新型M5胆碱受体选择性拮抗剂的发现及药理活性研究

目的:选择性拮抗M5胆碱受体可成为治疗药物成瘾的新途径,本文旨在利用虚拟筛选、结构优化、分子与细胞水平的药理活性评价,以期获得新型M5胆碱受体选择性拮抗剂。方法:通过虚拟筛选获得具有新型骨架的候选化合物,以该结构为基础进行结构优化;利用放射配基实验对系列化合物进行活性测定;进一步评价其亲和力对各胆碱受体亚型的选择性,并利用细胞内钙离子释放实验评价其拮抗活性。结果:通过虚拟筛选得到具有新型骨架的M5胆碱受体拮抗剂WXY-1-1,对其进行三轮结构优化共合成20个化合物;放射配基实验表明,其中化合物WXY-3-5对M5胆碱受体亚型具有亚微摩尔级亲和力(Ki=0.7μM)且具有亚型选择性(高出其他4个亚型9-35倍);钙流实验表明,化合物WXY-3-5对M5胆碱受体亚型具有拮抗活性(IC_(50)=6.1μM)。结论:通过虚拟筛选、结构优化以及药理活性评价,获得具有新骨架的M5胆碱受体选择性拮抗剂,为后续药物开发提供了新的先导结构。     

Aβ寡聚体调控M1胆碱受体下游信号转导通路的机制研究

摘要 目的：阿尔茨海默病（Alzheimer''s disease, AD）病理状态下M1胆碱受体功能受损,无法对配体做出正确响应。本研究模拟AD早期Aβ寡聚体富集的病理状态,考察对M1胆碱受体下游信号通路关键分子活性的影响,以期为AD病理状态下M1胆碱受体信号偶联障碍机制提供新的见解。方法：制备Aβ寡聚体（Aβ oligomers, AβOs）,1 μM AβOs预处理CHO-M1细胞30 min、24 h和48 h,加入胆碱受体激动剂乙酰胆碱或者卡巴胆碱激活M1胆碱受体,利用Ca²⁺检测实验、IP-One-Gq KIT检测实验及Western blot实验检测受体激动后下游关键信号分子Ca²⁺、IP1的释放量以及ERK磷酸化的动态变化。结果：AβOs预处理导致激活细胞内M1胆碱受体其下游Ca²⁺和IP1的释放量减少、ERK被更快激活。结论：AβOs预处理导致激活M1胆碱受体下游关键信号分子释放水平、磷酸化水平异常,揭示在阿尔茨海默病早期M1胆碱受体便出现功能障碍,无法正确激活信号转导通路。     

M1毒蕈碱乙酰胆碱受体同源建模模板的选取及验证

目的:探讨不同同源模板所获得M1毒蕈碱乙酰胆碱受体模型的合理性及可靠性。方法:以牛视紫红素受体、人源β2-肾上腺素受体、M2胆碱受体和M3胆碱受体为模板,分别对M1胆碱受体进行同源建模;采用分子对接获得各M1胆碱受体同源模板与配体的互作模式,并与已报道的M胆碱受体晶体结构进行静态比对,得到最佳M1胆碱受体同源模板;采用分子动力学模拟分析配体与关键残基距离的变化,对M1胆碱受体同源模板进行动态验证。结果:M2胆碱受体与M1胆碱受体的序列相似度较高,为67.9%;以Inactive M2胆碱受体为模板构建的M1胆碱受体(M1R_(inactive-M2R))与其他晶体结构间RMSD值的均值最低,为1.39;M1R_(inactive-M2R)别构位点K392及E397残基侧链与结合口袋距离更近,与配体结合构象更匹配;分子对接结果显示,双位点别构激动剂VU0184670与M1R_(inactive-M2R)别构结合位点Y85、Y381的距离分别为4.8、6.8,优于其他模型;分子动力学模拟后,配体与Q177残基的距离由7.4降至2.9,提示配体VU0184670向Q177方向偏转,与文献结果一致。结论:以Inactive M2受体结构为模板构建的M1胆碱受体模型最为合理,更接近M1胆碱受体的晶体结构。本研究为M1胆碱受体药物开发提供重要工具,为其他GPCRs受体同源建模提供创新范式。     

