Mechanisms Underlying Small Molecule Regulation of K2P Channel Activity for the Treatment of Pain

通过小分子选择性上调K2 P通道活性代表了用于治疗疼痛的有吸引力的策略。学生将参与一项关于小分子和其他生理相关配体如何调节K2 P通道活性的主要研究。我们最近确定了两个原型的人类K2 P通道（TREK 1和TREK 2）在不同的构象和复杂的状态依赖性抑制剂的晶体结构。这为K2 P通道如何工作提供了重要的见解，现在提出了一个令人兴奋的实验框架，以解决有关K2 P通道门控和调节的分子机制的更复杂的问题。我们目前正在结合膜蛋白结构生物学（LCP和X射线自由电子激光）的一些最新进展，以确定TREK 1和TREK 2通道的新构象，以及它们与许多（溴化）小分子结合剂（抑制剂和激活剂）的复合结构。根据这些信息，学生将参与多学科方法：1）通过定点突变和功能分析来验证这些配体结合位点。2）剖析配体作用的结构和生物物理机制。3）通过合理设计/合成新配体来改善/调节配体功效。4）检查新配体在不同K2 P通道中的选择性，并测试它们在模型细胞系统（例如培养的大鼠DRG和/或人干细胞衍生的感觉神经元）中的功效。这些目标将涉及异源表达的WT和突变体通道的电生理学分析。这将使我们能够探测配体结合如何影响通道门控和调节，以及不同的激活剂如何影响状态依赖性阻断剂的抑制作用。目前的结果已经确定了结构上不同的开放状态，拟议的研究将使我们能够将这些不同的状态与药物作用机制相关联。我们还将利用与工业界的合作，通过合理设计和合成新型配体以及高通量筛选配体库来提高配体功效。还将通过研究配体对不同克隆的K2 P通道（例如TREK/TRAAK/TRESK）以及对模型细胞类型（例如DRG和hES神经元，其中主要K2 P通道被认为是TREK 2和TRESK）中天然K2 P通道电流的影响来检查配体功效和选择性。关于所涉及的潜在风险，我们认为，由于该项目是一个更大的既定和成功的研究计划的一部分，而且我们有大量已发表的初步证据证明所提出的方法的可行性，因此这些风险得到了缓解。

The selective up-regulation of K2P channel activity by small molecules represents an attractive strategy for the treatment of pain. The student will be part of a major study of how small molecules and other physiologically relevant ligands regulate K2P channel activity. We have recently determined the crystal structures of two prototypical human K2P channels (TREK1 and TREK2) in different conformations and in complex with a state-dependent inhibitor. This has provided important insights into how K2P channels work, and now presents an exciting experimental framework to address more complex questions about the molecular mechanisms of K2P channel gating and regulation. We are currently combining some of the most recent advances in membrane protein structural biology (LCP and X-Ray Free Electron Lasers) to identify novel conformations of TREK1 and TREK2 channels and also their structures in complex with a number of (brominated) small molecule binders (both inhibitors and activators). Based upon this information, the student will be involved in a multidisciplinary approach to: 1) Validate these ligand binding sites via site-directed mutagenesis and functional analysis.2) Dissect the structural and biophysical mechanisms underlying ligand action.3) Improve/modulate ligand efficacy through the rational design/synthesis of novel ligands.4) Examine the selectivity of novel ligands amongst different K2P channels, and test their efficacy in model cellular systems (e.g. cultured rat DRG and/or human stem cell-derived sensory neurons). These aims will involve electrophysiological analysis of heterologously expressed WT and mutant channels. This will allow us to probe how ligand binding affects channel gating and regulation, and how different activators influence inhibition by state-dependent blockers. Current results have identified structurally distinct open states and the proposed studies will allow us to correlate these different states with mechanisms of drug action. We will also exploit our collaborations with industry to improve ligand efficacy through the rational design and synthesis of novel ligands and high throughput-screening of ligand libraries. Ligand efficacy and selectivity will also be examined by studying their effects on different cloned K2P channels (e.g. TREK/TRAAK/TRESK), as well as on native K2P channel currents in model cell types e.g. DRG and hES neurons where the principal K2P channels are thought to be TREK2 and TRESK. With regards to potential risks involved, we believe they are mitigated by the fact this project forms part of a larger established and successful research programme, and because we have extensive published and preliminary evidence demonstrating the feasibility of the proposed methods.