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学科主题: 生物化学与分子生物学
题名:
α7烟碱型乙酰胆碱受体激动剂和构象调节剂的筛选及生物活性评价研究
作者: 唐婧姝
答辩日期: 2015-04-21
导师: 李刚
专业: 生物化学与分子生物学
授予单位: 北京大学
授予地点: 北京大学深圳医院
学位: 硕士
关键词: α7 nAChR ; ACh ; 电生理 ; 激动剂 ; 构象调节剂 ; 突触传递 ; 学习认知
其他题名: Identification and evaluation of novel α7 nicotinic acethylcholine receptor agonists and positive allosteric modulators
分类号: R392
摘要:

    离子通道是众多疾病治疗药物作用的重要生物靶点,以离子通道为靶点的药物筛选是发现新药的重要途径之一。烟碱型乙酰胆碱受体α7亚型(α7 nAChR)是由五个相同亚基组成的配体门控离子通道,对钙离子有相当高的通透性,在介导神经递质释放和突触间快速信号传递中发挥重要的作用。α7 nAChR分布广泛,中枢神经系统中主要表达在大脑皮层、海马、杏仁核等与情绪和学习记忆相关的边缘系统,参与注意力、记忆力、认知功能和神经元保护等多种功能,与精神分裂症(Schizophrenia)、阿尔茨海默病(Alzheimer’s disease,AD)等的发病机制有着密切的关系。此外,近年来发现α7 nAChR在巨噬细胞、树突状细胞、淋巴细胞及内皮细胞等多种非神经元细胞上也有表达,参与迷走神经(vagus nerve stimulation, VNS)介导的“胆碱能抗炎通路”。因而,以α7 nAChR作为药物靶点,研发特异性的通道开放剂对于相关神经精神疾病、炎症反应的发病机制的理解及潜在治疗具有重要的意义。

    目前研究发现,能够增强α7受体功能的化合物主要分为两类:激动剂和正向构象调节剂 (positive allosteric modulator,PAM)。激动剂能够使α7通道开放,钙离子内流,引发一系列生物学效应;而正向构象调节剂则能增强内源性激动剂乙酰胆碱在α7受体上的效应,电生理方面表现为增强特异性电流或者延缓通道脱敏。

    本研究首先采用双电极电压钳(Two-Electrode Voltage Clamp, TEVC)电生理记录为基本筛选手段,筛选出能够增强α7受体功能的化学小分子调节剂;进一步通过电生理、分子生物学手段对筛选出的目标化合物进行相关体外生物学活性研究和初步体内活性研究,以期得到活性较好的靶向α7受体化合物用于深入探索α7 nAChR在神经精神疾病的发生发展中起到的作用,为精神分裂症、阿尔茨海默病的治疗提供候选化合物和新思路。

 

第一部分 α7 nAChR激动剂的筛选及代表性化合物Br-IQ17B的生物活性研究

    通过筛选近三十种不同母核的化合物,得到有激动活性新母核化合物两个,进一步评估其同系列类似物,发现激动剂Br-IQ17B具有高效激活α7通道的作用。结合双电极电压钳、全细胞膜片钳、脑片膜片钳等电生理技术,以及Western blot、钙荧光flexstation、放射性同位素标记的配体结合等分子生物学技术和Morris水迷宫手段对其进行体外、体内生物学效应评价研究,现得到以下结果和结论:

1. 利用双电极电压钳对化合物Br-IQ17B影响α7 nAChR电流的动力学进行了综合评估。 Br-IQ17B的EC50为1.8 μM,与文献报道的α7 nAChR激动剂的效价(potency)相比,属中等水平;Br-IQ17B引发的特征性电流可被阻断剂MLA拮抗、被正向构象调节剂PNU-120596放大、可浓度依赖性的引起α7 nAChR脱敏,故化合物Br-IQ17B符合α7 nAChR激动剂的动力学特征。

2. 放射性同位素配体结合实验表明Br-IQ17B与大鼠海马区内源性α7 nAChR有较强亲和力,Ki值为14.9 nM (n=3)。

3. 钙荧光Flexstation结果证实,Br-IQ17B可引起α7 稳转系GH3细胞钙内流。

4. 受体选择性实验显示,Br-IQ17B与α4β2 nAChR亚型无作用,但可抑制α3β4 nAChR,其IC50为381 μM (n=5)。

5. 全细胞膜片钳记录大鼠海马神经元发现,Br-IQ17B可激动神经元内源性α7 nAChR电流。

6. Western blot结果显示,Br-IQ17B可以增强PC12细胞的ERK1/2磷酸化水平。

7. 脑片膜片钳结果表明,Br-IQ17B可增强海马抑制性突触后电流(inhibitory post synaptic current, IPSC),增强GABA能突触传递。

