固定化乙酰胆碱酯酶的制备及其特性研究

本研究以期研制出能重复使用的固定化乙酰胆碱酯酶(AChE),为天然产物复杂体系中AchE抑制剂筛选新方法的发展奠定基础.以氨基化硅胶(APS-Si)微球为载体,戊二醛为交联剂对乙酰胆碱酯酶进行交联固定化,并研究了酶的最佳固定化条件和固定化酶的性质.结果表明,0.05 g氨基化硅胶微球载体,用戊二醛溶液活化6 h后,在给酶量5 U,28℃固定16 h条件下,得到固定化酶的活性最大.固定化酶在常温(20～40℃),以及较宽pH范围内(pH 6～10)均具有较高的活性,并且具有良好的保存稳定性和可重复利用率,为基于固定化靶酶亲和-色谱质谱联用分析快速筛选乙酰胆碱酯酶抑制剂新方法的发展奠定了基础.     

基于他克林的乙酰胆碱酯酶抑制剂的3D-QSAR研究及虚拟筛选

本文通过对58个他克林派生物乙酰胆碱酯酶抑制剂分子进行建模分析,研究其结构与活性的关系,并通过虚拟筛选方法获得一系列潜在AChE抑制剂双位点分子。首先将一系列他克林二联体化合物与AChE晶体结构对接,获得化合物的活性构象,以此进行建模分析,建立结构与活性之间的三维定量构效关系。所得模型CoMFA、CoMSIA、TopomerCoMFA的交叉验证系数分别为0.510、0.702、0.571,非交叉验证系数为0.998、0.988、0.794,测试集r_(pred)~2为0.750、0.742、0.766,所得模型具有良好的预测性,由此可以为设计高活性的新分子提供理论基础。然后,使用Topomer search对ZINC数据库中的125909分子进行虚拟筛选,得到891个具有潜在AChE抑制活性的分子。最后,对这891个分子进行分子对接,观察分子与晶体结构的结合情况,筛选得到66个具有高选择性的双位点AChE抑制剂分子。     

应用壳核纳米微球固定乙醇脱氢酶联用 HPLC对抑制剂筛选

对新型聚甲基丙烯酸甲酯/壳聚糖纳米微球进行制备、纯化和表征,采用该纳米微球对乙醇脱氢酶进行固定化。让多组分天然产物直接作用于固定化乙醇脱氢酶,通过高效液相色谱(HPLC)检测固定化酶吸附组分,研究多组分天然产物混合物对乙醇脱氢酶具有抑制活性的有效成分。结果表明:由HPLC检测到的被固定化乙醇脱氢酶从多组分化合物中直接吸附的组分对乙醇脱氢酶均有较强的抑制作用,说明该方法从多组分直接筛选抑制剂是可行的,同时,本文所检测到的7种天然化合物对乙醇脱氢酶的抑制活性还没有报道。     

毛秔子梢中天然高活性的神经氨酸酶抑制成分的研究

从天然产物中筛选高效低毒的神经氨酸酶抑制剂(NAI), 以期发现可抗流感病毒(H1N1)的化合物. 方法: 采用经典的NA活性检测实验(FL-MH-NANA method), 对从豆科植物毛秔子梢中分离得到10个化合物进行筛选, 检测这些化合物对NA的抑制活性. 结论: 化合物1, 2, 3, 5对流感病毒株的神经氨酸酶(NA)均具有抑制作用, 其中化合物1的抑制活性最强, 其IC₅₀值为16.76 μmol/L. 化合物1～4为新化合物, 化合物5～10为该植物中首次分得.     

