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

描述：离子通道在体内的每一个细胞中都有表达，并参与多种重要的生理过程，包括快速的神经传递、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了离子通道相关的病理，也导致了许多离子通道靶向治疗。虽然离子通道在过去三十年中一直是密集研究的目标，但关于特定离子通道在正常生理过程和疾病中的作用仍有许多需要了解。我们对特定离子通道过程的作用缺乏了解的一个重要原因是缺乏针对这些通道的有效和选择性的药理学工具。G蛋白偶联的内向整流钾通道(GIRK)是近20年来一直是研究热点的离子通道家族的主要例子，被认为是从心房颤动到镇痛的许多适应症的潜在靶点。然而，只有一个非常强大的 和选择性GIRK抑制剂，没有有效和选择性的GIRK激动剂。GIRK由四个亚基(GIRK 1-4)的同源和异构体组合组成。这些亚单位在中枢神经系统和周围的许多组织中都有表达。最近，我们使用基于铊(Tl+)助熔剂的筛选发现了由GIRK1/2亚基组成的GIRK通道的第一个有效和选择性的激活剂。令人惊讶的是，到目前为止评估的~100个GIRK 1/2激活剂中，没有一个在含有GIRK的非GIRK 1(GIRK 2，GIRK 2/3)上显示任何活性。因此，我们建议进行高通量筛选以发现GIRK 2/3亚基组合的第一个选择性激活子。与含有GIRK 1的GIRK相比，GIRK 2和GIRK2/3亚基组合在中脑结构中的表达模式相对受限，如腹侧被盖区(VTA)和离散的亚细胞定位。尽管有越来越多的研究表明这些通道与奖赏和成瘾相关的回路有关，但由于缺乏选择性的药理学工具，对GIRK作用的大多数理解仍然不确定。在我们利用TL+助熔剂高通量筛选成功发现GIRK 1/2小分子调节剂的基础上，我们建议筛选160,000个样本化合物集合，并使用一套完善的验证性、作用机制和选择性筛选来鉴定HITS，这些筛选旨在识别具有亚基选择性GIRK探针的化合物。这些探针将立即有助于开始了解GIRK1和含有GIRK1的非GIRK1之间的作用机制和选择性。这些探针还将作为体内探针开发的候选对象，以便能够研究GIRK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，包括成瘾性疼痛和癫痫。

DESCRIPTION: 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 shows 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.