脂蛋白受体激动剂调节COPD小鼠炎症反应机制研究

目的:探究脂蛋白受体激动剂BML-111调节COPD小鼠炎症反应的机制。方法:构建COPD小鼠模型,通过HE染色检测小鼠肺组织和血管周围炎性细胞侵润程度;通过ELISA检测小鼠支气管肺泡灌洗液(BALF)中TGF-β、TNF-α、IL-1β和IL-10的含量;通过Western blot检测小鼠肺组织中NLRP3、Cleaved-IL-1β、Cleaved-caspase-1和Nrf-2的表达。结果:番红染色结果显示,与对照组相比,COPD模型组显示出严重的炎症反应,炎症细胞侵润程度增加,肺泡囊和间隙增大,支气管壁增厚。与模型组相比,低BML、高BML和Dex组的肺组织和血管周围的炎性细胞浸润程度明显降低(P0.05)。ELISA检测结果显示,COPD模型组中TGF-β、TNF-α、IL-10和IL-1β的表达均明显高于对照组(P0.05);在高BML组中,TGF-β、TNF-α、IL-10和IL-1β的表达明显低于COPD模型组中的表达(P0.05)。与COPD模型组相比,Dex组的TNF-α、IL-10和IL-1β的表达显著下调(P0.05),TGF-β的表达无显著差异(P0.05)。Western blot检测结果显示,与对照组相比,COPD模型组中NLRP3、cleaved-IL-1β和cleaved-caspase-1的表达显著上调(P0.05);与COPD模型组相比,低剂量BML组、高剂量BML组和Dex组中NLRP3、cleaved-IL-1β和cleaved-caspase-1的表达显著下调(P0.05)。活性检测结果显示,与对照组相比,COPD模型组的SOD活性显著降低(P0.05),MDA活性显著增强(P0.05)。BML-111处理后,与模型组相比,10 mg/kg的BML-111和2 mg/kg的Dex显著提高SOD活性,并显著降低MDA活性(P0.05)。与对照组相比,COPD模型组的Nrf-2的表达显著下调;而低剂量BML组,高剂量BML组和Dex组中Nrf-2的表达明显高于COPD模型组(P0.05)。结论:BML-111对COPD小鼠的抗炎作用可能是通过调节NLRP3炎症小体激活和ROS的产生来介导。     

低心血管疾病风险选择性环氧合酶-2抑制剂的虚拟筛选

摘要 目的：寻找具有血栓素A2受体（Thromboxane A2 receptor,TP）抑制作用的选择性环氧合酶-2（Cyclooxygenase-2,COX-2）抑制剂,以期降低其心血管疾病风险。方法：本研究从公开数据库中获取了512种TP抑制剂,通过分子对接、分子动力学模拟和ADMET预测,筛选出化合物TP84。结果：分子对接结果显示,与先前获批的选择性COX-2抑制剂罗非昔布相比,TP84对COX-2的亲和力更高,对环氧合酶-1（Cyclooxygenase-1,COX-1）的亲和力更低;分子动力学模拟进一步表明,模拟过程中TP84与COX-1的结合不稳定,而TP84能稳定结合COX-2,与COX-2的结合自由能是COX-1的3倍;此外,根据ADMET预测,TP84的药物化学、吸收、分布、代谢、排泄和毒性处于类药物候选物的可接受范围内。结论：TP84是一种潜在的低心血管疾病风险选择性COX-2抑制剂。     

选择性雌激素受体β激动剂对子宫腺肌病模型小鼠孕激素受体和胰岛素样生长因子-1的影响

摘要 目的：探讨选择性雌激素β受体（Estrogen Receptor beta,ERβ）激动剂对子宫腺肌病模型小鼠孕激素受体和胰岛素样生长因子-1的影响。方法：子宫腺肌病小鼠(n=30)随机平分为三组-模型组、孕三烯酮组与选择性ERβ激动剂组。模型组每日灌胃蒸馏水0.4 mL/20 g体重,孕三烯酮组每日灌胃孕三烯酮水溶液0.008 mg/20 g体重,选择性ERβ激动剂组每日灌胃WAY-32255水溶液0.008 mg/20 g体重,连续口服14 d。结果：孕三烯酮组与选择性ERβ激动剂组治疗第7 d与第14 d的小鼠体重高于模型组（P<0.05）,选择性ERβ激动剂组高于孕三烯酮组（P<0.05）。孕三烯酮组与选择性ERβ激动剂组治疗第7 d与第14 d的子宫病理评分低于模型组（P<0.05）,选择性ERβ激动剂组低于孕三烯酮组（P<0.05）。孕三烯酮组与选择性ERβ激动剂组治疗第7 d与第14 d的血清孕激素受体（Progesterone receptor,PR）和胰岛素样生长因子（insulin like growth factor,IGF-1）含量低于模型组（P<0.05）,选择性ERβ激动剂组低于孕三烯酮组（P<0.05）。孕三烯酮组与选择性ERβ激动剂组治疗第7 d与第14 d的子宫RhoA和ROCK蛋白相对表达水平低于模型组（P<0.05）,选择性ERβ激动剂组低于孕三烯酮组（P<0.05）。结论：选择性ERβ激动剂对子宫腺肌病模型小鼠的应用能抑制血清PR和IGF-1的释放,降低RhoA和ROCK蛋白的表达,从而能改善子宫病理状况,提高小鼠体重。     