毛细管电泳法测定天麻素对乙酰胆碱酯酶活性的抑制

阿尔茨海默症(AD)是引起中老年人痴呆最常见的疾病。目前治疗AD的药物主要为乙酰胆碱酯酶抑制剂(AChEI)。建立快速地从天然产物中筛选AChEI的方法,将对临床治疗AD产生积极的作用。该研究建立了一种简单可靠的AChEI筛选新方法。通过海美溴铵在毛细管内壁形成一段正电荷涂层,再经过离子吸附作用制备1.5 cm长的乙酰胆碱酯酶(AChE)反应器。底物碘化乙酰硫代胆碱在0.015 MPa压力下进样10 s,在微酶反应器中停留1 min后采用毛细管电泳(CE)法对底物和酶解产物进行分离。天麻素是天麻的重要药效成分之一,对AChE具有抑制作用。该研究以天麻素为例,根据加入药物前后酶解产物峰面积的差异,完成天麻素对AChE活性的抑制能力的测定。结果表明,随着天麻素浓度的增加,产物峰面积逐渐减小,对AChE的活性抑制变大。该方法所建微酶反应器产物峰面积的RSD值小于5.3%,可连续使用300次。当天麻素浓度为5.24μmol/L时,对AChE活性抑制率达到64.8%。根据加入不同浓度天麻素时的抑制率,测定出天麻素的IC₅₀值为(2.26±0.14)μmol/L(R^...     

毛细管电泳结合高效液相色谱-质谱用于天然产物活性成分的筛选

发展了毛细管电泳(CE)和高效液相色谱-质谱(HPLC-MS)相结合的用于天然产物中活性成分筛选和鉴定的方法。该方法中,用HPLC半制备柱对天然产物粗提物进行分离纯化,再用CE对HPLC纯化后的组分进行活性测试。根据HPLC-MS/MS提供的二级质谱数据,即可确定活性成分的化学结构。以乙酰胆碱酯酶为实验模型,对我们发展的筛选方法进行了验证。从黄连粗提物中确定了药根碱、巴马汀等7种活性成分,并通过CE测定了它们的半抑制率(IC₅₀)值。与传统的天然产物分离纯化和活性筛选方法相比,该方法具有简单、微量、快速、准确的优点。本文建立的方法为天然产物粗提物中活性成分的筛选提供了新技术。     

基于毛细管电泳的天然产物中酶抑制剂筛选研究进展

人类疾病的发生往往与体内各种酶的功能失调密切相关,因此酶一直是目前新药研发的重要靶标。天然产物是发现新药的宝贵资源,但是由于成分复杂,活性筛选一直受制于耗时费力的分离纯化过程。毛细管电泳(CE)技术由于具有样品和试剂消耗少、灵活多样的分离模式且不受样品基质干扰的特点,可以直接从粗提物开始筛选活性成分,在复杂样品活性筛选中显示出独特的优势。该文综述了近十年来CE在天然产物中酶抑制剂筛选的研究进展。其中重点介绍了CE应用于重要药物靶标,包括转移酶(激酶)、水解酶以及氧化还原酶等方面的应用,总结了用于酶抑制剂筛选的电泳分离模式和酶动力学研究,并展望了CE用于天然产物中活性成分筛选的应用前景。     

齐墩果酸的PTP1B构效关系研究

蛋白质酪氨酸磷酸酯酶(PTP1B)是用于筛选Ⅱ型糖尿病药物的一个新靶点.为寻找天然的PTP1B抑制剂,运用高通量筛选技术对我们的天然产物化合物库进行筛选,发现齐墩果酸具有相对较好的PTP1B抑制活性.因此,我们在齐墩果酸基础上进行了构效关系研究,发现了若干个选择性、活性较好的化合物(IC50＜1μmol/L).     