8. Morris水迷宫行为学实验结果显示,Br-IQ17B可改善东莨宕碱对Wistar大鼠学习记忆的损害。

    综上所述,化合物Br-IQ17B是激动效应较高、选择性较好的α7 nAChR激动剂,可作用于内源性α7 nAChR,增强突触传递,改善东莨宕碱对Wistar大鼠学习记忆的损害。Br-IQ17B可作为研究α7通道结构功能、以及探索α7 nAChR与神经精神疾病关系的工具药,或进行全面的临床前动物实验,评估其作为临床候选药物的可能性,为治疗AD和精神分裂症患者认知缺陷提供新方案。

 

第二部分 α7 nAChR构象调节剂(allosteric modulator)的筛选和生物活性评估

    通过筛选近七十种不同母核的化合物,得到有构象调节活性新母核化合物三个,进一步评估其同系列类似物,发现LD486具有较高的增强内源性激动剂乙酰胆碱所引起电流幅度的能力。结合双电极电压钳、QPatch全自动膜片钳等电生理技术对LD486初步评估,现得到以下结果和结论:

1. 利用双电极电压钳对LD486调节α7 nAChR电流动力学的影响进行了综合评估。LD486可增强内源性激动剂ACh电流幅度3-4倍;导致ACh的量-效曲线左移,减小ACh的EC50;可延缓ACh对α7 nAChR的脱敏作用,加入化合物前后其τ值有显著性差异。

2. 受体选择性实验显示,LD486与α4β2 nAChR、α3β4 nAChR均无作用。

3. 全自动膜片钳结果表明,LD486对hERG钾通道无抑制作用。

4. 筛选以LD486为先导化合物的衍生物,通过结构改造与修饰获得两个活性较好的化合物JWX-1223 (增强ACh 16倍左右)、JWX-0108(增强ACh 20倍左右)。

    综上所述,化合物LD486是具有正向构象调节(positive allosteric modulation, PAM)作用、选择性较好的α7 nAChR的I型PAM,对hERG钾通道无作用,心脏安全性较好,以其作为先导物已发现活性更强的新结构,为靶向α7 nAChR构象调节剂的研发以及探讨PAM作为治疗神经精神类疾病认知缺陷的候选药物的可行性提供更多选择。

英文摘要:

    Ion channels have emerged as potential therapeutic targets for many diseases. Screening for compounds targeting specific ion channels is considered to be a promising approach for drug development. Nicotinic acetylcholine receptors (nAChRs) are pentameric integral membrane proteins belonging to a superfamily of Cys-loop ligand-gated ion channels, among which the homomeric α7 nAChR is characterized by its high calcium permeability and involved in numerous processes including modulation of neurotransmitter release and fast synaptic transmission between nerves. α7 nAChR subtype is highly expressed in critical brain regions associated with cognition, attention processing, and memory formation, particularly in the frontal cortex, hippocampus and amygdala. Disruption of α7 nAChR activity has been implicated in the pathophysiology of psychiatric and neurological conditions such as schizophrenia and Alzheimer’s disease. Moreover, in recent years, it has been proved that α7 nAChR is also widely expressed in dendritic cells, macrophages, lymphocytes, and endothelial cells, thus playing an important role in the “cholinergic anti-inflammatory pathway” mediated vagus nerve stimulation. Therefore, development of specific α7 nAChR openers may provide therapeutic potential for neuropsychiatric diseases and excessive inflammation, and promote our understanding of mechanisms underlying neuropsychiatric disorders.

    Nowadays, two types of compounds have been identified to enhance α7 nAChR function: agonist and positive allosteric modulator (PAM). Agonists of α7 nAChR can directly stimulate the target receptors, while PAMs modulate the activity of α7 nAChR by reinforcing the effect of endogenous agonist acetylcholine, resulting in augmented maximal agonist-evoked current or extended decay time of the evoked currents in the continuous presence of agonist, as determined from patch-clamp recordings. In the present study, we applied two-electrode voltage clamp (TEVC) as basic method to establish an electrophysiology platform for identification of α7 nAChR modulators. With the aid of multiple electrophysiology and molecular biology techniques, we evaluated in vitro and in vivo biological characterizations of selected compounds, hoping to identify compounds that can be served as a suitable tool for characterizing the role of α7 nAChR in CNS function, and provide new strategies to treat several neurological and psychiatric disorders

 

 