人蛋白酶体亚基PSMB1的重组表达、纯化及在蛋白酶体抑制剂体外筛选中的应用

蛋白酶体是真核细胞中的一类多亚基蛋白酶复合物,它在胞内蛋白质降解的泛素-蛋白酶体通路中起关键作用。重组表达蛋白酶体的活性亚基可以用于在体外筛选、寻找具有蛋白酶体抑制剂作用的化合物。将人蛋白酶体催化亚基(PSMB1)cDNA的编码区(全长726 bp)克隆至原核表达载体pET28a(+),构建重组质粒pET28a-PSMB1,转化大肠杆菌BL21(DE3),通过1 mmol/L IPTG,20℃过夜诱导,获得相对分子量约为27 kDa的重组蛋白,采用IMAC亲和层析柱纯化重组蛋白,纯化后的重组蛋白纯度超过95%。重组蛋白酶解后经NanoLC-MS/MS鉴定表明所表达的融合蛋白氨基酸序列完全正确。在体外BIAcore分析中,重组蛋白表现出对不同化合物的选择性结合能力,其中与蛋白酶体抑制剂雷公藤红素的结合较强,10μmol/L的雷公藤红素与重组蛋白的结合达到27 RU,并且具有良好的浓度依赖型。本研究建立了表达、纯化人蛋白酶体催化亚基PSMB1的方法,并应用于具有蛋白酶体抑制活性化合物的体外筛选。     

BRCA1相关蛋白1对肾癌增殖和侵袭的实验研究

目的:研究BRCA1相关蛋白(BRCA1-associated protein-1,BAP1)对肾癌细胞增殖和侵袭的影响。方法:通过含有不同BAP1和HAT1载体的慢病毒感染后用嘌呤霉素筛选的方法在肾癌细胞系中构建BAP1稳定低表达、HAT1稳定高表达、HAT1稳定低表达以及BAP1低表达且HAT1低表达的慢病毒细胞稳转株。通过WB和PCR验证BAP1在肾癌细胞系中对于HAT1的调控。使用CCK-8细胞增殖和TRANSWELL侵袭实验检测对于肾癌细胞增殖和侵袭能力的影响。利用免疫组化和临床回访数据分析其临床意义。结果:肾癌细胞系中BAP1表达降低后HAT1表达升高。BAP1低表达部分通过了HAT1表达升高促进肾癌的增殖和侵袭能力。在肾癌组织中BAP1的表达与HAT1表达具有相关性(P0.05)。结论:BAP1敲低的肾癌细胞中HAT1特异性的高表达可能是导致肾癌细胞增殖和侵袭能力增强的原因。BAP1与HAT1在肾癌组织中的表达具有显著相关性。     

基于天然产物的蛋白酪氨酸磷酸酶1B 抑制剂的虚拟筛选

蛋白酪氨酸磷酸酶1B(protein tyrosine phosphatase 1B,PTP1B)是治疗Ⅱ型糖尿病的靶点之一,筛选PTP1B抑制剂具有十分重要的意义.本文采用分子对接虚拟筛选方法,构建共含有42 296个小分子的天然产物库,分别与PTP1B靶点蛋白进行分子对接,以原配体的结合能量为阈值,经过三轮筛选选取打分值高于阈值的小分子进行药代动力学参数和毒性参数预测,最终筛选出3个PTP1B抑制剂,对苯醌类化合物7、异香豆素类衍生物10和Clavepictine类似物11.结合方式研究表明,3个候选抑制剂类药性良好,均具有较好的PTP1B抑制活性,其中化合物10和11的PTP1B抑制活性未见报道.对化合物10进行体外抑制活性检测,其IC50为(74.58±1.23)μmol/L,可作为潜在Ⅱ型糖尿病治疗药物.     

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M₁ muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M₁ mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M₁ mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M₁ mAChRs, we show that once internalized, M₁ mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M₁ mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.     