基于药效团模型的DHODH抑制剂构效关系研究

利用药效团模型研究二氢乳清酸脱氢酶(Dihydroorotate dehydrogenase,DHODH)抑制剂的构效关系,为DHODH抑制剂的虚拟筛选提供新的方法.以31个具有DHODH抑制活性的化合物为训练集化合物,半数抑制浓度(IC50)范围为7~63000 nmol/L,利用Catalyst/HypoGen算法构建DHODH抑制剂药效团模型,通过对训练集化合物多个构象进行叠合,提取药效团特征及三维空间限制构建药效团模型.利用基于CatScramble的交叉验证方法及评价模型对已知活性化合物的活性预测能力,确定较优药效团模型.模型包含1个氢键受体、3个疏水中心,表征了受体配体相互作用时可能发生的氢键相互作用、疏水相互作用和π-π相互作用,4个药效特征在三维空间的排列概括了DHODH抑制剂产生活性的结构特点.所得较优模型对训练集化合物及测试集化合物的计算活性值与实验活性值的相关系数分别为0.8405和0.8788.利用药效团模型对来源于微生物的系列化合物进行虚拟筛选,筛选出59个预测活性较好的化合物,可作为进一步药物研发的候选化合物.     

HMG-CoA还原酶抑制剂三维药效团的构建

以作用于鼠肝脏细胞的21个3-羟基-3-甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂(RI)为训练集, 训练集化合物具备结构多样性, 来源于相同药理模型, 活性值IC50范围在0.3-8000 nmol·L-1. 利用Catalyst 计算HMG-CoA还原酶抑制剂最优药效团由一个氢键受体, 一个氢键给体, 一个疏水基团和一个芳香环特征组成. 药效团模型Fixed cost值, Total cost值和Configuration cost值分别为88.75、111.5 和16.98. 训练集化合物活性计算值与实测值相关系数为0.8883, 偏差值为1.269, 交叉验证结果表明, 药效团模型具有较高的置信度, 对测试集化合物活性值的预测结果显示有较好的预测能力, 可用于数据库搜索发现新的具有该活性的化合物, 也可用于中药或天然产物药物的研究开发.     

Rapid on‐line system for preliminary screening of lipase inhibitors from natural products by integrating capillary electrophoresis with immobilized enzyme microreactor

An on‐line system for preliminary screening lipase inhibitors from natural products with an immobilized lipase microreactor coupled to capillary electrophoresis was established. In this research, the lipase was anchored on the amino activated capillary inner wall using glutaraldehyde as a homobifunctional linker through Schiff base reaction. The immobilized lipase activity was evaluated by measuring the peak area of the hydrolyzate of p‐nitrophenyl acetate. In order to maintain the enzymatic activity of immobilized lipase, the acetonitrile content and the pH of the reaction solution were also optimized. Under the optimized reaction conditions, the Michaelis–Menten constant of the immobilized lipase and the half maximal inhibitory concentration for orlistat were studied, which were consistent with previous literature data. Furthermore, the developed method was applied to screen lipase inhibition activity from ten natural products. As a result, we found that six natural products have inhibitory effect on the activity of lipase, among which the inhibitory effect of Rhizoma atractylodis extract has never been reported before.     

On-line immobilized acetylcholinesterase microreactor for screening of inhibitors from natural extracts by capillary electrophoresis

In this study we developed a simple capillary electrophoresis (CE) method with an on-line acetylcholinesterase (AChE) microreactor at the inlet of capillary for inhibitor screening. The fused-silica capillary surface was modified with a polycationic polyethylenimine coating. Solutions of the enzyme and chitosan were then injected to immobilize the enzyme in approximately 2.9?cm of the capillary inlet (total length of capillary 60.2?cm) by electrostatic interaction and the film overlay technique. Separation of enzyme reaction product (thiocholine, ThCh) and unreacted substrate (acetylthiocholine, AThCh) was achieved within 3.0?min. The conditions affecting the efficiency of reaction of the enzyme were optimized by measuring the peak area of ThCh. Under the optimum conditions, using Huperzine-A as model inhibitor, K (i) and IC (50) were 0.551?μmol?L(-1) and 1.52?μmol?L(-1), respectively, for immobilized AChE. Finally, screening of a small compound library containing two known AChE inhibitors and 30 natural extracts was conducted, and species with inhibition activity were directly identified. Compared with previous publications on screening for AChE inhibitors in natural products based on CE methods, the method developed in this work has the advantages of lower cost per analysis, less leakage, and better bioaffinity for the immobilized enzyme because of the unique properties of sodium alginate and chitosan.     