Part One: Identification and biological evaluation of selective α7 nAChR agonist

  To identify novel α7 agonists, we selected nearly 30 compounds with different core structure from virtual screening and identified two novel core structure with agonist activity. Further evaluation of their derivatives, one compound, named Br-IQ17B was finally identified as an ideal lead based on the criteria that met both the minimal EC50 and cutoff efficacy of 60%. Using combined TEVC, whole-cell patch clamp, brain slice electrophysiology, Western blot, Flexstation-3 and Radio-ligand binding assays, we characterized the Br-IQ17B compound. Based on the results obtained from this study, we have made the following conclusions:

1. Two-electrode voltage clamp recordings were used to assess the properties of Br-IQ17B on recombinant α7 nAChR expressed in oocytes. Br-IQ acted as a partial agonist, with an EC50 of 1.8 μM. Currents evoked by Br-IQ17B can be blocked by specific blocker MLA and augmented in the presence of positive allosteric modulator PNU-120596. Moreover, Br-IQ17B also induced α7 nAChR desensitization upon sustained exposure to low concentration. Therefore compound Br-IQ17B possesses a profile of α7 agonist characteristic.

2. Radio-ligand binding analysis revealed that Br-IQ17B was able to displace the binding of specific competitive antagonist [3H]-MLA to rat hippocampus α7 nAChR, yielding a Ki value of 14.9 nM (n=3).

3. Both in GH3 cells stably expressing human α7 nAChR, intracellular calcium measured by Flexstation3 was increased significantly after application of Br-IQ17B.

4. Selectivity evaluation by TEVC showed that Br-IQ17B displayed a much lower potency for other subtypes of nAChRs such as α4β2 and α3β4.

5. Whole-cell patch clamp recordings of hippocampus neurons indicated that Br-IQ17B was able to induce native characteristic α7 currents.

6. Western blot analysis indicated that Br-IQ17B enhanced ERK1/2 phosphorylation that was MLA-sensitive in PC12 cells.

7. In brain slice recordings, Br-IQ17B resulted in enhanced inhibitory postsynaptic current (IPSC) from CA1 pyramidal neurons and enhanced GABAergic synaptic transmission in hippocampus neurons.

8. In Morris water maze, Br-IQ17B counteracted (-)-scopolamine-induced deficit in acquisition of a water-maze learning task in Male Wistar rats.

  Taken together, our findings show that Br-IQ17B is a relatively potent and selective α7 nAChR agonist, which can activate native α7 nAChR, enhance synaptic transmission and ameliorate the cognitive and mnemonic deficits in rats. Br-IQ17B can be applied as a tool compound for investigating the role of α7 nAChR in neurodegenerative and psychiatric disorders with associated cognitive dysfunction. In addition, Br-IQ17B can be served as a candidate for further in vivo evaluation for potential utility of Br-IQ17B in preclinical study.

 

Part Two: Identification and biological evaluation of selective α7 nAChR PAM

  To identify novel α7 agonists, we selected nearly 70 compounds with different core structure from virtual screening and identified three novel compounds with allosteric modulator activity. Further evaluations of their derivatives, one compound, named LD486 was finally identified as an ideal lead. Using TEVC and automated patch clamp system QPatch-16, we studied the compound’s in vitro biological properties. Based on the results obtained from this study, we have made the following conclusions:

1. Two-electrode voltage clamp recordings of oocytes indicated that LD486 enhanced both the efficacy as well as the potency of ACh without activating the receptor on its own, and LD486 had marginal effects on desensitization.

2. Selectivity evaluation by TEVC showed that LD486 did not potentiate other subtypes of nAChRs such as α4β2 and α3β4.

3. Automated QPatch revealed that LD486 slightly inhibited hERG channel expressed in CHO stable cell line.

4. Screening derivations of LD486, we found that compounds JWX-1223 and JWX-0108 enhance amplitude of ACh currents (16 and 20 fold change respectively) on α7 nAChR.

  Taken together, LD486 is an efficacious and relatively selective modulator of α7 nAChR. LD486 possesses favorable cardiac safety and can be used for further investigation of its PK properties. In addition, two derivations of LD486 were identified with more potent efficacy to enhance ACh currents on oocytes, providing us more choices to testify the utility of allosteric modulation of α7 nAChR as a novel therapeutic principle for treating cognitive dysfunction associated with various forms of dementia and schizophrenia.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125375
Appears in Collections:北京大学深圳医院_学位论文

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作者单位: 北京大学深圳医院

Recommended Citation:
唐婧姝. α7烟碱型乙酰胆碱受体激动剂和构象调节剂的筛选及生物活性评价研究[D]. 北京大学深圳医院. 北京大学. 2015.
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