M1乙酰胆碱受体对AMPA受体GluA1-Ser831 位点的调控作用

目的:从海马神经元谷氨酸离子型受体--AMPA受体亚基GluA1的831位丝氨酸(GluA1Ser831)磷酸化角度,探讨M1乙酰胆碱受体对AMPA受体GluA1亚基的调控作用及作用机制。方法:本研究以成熟的原代海马神经元为实验对象,用不易被降解的卡巴胆碱(Carbachol,CCh)作为胆碱受体激动剂,以免疫印迹法作为蛋白和磷酸化蛋白的主要检测手段,结合不同蛋白抑制剂研究M1受体调控AMPA受体GluA1亚基的关键信号分子及其机制。结果:1与对照组相比,CCh组Ser831的磷酸化水平显著升高。2CCh促进Ser831磷酸化的现象在M1受体选择性拮抗剂哌仑西平(Pirenzepine)+CCh组消失,CCh升高GluA1-Ser831磷酸化水平的作用由M1受体介导。3蛋白激酶C(ProteinkinaseC,PKC)抑制剂白屈菜红碱(Chelerythrinechloride,CHCL)能对抗CCh促进GluA1-Ser831位点磷酸化的作用,而钙/钙调素依赖性蛋白激酶II(Calcium/calmodulin-dependentkinaseII,CaMKII)抑制剂KN62不能对抗CCh的作用。4为检测体内GluA1-Ser831的磷酸化情况,用小鼠海马组织定位注射CCh和CHCL,CCh组小鼠海马组织GluA1-Ser831位点的磷酸化水平升高,CHCL能对抗这种作用,PKC介导了M1受体激活所导致的GluA1-Ser831磷酸化水平的升高。结论:M1受体通过激活PKC促进GluA1-Ser831的磷酸化。     

Elongation Factor 1A Family Regulates the Recycling of the M4 Muscarinic Acetylcholine Receptor

In this study, we tested the hypothesis that the elongation 1A (eEF1A) family regulates the cell surface density of the M4 subtype of the muscarinic acetylcholine receptors (mAChR) following agonist-induced internalization. Here, we show that mouse brains lacking eEF1A2 have no detectable changes in M4 expression or localization. We, however, did discover that eEF1A1, the other eEF1A isoform, is expressed in adult neurons contrary to previous reports. This novel finding suggested that the lack of change in M4 expression and distribution in brains lacking eEF1A2 might be due to compensatory effects of eEF1A1. Supporting this theory, we demonstrate that the overexpression of either eEF1A1 or eEF1A2 inhibits M4 recovery to the cell surface after agonist-induced internalization in PC12 cells. Furthermore, eEF1A1 or eEF1A2 had no effect on the recovery of the M1 subtype in PC12 cells. These results demonstrate the novel ability of the eEF1A family to specifically regulate the M4 mAChR.     

Structural Determinants of Allosteric Agonism and Modulation at the M4 Muscarinic Acetylcholine Receptor: IDENTIFICATION OF LIGAND-SPECIFIC AND GLOBAL ACTIVATION MECHANISMS*

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M₄ mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M₁/M₄ mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M₄ mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile⁹³ and Lys⁹⁵ as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe¹⁸⁶ in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp⁴³² in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr⁴³⁹ and Tyr⁴⁴³, were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Increased Insulin Secretion by Muscarinic M1 and M3 Receptor Function from Rat Pancreatic Islets in vitro

Parasympathetic system plays an important role in insulin secretion from the pancreas. Cholinergic effect on pancreatic beta cells exerts primarily through muscarinic receptors. In the present study we investigated the specific role of muscarinic M1 and M3 receptors in glucose induced insulin secretion from rat pancreatic islets in vitro. The involvement of muscarinic receptors was studied using the antagonist atropine. The role of muscarinic M1 and M3 receptor subtypes was studied using subtype specific antagonists. Acetylcholine agonist, carbachol, stimulated glucose induced insulin secretion at low concentrations (10⁻⁸–10⁻⁵ M) with a maximum stimulation at 10⁻⁷ M concentration. Carbachol-stimulated insulin secretion was inhibited by atropine confirming the role of muscarinic receptors in cholinergic induced insulin secretion. Both M1 and M3 receptor antagonists blocked insulin secretion induced by carbachol. The results show that M3 receptors are functionally more prominent at 20 mM glucose concentration when compared to M1 receptors. Our studies suggest that muscarinic M1 and M3 receptors function differentially regulate glucose induced insulin secretion, which has clinical significance in glucose homeostasis.     

In Vivo Striatal Dopamine Release by M1 Muscarinic Receptors Is Induced by Activation of Protein Kinase C

Islet-activating protein was unilaterally microinjected into rat striatum, and a dialysis cannula was implanted into the same area under anesthesia. After 2 days, various agents were perfused continuously into the striatum through the dialysis membrane, under freely moving conditions. Islet-activating protein (2 micrograms/2 microliters) treatment alone did not change in vivo striatal dopamine (DA) release and metabolism, but completely abolished the increase of striatal DA release evoked in vivo by the M1-selective agonist McN-A-343 (10(-7) M). Forskolin (10(-5) M), an adenylate cyclase activator, increased DA release and showed an additive effect on the DA release evoked by McN-A-343. Polymyxin B, a rather selective inhibitor of protein kinase C, decreased DA release and completely blocked the effect of McN-A-343. These results suggest that in vivo striatal DA release elicited by M1 muscarinic receptors is coupled with interaction with a Go protein and is induced by activation of protein kinase C.