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis–Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half‐maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.     

Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies

The development and characterization of a human recombinant acetylcholinesterase (hrAChE) micro-immobilized-enzyme reactor (IMER), prepared by using an in situ immobilization procedure is reported. hrAChE was covalently immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk (12 mm x 3 mm i.d.), previously derivatized with glutaraldehyde. The optimal conditions for the immobilization were: 12 microg of enzyme dissolved in 800 microl of phosphate buffer (50 mM, pH 6.0). The mixture was gently agitated overnight at 4 degrees C. The resulting Schiff bases were reduced by cyanoborohydride and the remaining aldehydic groups were condensed with monoethanolamine. Under these conditions, 0.22 U of hrAChE were immobilized with retention of 3.0% of the initial enzymatic activity. The activity of the immobilized hrAChE was stable for over 60 days. The activity and kinetic parameters of the hrAChE micro-IMER were investigated by inserting the micro-IMER in a HPLC system and it was demonstrated that the enzyme retained its activity. The micro-IMER was characterized in terms of units of immobilized enzyme and best conditions for immobilization yield. IMERs were compared for their relative enzyme stability, immobilized units, yield and aspecific matrix interactions. The effect of AChE inhibitors was evaluated by the simultaneous injection of each inhibitor with the substrate. The relative IC50 values were found in agreement with those derived by the conventional kinetic spectrophotometric method. In comparison with previously developed AChE-based IMERs, AChE monolithic micro-IMER showed advantages in terms of reduction of analysis time (2 min), lower aspecific matrix interactions and lower backpressure. Included in a HPLC system, it can be used for the rapid screening of new compounds' inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analyzed in continuous.     

Screening of tyrosinase inhibitors by capillary electrophoresis with immobilized enzyme microreactor and molecular docking

A new method for screening tyrosinase inhibitors from traditional Chinese medicines (TCMs) was successfully developed by capillary electrophoresis with reliable online immobilized enzyme microreactor (IMER). In addition, molecular docking study has been used for supporting inhibition interaction between enzyme and inhibitors. The IMER of tyrosinase was constructed at the outlet of the capillary by using glutaraldehyde as cross‐linker. The parameters including enzyme reaction, separation of the substrate and product, and the performance of immobilized tyrosinase were investigated systematically. Because of using short‐end injection procedure, the product and substrate were effectively separated within 2 min. The immobilized tyrosinase could remain 80% active for 30 days at 4°C. The Michaelis–Menten constant of tyrosinase was determined as 1.78 mM. Kojic acid, a known tyrosinase inhibitor, was used as a model compound for the validation of the inhibitors screening method. The half‐maximal inhibitory concentration of kojic acid was 5.55 μM. The method was successfully applied for screening tyrosinase inhibitors from 15 compounds of TCM. Four compounds including quercetin, kaempferol, bavachinin, and bakuchiol were found having inhibitory potentials. The results obtained in this work were supported by molecular docking study.     

靶向亲和-液相色谱-质谱联用技术快速筛选黄藤总生物碱中乙酰胆碱酯酶抑制剂

采用靶向亲和-液相色谱-质谱联用技术(Target molecule affinity-LC-ESI-MSn)快速筛选黄藤总生物碱中能够抑制乙酰胆碱酯酶活性的成分,共筛选出12种具有潜在抑制乙酰胆碱酯酶的活性成分,并鉴定了6种成分,分别为黄藤素(Palmatine)、小檗碱(Berberine)、药根碱(Jatrorrhizine)、巴马汀红碱(Palmatrubine)、7,8-二氢-8-羟基小檗碱(7,8-Dihydro-8-hydroxyberberine)、Groenlandicine,结合体外酶学实验对这6种化合物进行了活性验证实验.结果表明,黄藤素抑制活性最强,其抑制作用强于阳性对照药盐酸多奈哌齐,说明黄藤素具有开发成抗阿尔茨海默症药物的潜力.本方法简单、快速、准确地从复杂的中药提取物中筛选出具有抑制乙酰胆碱酯酶活性的成分,适用于复杂体系中的高通量筛选.     

