与睡眠调节相关的三种G蛋白偶联受体探针对化合物活性的筛选研究

发布时间：2018-03-11 06:00

本文选题：失眠　切入点：G蛋白偶联受体　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：失眠(insomnia)是一种常见的睡眠障碍性疾病,在人群中患病率极高,不仅影响人们生活质量,而且与忧郁、焦虑等疾病密切相关。进一步发展能够引发多种代谢疾病,如:2型糖尿病。人体的睡眠由一系列受体调控,它们主要包括:GABAA受体、褪黑素受体(Melatonin受体)、五羟色胺受体(Serotonin受体)、食欲肽受体(Orexin受体)、组胺受体(Histamine受体)、腺苷受体(Adenosine受体)和多巴胺受体(Dopamine受体)。除GABAA受体外,其余的几种受体都属于G蛋白偶联受体。市售治疗失眠药物主要针对GABAA受体来发挥作用,因为GABAA受体在神经系统中弥散性分布,所以此类药物弥散性地抑制中枢神经系统并阻断了脑干网状结构上行激活系统的传导功能,从而使大脑皮质细胞由兴奋转为抑制。但是,此类药物有的会引起戒断综合症以及具有白天残留效应等不良影响。目前治疗失眠的新型药物比如褪黑素(MT1/MT2)受体激动剂、五羟色胺(5HT2A)受体调节剂和食欲肽(OXR)受体拮抗剂都是特异性的作用于G蛋白偶联受体(GPCR),不影响GABAA受体,在调节睡眠行为时副作用较少。本研究利用荧光共振能量转移的技术(fluorescence resonance energy transfer,FRET)构建以GPCR空间结构变化的分子探针以及诱导表达受体的方法,可以鉴定化合物是否对GPCR有特异性作用,从而筛选出可能具有镇静安神作用的化合物来进行药物开发。本课题选用五羟色胺5HT2A/1B受体、多巴胺D2/D3受体和食欲肽OX2受体三种与睡眠调节相关的G蛋白偶联受体进行研究。首先,构建多巴胺D2/D3受体表达载体和带有荧光基团的5HT2A/1B受体分子探针载体,为构建稳定细胞株做基础。然后,建立诱导表达多巴胺D2/D3受体和五羟色胺5HT2A/1B受体探针的稳定细胞株,从而构建出以多巴胺D2/D3受体和五羟色胺5HT2A/1B受体为预设靶点的药物筛选平台。最后,我们利用实验室已有的诱导表达食欲肽OX2受体的稳定细胞株对一系列化合物活性进行了筛选。研究结果表明,成功构建了以多巴胺D2/D3受体和五羟色胺5HT2A/1B受体为预设靶点的药物筛选平台,为治疗失眠药物的开发提供了理论支持。另外,利用诱导表达食欲肽OX2受体的稳定细胞株对化合物活性进行筛选,从一系列食欲肽受体拮抗性化合物(与中科院上海药物所合作)中筛选出了多种可能具有镇静安神作用的有效成分,为靶向食欲肽受体的治疗失眠药物研究提供了数据支持。
[Abstract]:Insomnia omniais is a kind of common sleep disorder disease, which has a high prevalence rate in the crowd. It not only affects people's quality of life, but also is closely related to depression, anxiety and other diseases. Further development can lead to a variety of metabolic diseases. For example, in type 2 diabetes, sleep in the body is regulated by a series of receptors, which mainly include the receptor: GABAA. Melatonin receptor, serotonin receptor, orexin receptor, histamine receptor histamine receptor, adenosine receptor, dopamine receptor, and dopamine receptor, with the exception of GABAA receptor, melatonin receptor, serotonin receptor, orexin receptor, histamine receptor, adenosine receptor and dopamine receptor. The rest of the receptors are G-protein-coupled receptors. Marketed insomnia drugs mainly target GABAA receptors, because GABAA receptors are distributed diffusely in the nervous system. So this kind of drug diffusely inhibits the central nervous system and blocks the conduction function of the upstream activation system of the brain stem reticular structure, which changes the brain cortex cells from excitement to inhibition. Some of these drugs have adverse effects such as withdrawal syndrome and daytime residual effects. New drugs for insomnia, such as melatonin 1 / MT2) receptor agonists, Both serotonin 5HT2A) receptor modulators and orexin OXR receptor antagonists are specific to the G protein-coupled receptor (GPC) receptor, and do not affect the GABAA receptor. There are few side effects in regulating sleep behavior. In this study, fluorescence resonance energy transfer technique was used to construct molecular probes with spatial structure changes of GPCR and to induce expression of receptors. It is possible to identify whether the compounds have specific effects on GPCR and to screen out compounds that may have calming and soothing effects for drug development. This study selected serotonin 5HT2A / 1B receptors. Dopamine D _ 2 / D _ 3 receptor and appetite peptide OX2 receptor were used to study three G protein-coupled receptors associated with sleep regulation. Firstly, the dopamine D _ 2 / D _ 3 receptor expression vector and the 5HT _ 2A / 1B receptor molecular probe vector with fluorescence group were constructed. In order to construct a stable cell line, a stable cell line was established to induce the expression of dopamine D 2 / D 3 receptor and serotonin 5 HT 2 A / 1B receptor probe. So we build a drug screening platform with dopamine D2 / D3 receptor and serotonin 5HT2A / 1B receptor as the preset target. A series of compounds were screened using stable cell lines that induce orexin OX2 receptor expression in our laboratory. A drug screening platform with dopamine D _ 2 / D _ 3 receptor and serotonin _ 5HT _ 2A / 1B receptor as preset target was successfully constructed, which provides theoretical support for the development of drugs for treating insomnia. The activity of the compound was screened by the stable cell line which induced the expression of orexin OX2 receptor. From a series of orexin receptor antagonistic compounds (in cooperation with Shanghai Institute of Pharmacology, Chinese Academy of Sciences), a variety of active components with calming and tranquilizing effect were selected, which provided data support for the study on the treatment of insomnia by targeting appetite peptide receptor.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R740

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