【作者】 叶姗姗；

【导师】 陈枢青；

【作者基本信息】 浙江大学 ， 微生物与生化药学， 2008， 硕士

【摘要】 I孕烷X受体配体筛选模型的构建孕烷X受体（pregnane X receptor，PXR），核受体NR1I亚家族成员之一。PXR由位于N端的DNA结合域（DNA binding domain，DBD）和C端的配体结合域（ligand binding domain，LBD）组成。PXRLBD形成巨大、球状、可伸缩的配体结合口袋，能被大量外源性物质和内源性甾体活化。PXRLBD结合配体后，被活化而发生构象变化，招募辅调控因子（如人甾体受体辅活化因子-1，steroidreceptor coactivator-1，SRC-1）形成复合物，通过其DBD结合到药物代谢酶基因启动子的特定DNA序列上，从而调控CYP3A4等药物代谢酶基因的表达。外源药物通过充当配体结合并活化PXR，诱导药物代谢酶基因的表达，从而影响联合用药的代谢，导致临床上药物-药物相互作用。因此，PXR和药物的相互作用是药物代谢酶诱导作用和药物相互作用的重要分子基础之一。PXR与药物相互作用研究模型的构建和应用将有助于核受体介导的药酶诱导作用和药物代谢性相互作用的研究。本研究利用共表达载体pETDuet-1-SRC88-PXRLBD在Escherichia coli中共表达SRC88（SRC-1的88个氨基酸）和PXRLBD，获得了可溶性表达的PXRLBD，并根据PXR与配体的结合特性，采用平衡透析模型和高效液相色谱法，建立了体外研究PXR和药物相互作用的新方法，为体外筛选PXR的配体药物提供了新模型。一PXRLBD蛋白的表达1 pGEX-4T-1-PXRLBD Rosetta（DE3）表达菌株的构建PXRLBD片段通过BamHⅠ和XhoⅠ位点的介导插入pGEX-4T-1载体的多克隆位点。此亚克隆过程在Ecoli DH5α中完成，经限制性内切酶消化。确证和DNA序列测定确证后，转化表达宿主Ecoli Rosetta（DE3）。2 GST-PXRLBD蛋白的表达与鉴定500mL 2×YT培养基中，接种带有pGEX-4T-1-PXRLBD重组子的Rosetta（DE3），培养至OD₆₀₀为0．6左右，加IPTG（终浓度50μmol／L），20-25℃诱导培养10h。收集细菌，对细菌破碎液，细菌破碎液上清和细菌破碎液沉淀进行SDS-PAGE，结果表明GST-PXRLBD蛋白主要以不可溶的包涵体形式存在。构建新的表达菌株，利用SRC88与PXRLBD的共表达实现PXRLBD的可溶性表达。3 pETDuet-1-SRC88-PXRLBD Rosetta（DE3）表达菌株的构建SRC88片段通过NcoⅠ和HindⅢ位点的介导插入pETDuet-1载体的多克隆位点-1，PXRLBD片段通过NdeⅠ和XhoⅠ位点的介导插入pETDuet-1载体的多克隆位点-2（PXRLBD C-端设计His-tag以便于蛋白纯化）。此亚克隆过程在E．coli DH5α中完成，经限制性内切酶消化确证和DNA序列测定确证后，转化表达宿主E.coli Rosetta（DE3）。4 PXRLBD和SRC88蛋白共表达500mL含1％葡萄糖的LB培养基中，接种带有pETDuet-1-SRC88-PXRLBD重组子的Rosetta（DE3）。培养至OD₆₀₀为0．6左右，离心去除上清，用500 mL含50μmol／L IPTG的2×YT培养基重悬细菌沉淀，20-25℃诱导培养10h。5 PXRLBD蛋白的纯化与鉴定表达菌液离心去除上清培养液，用1×PBS重悬细菌沉淀物。细菌经Frenchpressure cell press破碎后，超声破碎，随即高速离心取上清用His Bind Resin纯化后获得纯化液8mL。纯化液进行SDS-PAGE，在分子量34KD附近出现明显的PXRLBD表达条带，在分子量10KD附近出现明显的SRC88表达条带。纯化液进行Western blot分析，一抗为人PXR101-260aa的多克隆抗体，在分子量34KD附近出现明显的PXRLBD表达条带。纯化液经BCA蛋白浓度测定试剂盒测得纯化液蛋白浓度为（0．411±0．005）g／L。二PXR配体筛选模型的构建将透析袋A和B放入盛有18mL含克霉唑（11．1μmol／L）的1×PBS缓冲液的试管中，透析袋C和D放入盛有18mL含地塞米松（11．1μmol／L）的1×PBS缓冲液的试管中，透析袋A和C内均加入2mL纯化的PXRLBD溶液，透析袋B和D内均加入2mL His·Bind Resin Elute Buffer作为对照组。4℃透析，A和B透析12h达到平衡，C和D透析24h达到平衡。分别取透析袋袋内外的溶液，其中A和C溶液加蛋白酶K（终浓度10μg／mL）4℃消化1h后加甲醇（1：1）沉淀蛋白，并用高效液相色谱法进行含量分析，B和D溶液不加蛋白酶K直接加甲醇（1：1）进行含量分析。透析平衡后，袋内游离药物浓度等于袋外游离药物浓度（即袋外药物总浓度），因此透析袋袋内与PXRLBD结合的药物浓度等于袋内药物总浓度减去袋内游离药物浓度即袋内药物总浓度减去袋外药物总浓度。以袋内药物峰面积与袋外药物峰面积的比值A作为表征，结果表明克霉唑组A=1．382±0．086，表明袋内药物总浓度高于袋外药物浓度，即PXRLBD与克霉唑发生结合，地塞米松组A=1．004±0．012，表明袋内药物总浓度等于袋外药物浓度，即PXRLBD与地塞米松结合作用不明显，模型建立成功。Ⅱ乙酰胆碱酯酶抑制剂筛选模型的构建乙酰胆碱酯酶（acetylcholinesterase，AChE）在神经肌肉接头和脑胆碱能突触水解神经递质乙酰胆碱，来终止神经递质对突触后膜的兴奋作用，保证神经信号的正常传递。乙酰胆碱酯酶抑制剂能够选择性抑制脑内乙酰胆碱酯酶的活性，延长乙酰胆碱在大脑内的存留时间，提高阿尔茨海默氏病（Alzheimer’s Disease，AD）病人的胆碱神经功能，减轻病人认知能力的下降。目前乙酰胆碱酯酶抑制剂已经成为治疗轻、中度阿尔茨海默氏病的主流产品。经FDA批准上市的乙酰胆碱酯酶抑制剂共5种：他克林（Tarcrine）、多奈哌齐（Donepezil，E2020）、利伐司替明（Rivastigmine）、加兰他敏（Galanthamine）和石杉碱甲（Huperzine A），但是已有的乙酰胆碱酯酶抑制剂还存在半衰期短、较严重的外周胆碱能系统副作用等缺点，它们的临床应用受到限制。因此，寻找作用时间长、副作用小的新一代乙酰胆碱酯酶抑制剂仍是目前AD药物研究的热点。