糖苷酶抑制劑Acarbose(IC50=51.32 μM)的5倍,對PTP1B的抑制活性為IC50= 13.46 μM,也好于其陽性對照熊果酸(IC50= 14.50 μM)。構(gòu)效關(guān)系的研究顯示,含有苯并唑環(huán)結(jié)構(gòu)的化合物(包括苯并惡唑衍生物和苯并噻唑衍生物)比相應(yīng)的噻唑啉衍生物具有更強的α糖苷酶/PTP1B雙靶點抑制活性;與脂肪族伯胺相比,芳香性伯胺對提高α葡萄糖苷酶/PTP1B雙靶點抑制活性更有利;連接吸電子取代基的芳伯胺比無取代的苯胺具有更強的α糖苷酶抑制活性;適當延長linker能更有效地抑制α糖苷酶的活性。(4)在藥物-蛋白作用機制探究部分,雙倒數(shù)作圖法Lineweaver-Burk plot與透析試驗結(jié)果顯示,小分子5j是α葡萄糖苷酶的可逆性混合型抑制劑。分子對接研究顯示,化合物5j與α糖苷酶和PTP1B的活性口袋在空間構(gòu)象上均可以很好地相匹配,并以強烈的藥物-蛋白相互作用(包括氫鍵,范德華,電荷相互作用和Pi-cation相互作用等)相結(jié)合。結(jié)論:本文以2型糖尿病的兩個治療靶點α葡萄糖苷酶與PTP1B的抑制活性為檢測指標,利用計算機輔助藥物設(shè)計手段,成功設(shè)計并合成了一系列具有α葡萄糖苷酶與PTP1B雙靶點抑制活性的小分子,其中化合物5j的體外活性優(yōu)于分別作用于α葡萄糖苷酶和PTP1B兩個靶點的陽性對照,抑制動力學(xué)顯示5j是α葡萄糖苷酶的可逆性混合型抑制劑。構(gòu)效關(guān)系與對接分析的研究將指導(dǎo)我們對該類化合物進行深入的結(jié)構(gòu)修飾與改造,以進一步改善其活性并降低副作用。本研究將為多靶點治療藥物的開發(fā)提供行之有效的方法,為T2DM的治療提供新思路。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R914;R96
本文編號：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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R914;R96
【引證文獻】
相關(guān)期刊論文 前1條
1 徐昆侖;余蘭;;新型天麻素衍生物的設(shè)計、合成及抗腫瘤活性研究[J];合成化學(xué);2018年02期
，本文編號：2204239
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