TREK-1通道的分子动力学行为研究及其配体的发现

发布时间：2018-03-08 12:03

本文选题：抗抑郁　切入点：TREK-1通道　出处：《中国科学院大学(中国科学院上海药物研究所)》2017年博士论文　论文类型：学位论文

【摘要】：当前抑郁症已严重的影响到了人们的生活。抑郁症的治疗方法包括药物治疗、心理治疗以及物理治疗等。70%的抑郁症患者经过抗抑郁药物治疗后都可缓解症状,因而高效安全的抗抑郁药物已成为药物研发的热点。TREK-1通道属于双孔钾离子通道(K2P)。TREK-1通道在人的海马、皮质、杏仁核等脑区大量存在,与单胺神经递质系统高度相关。抗抑郁动物模型和基因敲除研究表明抑制TREK-1通道可以治疗抑郁,TREK-1通道已成为新型抗抑郁药物设计的重要靶标。但是目前我们对TREK-1通道的失活机制以及与小分子的结合方式仍缺乏了解。分子动力学(Molecular dynamics,MD)模拟是一种研究蛋白质动态行为和功能关系的有效方法,它可在原子水平上模拟蛋白质的动态行为,提供构象变化的细节,为蛋白质的功能研究提供重要信息。本论文运用分子动力学模拟和分子对接方法研究了TREK-1通道的结构与功能,阐明了胞外p H调控TREK-1通道失活机制、小分子与TREK-1通道的胞外Cap区的结合方式以及TREK-1通道跨膜区域的动态行为。TREK-1通道是酸敏感性钾离子通道。TREK-1通道胞外侧loop上的残基His126是感应胞外酸环境的主要位点。为了研究胞外p H调控TREK-1通道的分子机制,我们利用MD技术观察了His126质子化和非质子化条件下,TREK-1通道在POPC膜中的动态行为差异。我们发现在这两种条件下,TREK-1通道的“选择性滤器”结构倾向于不同的构象。His126残基的质子化通过稳定“选择性滤器”结构周围的氢键网络而使TREK-1通道处于不传导状态。K2P通道的结构上的一个明显特征是胞外侧形成一个有四个螺旋结构组成的Cap区。一直以来有关K2P通道的Cap区的功能不是太清楚。在本论文中,我们通过以我们发现的TREK-1通道的抑制剂小分子TKDC为探针,结合计算,突变以及电生理等相关技术表明TREK-1通道的Cap区是小分子结合的潜在位点。分子动力学模拟发现小分子TKDC可以诱导Cap区的构象变化使TREK-1通道胞外的离子通路阻塞。尽管很多研究表明TREK家族通道的门控位点在“选择性滤器”结构上,但是TREK家族通道的跨膜区域对于调控通道的活性仍然很重要。目前的晶体结构揭示TREK家族通道的结构主要存在两种构象,Down构象和Up构象。突变,化学交联以及药理实验等相关研究表明Up构象有利于TREK家族通道维持激活状态。我们利用MD模拟揭示了TREK-1通道由Down状态转变成Up状态的过程”。通过对该转变过程的分析,我们预测TREK-1通道的一个亚基M4螺旋与另一个亚基的M2螺旋之间(Site1),以及同一个亚基上M2、M3以及M4的胞内侧螺旋之间(Site2)存在抑制剂结合位点。Site1位点已被TREK-2通道的晶体结构所证实。我们通过对Site2位点进行虚拟筛选,发现该位点也可以结合小分子抑制剂。
[Abstract]:At present, depression has a serious impact on people's lives. The treatment of depression includes medication, psychotherapy and physical therapy, and .70% of depression patients can relieve their symptoms after treatment with antidepressant drugs. Therefore, high efficient and safe antidepressants have become a hot spot in drug research and development. TREK-1 channel belongs to the double pore potassium channel. K2PU. TREK-1 channel is abundant in the hippocampus, cortex, amygdala and other brain regions of human beings, such as hippocampus, cortex, amygdala and so on. Antidepressant animal model and gene knockout study indicate that inhibiting TREK-1 channel can treat depression and TREK-1 channel has become an important target for the design of new antidepressant drugs. The mechanism of channel inactivation and the way of binding to small molecules are still not well understood. Molecular dynamics molecular dynamics (MD) simulation is an effective method to study the dynamic behavior and functional relationship of proteins. It can simulate the dynamic behavior of protein at atomic level, provide the details of conformation change, and provide important information for the study of protein function. In this paper, molecular dynamics simulation and molecular docking method are used to study the structure and function of TREK-1 channel. The mechanism of TREK-1 channel inactivation regulated by extracellular pH was elucidated. The binding mode of small molecules to the extracellular Cap region of TREK-1 channel and the dynamic behavior of the transmembrane region of TREK-1 channel. TREK-1 channel is the main site of acid-sensitive potassium channel. TREK-1 channel on the extracellular loop is the main site of acid-sensitive potassium channel. In order to study the molecular mechanism of TREK-1 channel regulated by extracellular pH, The dynamic behavior of TREK-1 channel in His126 membrane was observed by MD technique. We found that the "selective filter" structure of TREK-1 channel tended to be different conformation under these two conditions. His126. The protonation of the residue makes the TREK-1 channel unconductive by stabilizing the hydrogen bond network around the "selective filter" structure. An obvious feature of the structure of the K2P channel is the formation of a four-helix structure on the outside of the cell. Cap region. The function of Cap region of K2P channel is not very clear all the time. We used a small molecule of TKDC, an inhibitor of the TREK-1 channel, that we discovered as a probe, combined with calculations. Mutation and electrophysiological techniques showed that the Cap region of TREK-1 channel was the potential site for small molecule binding. Molecular dynamics simulation showed that small molecule TKDC could induce conformation change of Cap region and block the extracellular ion pathway of TREK-1 channel. Although many studies have shown that the gated site of the TREK family channel is in the "selective filter" structure, However, the transmembrane region of the TREK family channel is still very important for regulating the channel activity. The current crystal structure of the TREK family channel reveals that there are two main conformational conformations: down conformation and up conformation. Chemical crosslinking and pharmacological experiments have shown that up conformation is beneficial to maintain the activated state of TREK family channels. We use MD simulation to reveal the process of TREK-1 channel transition from Down state to up state. We predict the existence of an inhibitor binding site, Site1, between one subunit M4 helix of the TREK-1 channel and the M2 helix of another subunit, and the Site1 site on the same subunit between M2M3 and M4 medial helix. The Site1 site has been formed by the crystal junction of the TREK-2 channel. We do virtual screening of Site2 loci, It was also found that this site could bind to small molecular inhibitors.
【学位授予单位】：中国科学院大学(中国科学院上海药物研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R91

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