本研究利用HEK293细胞表达重组人乙酰胆碱酯酶，通过酶动力学研究考察酶活性，建立乙酰胆碱酯酶抑制剂的筛选模型，并应用该模型测定一系列新化合物的抑制活性。一HEK293细胞表达重组人乙酰胆碱酯酶（rhAChE）pCMV-AChE由Hebrew University的Hermona Soreq教授赠送。pCMV-AChE经DNA序列测定确证AChE部分与AChE结构基因序列（GenBank Accession No：NM₀00665）一致。利用阳离子脂质体lipofectamine^TM2000介导pCMV-AChE瞬时转染HEK293细胞，rhAChE分泌表达至细胞培养液中，收集细胞培养液作为酶液分析AChE活性。二酶活性研究采用改进的Ellman方法：反应体系中加入酶液和显色剂DTNB（5,5’-二硫代双-2-二硝基苯甲酸），37℃孵育5min，加入底物ATCh（碘化硫代乙酰胆碱）37℃孵育一定时间，加入SDS终止反应，用酶标仪在412nm处测定吸光度（OD值），酶反应速率v用OD／min表示。1酶底物动力学研究反应速率对不同底物浓度作图得底物浓度曲线，通过底物浓度曲线确定反应体系中底物浓度条件。反应速率对不同酶量作图得酶量曲线，通过酶量曲线确定反应体系中酶量条件。反应速率对不同反应时间作图得反应时间曲线，通过反应时间曲线确定反应体系中孵育时间条件。米氏常数K_m测定的酶促反应条件：pH7．4，酶量4μL，底物浓度：0．075，0．1，0．125，0．175，0．25，0．3μmol／L，反应温度37℃，反应时间15min，酶反应速率v用OD／min表示，1／v对1／[S]作图获得Lineweaver-Burk图并回归分析得到K_m值为151．9μmol／L。2酶活力测定酶活力计算公式：U（μmol／min）=V×A／（ε×L）计算，A表示吸光度随时间的变化率（1／min），V表示反应总体积，消光系数ε=13．6L／mmol／mm，光程L=10mm。1．6mL反应体系：pH7．4，酶量32μL，底物浓度1．25mmol／L，反应温度37℃，在412nm处测定OD值，每隔0．5min读数，连续测定3min。反应测得A=0．05934／min，rhAChE酶活力为0．698mU（μmol／min）。3酶抑制作用动力学研究多奈哌齐是AChE的可逆性抑制剂，多奈哌齐对AChE的抑制作用为竞争性和非竞争性结合的混合类型，酶抑制常数K_i和K’_i通过二次作图法获得：不同抑制剂浓度下进行Lineweaver-Burk作图（一次作图）获得一系列曲线，一次作图各系列的斜率对[I]进行二次作图获得K_i，纵轴截距对[I]进行二次作图获得K’_i。反应体系条件：pH7．4，酶量4μL，多奈哌齐浓度：0，2．5，5．0，10，20，40，80nmol／L，底物浓度：0．065，0．125，0．25，0．5μmol／L，反应温度37℃，反应时间15min，通过二次作图法测得K_i=16．03nmol／L，K’_i=18．36nmol／L。三酶抑制剂筛选模型的建立和应用1多奈哌齐IC₅₀的测定多奈哌齐是AChE的可逆性抑制剂，文献报道IC₅₀为14．0nmol／L。选择7个浓度测定多奈哌齐对rhAChE的抑制率，以化合物摩尔浓度的负对数与酶抑制率进行线性回归求取IC₅₀值。反应体系条件：pH7．4，酶量4μL，多奈哌齐浓度：0，1．25，2．5，5．0，10．0，20．0，40．0，80．0nmol／L，底物浓度：0．125mmol／L，反应温度37℃，反应时间15min。结果测得多奈哌齐IC₅₀=14．0nmol／L。2新化合物IC₅₀的测定新化合物来源：多奈哌齐中的二甲氧基茚酮（与AChE外周位点结合）和利伐司替明的苄基胺（与AChE中心位点结合）用氧原子连接，并且通过苄基胺结构中胺烷基的位置、种类变化，设计并合成了一系列苯氧茚酮类衍生物2a-m，3a-m。每种化合物根据预试结果，选择6-8个浓度测定该化合物对rhAChE的抑制率，以化合物摩尔浓度的负对数与酶抑制率进行线性回归求取IC₅₀值。更多还原

【Abstract】 I Construction of pregnane X receptor ligand screening modelThe nuclear pregnane X receptor [PXR; NR112, also known as SXR and PAR] is a member of the nuclear receptor （NR） family of ligand-dependent transcriptional factors and a key regulator of genes involved in xenobiotic and endobiotic metabolism. PXR is composed of four functional modules including the modulator domain, the DNA-binding domain （DBD）, the hinge region, and the ligand-binding domain （LBD）. Elucidation of the three-dimensional structure of the PXR ligand binding domain reveals that it has a large, spherical ligand binding cavity that allows it to interact with a wide range of endogenous and exogenous chemicals. PXR binds with coregulatory proteins in the absence of ligands to DNA response elements in the regulatory regions of its target genes to modulate transcription of its target genes including cytochrome P450 3A monooxygenase genes and a number of other genes involved in the metabolism and elimination of xenobiotics from the body. The