An HTS to Discover Novel Modulators of the GIRK 2/3 Potassium Channel

项目摘要 离子通道在体内的每个细胞中都有表达，并且参与了关键和多种生理过程。 这些过程包括快速神经传递、建立和维持心律以及溶质平衡。 离子通道功能的重要性和多样性不仅导致离子通道相关的病理，而且还导致 一系列离子通道靶向疗法。虽然离子通道一直是深入研究的目标 在过去的三十年里，关于特定离子通道在细胞内的作用，还有很多东西有待了解。 正常的生理过程和疾病。一个重要的原因，我们缺乏了解的作用， 特异性离子通道过程是缺乏有效的和选择性的药理学工具靶向这些 渠道G蛋白偶联内向整流钾离子通道（GIRK）是钾离子通道的主要例子。 近二十年来，离子通道家族一直是研究的焦点，被认为是潜在的 从房颤到镇痛的多种适应症的靶点。然而，只有一个高度 有效的和选择性的GIRK抑制剂和没有有效的和选择性的GIRK激活剂。 GIRK由四个亚基（GIRK 1-4）的同源和异源组合组成。这些亚单位是 在CNS和外周的许多组织中表达。最近，我们使用了铊（Tl+）助熔剂- 基于筛选，以发现由GIRK 1/2组成的GIRK通道的第一个有效和选择性激活剂 亚单位。令人惊讶的是，迄今为止评估的约100种GIRK 1/2激活剂中没有一种在非GIRK激活剂中显示出任何活性。 1个含有GIRK（GIRK 2，GIRK 2/3）。因此，我们建议进行高通量筛选，以发现 GIRK 2/3亚基组合的第一选择性激活剂。GIRK 2和GIRK 2/3亚基组合 在中脑结构如腹侧被盖区（VTA）中具有相对受限的表达模式 和离散的亚细胞定位相比，含有GIRK 1的GIRK。虽然有建筑研究 这暗示了这些通道与奖赏和成瘾相关的回路，大多数对GIRK作用的理解 由于缺乏选择性的药理学工具，仍然不确定。在我们成功发现 GIRK 1/2的小分子调节剂，使用基于Tl+通量的高通量筛选，我们建议 筛选160，000个样品化合物集合，并使用一套完善的 验证性、作用机制和选择性筛选，旨在鉴定具有以下能力的化合物： 作为亚基选择性GIRK探针开发。这些探测器将立即用于开始 了解GIRK 1和不含GIRK 1的GIRK之间的作用机制和选择性。这些 探针也将作为体内探针开发的候选者，以使GIRK的研究成为可能。 在生理学中的作用，并探索其对各种重要适应症的治疗潜力，包括 成瘾、疼痛和癫痫

Project Summary Ion channels are expressed in every cell in the body and are involved in critical and diverse physiological processes including fast neurotransmission, establishing and maintaining cardiac rhythms, and solute balance. The importance and diversity of ion channel function results both in ion channel-related pathologies but also in a number of ion channel-targeted therapies. Though ion channels have been the targets of intense research over the last three decades, much remains to be understood regarding the roles of specific ion channels in normal physiological processes and disease. A substantial reason for our lack of understanding of the roles of specific ion channels process is the lack of potent and selectively pharmacological tools targeting these channels. The G-protein Coupled Inward Rectifying Potassium K+ Channels (GIRK) are prime examples of an ion channel family that has been the focus of research for nearly two decades and are thought to be potential targets for numerous indications spanning from atrial fibrillation to analgesia. However, there is but one highly potent and selective GIRK inhibitor and no potent and selective GIRK activators. GIRKs are comprised of homo and heteromeric combinations of four subunits (GIRK 1-4). These subunits are expressed in numerous tissues in the CNS and the periphery. Very recently we have used thallium (Tl+) flux- based screening to discover the first potent and selectively activators of a GIRK channel comprised of GIRK1/2 subunits. Surprisingly, none of the ~ 100 GIRK 1/2 activators evaluated thus far show any activity at non-GIRK 1 containing GIRKs (GIRK 2, GIRK 2/3). Thus, we propose to perform a high-throughput screen to discover the first selective activators of the GIRK 2/3 subunit combination. The GIRK 2 and GIRK2/3 subunit combinations have a relatively restricted expression pattern in mid-brain structures such as the ventral tegmental area (VTA) and discrete subcellular localization compared to GIRK 1-containing GIRKs. Though there is building research that implicates these channels in reward and addiction-related circuitry, most of understanding of GIRKs role remains uncertain due to the lack of selective pharmacological tools. Building upon our success in discovering small molecule modulators of GIRK 1/2 using Tl+ flux-based high-throughput screening, we are proposing to screen a 160,000 sample compound collection and characterize hits using a well-developed set of confirmatory, mechanism of action, and selectivity screens designed to identify compounds with the capacity to be developed as subunit-selective GIRK probes. These probes will be immediately useful to begin understanding mechanisms of action and selectivity between GIRK 1 and non-GIRK1 containing GIRKs. These probes will also advance as candidates for in vivo probe development to enable the investigation of GIRK's roles in physiology and exploration of its therapeutic potential for a variety of important indications including addiction, pain, and epilepsy.