Development of Small-molecule Potentiators of Kir4.1/5.1 Potassium Channels

项目摘要/摘要 由Kir4.1(KCNJ10)和Kir5.1(KCNJ16)组成的异四聚体内向整流钾通道(KIR) 远曲小管在钠、钾和水平衡的调节中起关键作用 肾小管。KCNJ10功能缺失突变导致EAST综合征，其特征为 神经功能障碍(如癫痫、共济失调和感觉神经性耳聋)和肾脏盐耗。虽然遗传 方法对于理解肾脏Kir4.1/5.1的生理学非常有价值，Kir4.1/5.1是药理学工具 为了探索这些通道的治疗潜力，一直局限于非特异性抑制剂。作为计划的一部分 NIDDK资助的高通量筛查计划旨在开发有效和特异的Kir4.1/5.1小分子筛查 分子调节剂，我们最近发现了第一个已知的Kir4.1/5.1的增强子/激活子。少校 我的F32奖学金的重点将是优化其中一种增效剂的效力和选择性，称为 VU206，了解其分子作用机制，检测其挽救SELECT EAST功能的能力 变异频道。在目标1中，将使用药物化学来创建VU206的迭代模拟库 评价化学支架官能团的变化与效价的关系 对Kir4.1/5.1的选择性。建立的铊通量定量分析方法将用于筛查VU206和 针对Kir4.1/5.1和10个不同KIR通道的模拟库。最具效力的增效剂和 选择性将使用“金标准”手动膜片钳电生理学进行确认。最后，VU206和/或 优化的增强剂将在天然大鼠远端曲管Kir4.1/5.1通道上进行测试 开发一种体内活性的通道增强剂。在目标2中，我们将确定分子机制 VU206的行动。我将使用单通道分析来检验VU206增强Kir4.1/5.1的假设 通过增加的开态几率(Po)而不是增加的单位电导进行的活动。VU206的影响 On Po与螺旋束交叉(HBC)门的打开是一致的，这是一个公认的机制 通过细胞内pH的变化来调节通道。接下来，我们将对其敏感性进行系统评估。 EAST门控突变R65P、A167V和R175Q对细胞内pH和VU206的影响 通道功能障碍是否通过保守的分子机制发生。成功完成这些任务 AIMS将导致KIR4.1/5.1的一流增强剂的开发，为 如何从药理上打开Kir4.1/5.1通道，并为评估提供一个不断增长的“工具箱” Kir4.1/5.1通道在肾小管调节中的综合生理和治疗潜力 功能。重要的是，这一团契经历将为我作为一名独立人士的成功职业生涯做好准备 调查员。

PROJECT SUMMARY/ABSTRACT Heterotetrameric inward rectifier potassium (Kir) channels composed of Kir4.1 (KCNJ10) and Kir5.1 (KCNJ16) play key roles in regulation of sodium, potassium, and water balance by the distal convoluted tubule (DCT) of the renal tubule. Loss-of- function mutations in KCNJ10 lead to EAST syndrome, which is characterized by neurological dysfunction (e.g. epilepsy, ataxia, and sensorineural deafness) and renal salt wasting. While genetic approaches have been invaluable for understanding the physiology of renal Kir4.1/5.1, the pharmacological tools for probing the therapeutic potential of these channels have been limited to non-specific inhibitors. As part of a NIDDK-funded high-throughput screening program aimed at developing potent and specific Kir4.1/5.1 small- molecule modulators, we have recently discovered the first known potentiators/activators of Kir4.1/5.1. The major focus of my F32 fellowship will be to optimize the potency and selectivity of one of these potentiators, termed VU206, to understand its molecular mechanism of action and test its ability to rescue the function of select EAST mutant channels. In Aim 1, medicinal chemistry will be employed to create iterative analog libraries of VU206 to evaluate the relationship between changes on the functional groups of the chemical scaffold and potency and selectivity toward Kir4.1/5.1. An established quantitative thallium flux assay will be used to screen VU206 and analog libraries against Kir4.1/5.1 and ten different Kir channels. Potentiators with the greatest potency and selectivity will be confirmed using “gold standard” manual patch clamp electrophysiology. Finally, VU206 and/or optimized potentiators will be tested against native rat distal convoluted tubule Kir4.1/5.1 channels as a step towards developing an in vivo-active channel potentiator. In Aim 2, we will determine the molecular mechanism of action of VU206. I will use single channel analysis to test the hypothesis that VU206 potentiates Kir4.1/5.1 activity via increased open-state probability (Po), as opposed to increased unitary conductance. Effects of VU206 on Po would be consistent with opening of the helix-bundle crossing (HBC) gate, a well-established mechanism for regulating the channel by changes in intracellular pH. Next, we will systematically evaluate the sensitivity of EAST gating mutations, R65P, A167V, and R175Q, to changes in intracellular pH and VU206 to determine whether channel dysfunction occurs via a conserved molecular mechanism. Successful completion of these Aims will lead to the development of first-in-class potentiators of Kir4.1/5.1, provide important new insights into how Kir4.1/5.1 channels can be opened pharmacologically, and contribute to a growing “toolkit” for evaluating the integrative physiology and therapeutic potential of Kir4.1/5.1 channels in the regulation of renal tubule function. Importantly, this fellowship experience will prepare me for a successful career as an independent investigator.