interaction between PXR and drug provides a molecular basis for the reported induction of drug metabolism enzyme gene expression and interactions between various prescription drugs.In this study, pETDuet-1-SRC88-PXRLBD expression plasmid was constructed and transformed into Escherichia coli Rosetta （DE3） to coexpress PXRLBD and SRC88, and equilibrium dialysis model was constructed to study the interaction between PXR and drugs, which may be used in the screening of PXR ligands in vitro.1. Expression of PXRLBD protein 1) construction of the expression vector pGEX-4T-1-PXRLBDThe expression vector pGEX-4T-1-PXRLBD was generated by PCR amplification and subcloning of nucleotides 390-1302 of the hPXR into the BamHⅠand XhoⅠsites of the pGEX-4T-1 expression vector. The expression vector was confirmed by electrophoretic analysis and sequencing.2) expression and identification of GST-PXRLBDThe pGEX-4T-1-PXRLBD plasmid was transformed into the Rosetta （DE3） strain of Escherichia coli and expressed with isopropyl-β-D-thiogalactopyranoside （50μmol/L） for 10h in shaker flasks at 20-25℃.Cell pellets were resuspended, lysed by French pressure cell press and sonication, and clarified by centrifugation. Total-cell lysated, soluble fraction of cell lysated and insoluble fraction of cell lysated were assessed by SDS-polyacrylamide gel electrophoresis （PAGE） with Coomassie blue staining. The result revealed the GST-PXRLBD was expressed as inclusion bodies.3) Construction of the expression vector pETDuet-1-SRC88-PXRLBDThe expression vector pETDuet-1-SRC88-PXRLBD was generated by insertion of nucleotides 1867-2130 of steroid receptor coactivator-1 （SRC88） by NcoⅠand HindⅢsites into the multiple cloning site 1 of pETDuet-1 expression vector, and nucleotides 388-1302 of hPXR with a polyhistidine-tagged C-terminal of PXRLBD by NdeⅠand XhoⅠsites into the multiple cloning site 2 of pETDuet-1 expression vector. The expression vector was confirmed by electrophoretic analysis and sequence analysis, and transformed into Escherichia coli Rosetta （DE3）.4) Coexpression of PXRLBD and SRC88pETDuet-1-SRC88-PXRLBD Rosetta （DE3） were grown in 500mL LB with 1% glucose to an OD₆₀₀ of 0.6 at 37℃. The cells were harvested and grown in 2×YT with 50μmol/L IPTG for 10h at 20-25℃.5) Purification and identification of PXRLBDCell pellets were resuspended, lysed by French pressure cell press and sonication, and clarified by centrifugation. The cleared supernatant was loaded onto His·Bind Resin. purified protein was eluted by 8mL elute buffer and then analysed by SDS-PAGE and Western blot. Two single protein bands of PXRLBD and SRC88 were detected on gel which revealed that SRC88 binded to PXRLBD as complex, and the concentration of purified protein was （0.411±0.005） g/L measured by BCA Protein Assay Kit.2. Construction of PXR ligand screening modelThe PXRLBD/SRC88 complex and drugs （clotrimazole or dexamethasone） were respectively added to the inside and outside of the dialysis bag. PXRLBD interacted with drugs infiltrating into bag filter. After dialysis equilibrium the buffer inside and outside of the dialysis bag was digested with 10μg/mL protease K for 1h, methanol was then added to precipitate protein and drug concentration was measured by HPLC.The HPLC data, that A （defined as ratio of drug peak area between the inside and outside of dialysis bag） of clotrimazole group equaled to 1.382±0.086 and A of dexamethasone group equaled to 1.004±0.012 indicated that clotrimazole binded to PXRLBD, while dexamethasone did not bind to PXRLBD, which coincided with the property of PXR. The equilibrium dialysis model for studying the interaction between PXR and drugs was established successfully and may be used in the screening of PXR ligands in vitro. ⅡConstruction of acetylcholinesterase inhibitor screening modelAcetylcholinesterase （AChE） hydrolyzes the neurotransmitter, acetylcholine at neuromuscular junctions and brain cholinergic synapses, and thus terminates signal transmission. AChE inhibitors can selectively inhibit the activity of AChE in the brain, which improved cholinergic nerve function of Alzheimer’s disease （Alzheimer’s Disease, AD） patients and reduced the decline in cognitive ability.FDA has approved five acetylcholinesterase inhibitors, including Tarcrine, Donepezil, Rivastigmine, Galanthamine and Huperzine A, which have been the main therapeutics of mild to moderate Alzheimer’s disease. However, the clinical application of AChE inhibitors were limited because of shortcomings such as short half-life, serious peripheral cholinergic system side effects, and scientists focus on a new generation of AChE inhibitors with small side-effect but more effective.In this study, recombinant human acetylcholinesterase （rhAChE） was expessed by HEK293 cells, enzyme kinetics of the rhAChE was investigated, and AChE inhibitors screening model was established and applicated to assay the inhibition activity of new compounds.1. rhAChE was expressed by HEK293 cellsPlasmid of pCMV-AChE was kindly provided by Prof. Hermona Soreq in Hebrew University, and the DNA sequence of AChE was confirmed to be correct （GenBank Accession No: NM₀00665）. HEK293 Cells were transfected with plasmid of pCMV-AChE, by utilizing the Cationic liposome lipofectamine^TM2000. rhAChE was secreted to cell culture medium and was collected for AChE activity assay.2. Assay of AChE Kinetic determinationsAChE activity was assayed according to modified Ellman method. The reaction mixture contained rhAChE, 5:5’-dithiobis （2- nitrobenzoic acid） （DTNB）, acetylthiocholine iodide （ATCh）. The assay was performed at 37℃and terminated by SDS. The reaction was monitored by recording the increase in absorbance at 412nm. Activity was monitored as OD/min.1) Kinetic parameters of the interaction between ATCh and rhAChE ATCh concentration was determined from the plot representing initial rhAChE enzyme velocity vs. ATCh concentration, rhAChE content was determined from the plot representing initial rhAChE enzyme velocity vs. rhAChE content, and incubation time was determined from the plot representing initial rhAChE enzyme velocity vs. incubation time.The reaction mixture contained rhAChE 4μL, 0.025%DTNB, different concentrations of ATCh （0.075, 0.1, 0.125, 0.175, 0.25, 0.3 mmol/L） and the assay was performed for 15 min at 37℃and terminated by SDS. K_m was calculated from the Lineweaver-Burk plot representing reciprocals of initial rhAChE enzyme velocity vs. reciprocal of ATCh concentration. K_m value is 151.9μmol/L.2) AChE activityThe reaction mixture contained rhAChE 32μL, 0.025%DTNB, 1.25mmol/L ATCh and the assay was performed at 37℃. The reaction was monitored by recording the increase in absorbance at 412nm every 0.5min for 6 times. The value of AChE activity is 0.698mU （μmol/min） according to the formula: U （μmol/min）=V×A/（ε×L）.3) Kinetic parameters of the interaction between Donepezil and rhAChEThe reversible noncovalent inhibitor of AChE Donepezil （E2020） was shown to inhibit AChE with high affinity in a mixed competitive-non-competitive way. K_i was calculated by the plot representing slope rate of Lineweaver-Burk plot vs. different concentrations of Donepezil and K’_i was calculated by the plot representing Y-axis intercept of Lineweaver-Burk plot vs. different concentrations of Donepezil.The reaction mixture contained rhAChE 4μL, 0.025%DTNB, different concentrations of Donepezil （0, 2.5, 5.0,10, 20,40, 80nmol/L）, different concentrations of ATCh （0.065, 0.125, 0.25, 0.5 mmol/L） and the assay was performed for 15min at 37℃. The value of K_i is 16.03nmol/L and K’_i is18.36nmol/L.3. Establishment and application of AChE inhibitor screening modelThe reaction mixture contained rhAChE 4μL, 0.025%DTNB, different concentrations of Donepezil （0,1.25,2.5, 5.0,10,20,40, 80nmol/L）, 0.125mmol/L ATCh and the assay was performed for 15min at 37℃. The value of IC₅₀14.0nmol/L coinsistent with the report was obtained through linear regression of inhibition ratio vs. different concentrations of Donepezil.Novel AChE inhibitors were designed and synthesized through the condensation of 2-Bromo-5,6-dimethoxy-indan-l-one with various aminoalkyl phenols. IC₅₀values of the rhAChE inhibitors were obtained and the AChE inhibition activity was compared.更多还原