Evaluation of Enzyme Inhibitory Activity of Flavonoids by Polydopamine-Modified Hollow Fiber-Immobilized Xanthine Oxidase

In this study, a polydopamine (PDA)-modified hollow fiber-immobilized xanthine oxidase (XOD) was prepared for screening potential XOD inhibitors from flavonoids. Several parameters for the preparation of PDA-modified hollow fiber-immobilized XOD, including the dopamine concentration, modification time, XOD concentration and immobilization time, were optimized. The results show that the optimal conditions for immobilized XOD activity were a dopamine concentration of 2.0 mg/mL in 10.0 mM Tris-HCl buffer (pH 8.5), a modification time of 3.0 h, an XOD concentration of 1000 μg/mL in 10.0 mM phosphate buffer (pH 7.5) and an immobilization time of 3.0 h. Subsequently, the enzymatic reaction conditions such as the pH value and temperature were investigated, and the enzyme kinetics and inhibition parameters were determined. The results indicate that the optimal pH value (7.5) and temperature (37 °C) of the PDA-modified hollow fiber-immobilized XOD were consistent with the free enzyme. Moreover, the PDA-modified hollow fiber-immobilized XOD could still maintain above 50% of its initial immobilized enzyme activity after seven consecutive cycles. The Michaelis–Menten constant (K_m) and the half-maximal inhibitory concentration (IC₅₀) of allopurinol on the immobilized XOD were determined as 0.25 mM and 23.2 μM, respectively. Furthermore, the PDA-modified hollow fiber-immobilized XOD was successfully applied to evaluate the inhibitory activity of eight flavonoids. Quercetin, apigenin, puerarin and epigallocatechin showed a good inhibition effect, and their percentages of inhibition were (79.86 ± 3.50)%, (80.98 ± 0.64)%, (61.15 ± 6.26)% and (54.92 ± 0.41)%, respectively. Finally, molecular docking analysis further verified that these four active compounds could bind to the amino acid residues in the XOD active site. In summary, the PDA-modified hollow fiber-immobilized XOD is an efficient method for the primary screening of XOD inhibitors from natural products.     

氧化石墨烯基质毛细管电色谱胰蛋白酶微反应器的性能研究

基于石墨烯优良的物化性能,利用层层组装法将氧化石墨烯修饰于石英毛细管内壁,制备了氧化石墨烯基质的毛细管电色谱,通过电渗流、拉曼光谱等对其进行表征。在此基础上,基于离子键合法将胰蛋白酶固定于毛细管电色谱柱头,制备胰蛋白酶微反应器。两者结合构成毛细管电色谱胰蛋白酶微反应器。实验结果显示,氧化石墨烯作为基质既可提高样品的分离效率,还能促进胰蛋白酶的催化性能。氧化石墨烯修饰的毛细管电色谱对N-苯甲酰-L-精氨酸乙酯盐酸盐(BAEE)和N-苯甲酰-L-精氨酸(BA)混合物的分离度从裸毛细管的3.70提升至4.71,而其固定化酶活性(米氏常数K_m=1.10 mmol/L,最大反应速率V_(max)=0.32 mmol·L~(-1)·s~(-1))也明显优于裸毛细管(K_m=109.77 mmol/L,V_(max)=0.000 46 mmol·L~(-1)·s~(-1))。利用所制备的微反应器从10种中药材中筛选胰蛋白酶抑制剂活性成分的药材,结果发现三七和大黄中均存在胰蛋白酶抑制剂活性成分。