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

项目总结/摘要 由Kir4.1（KCNJ10）和Kir5.1（KCNJ16）组成的异四聚体内向整流钾（Kir）通道 在调节钠，钾和水平衡的远曲小管（DCT）的关键作用， 肾小管KCNJ10的功能缺失突变导致EAST综合征，其特征是： 神经功能障碍（例如癫痫、共济失调和感觉神经性耳聋）和肾性盐耗。而遗传 这种方法对于理解肾脏Kir4.1/5.1的生理学是非常宝贵的， 用于探测这些通道的治疗潜力的药物仅限于非特异性抑制剂。的一部分 NIDDK资助的高通量筛选计划旨在开发有效和特异性的Kir4.1/5.1小分子- 作为分子调节剂，我们最近发现了第一个已知的Kir4.1/5.1的增效剂/活化剂。主要 我的F32奖学金的重点将是优化这些增效剂之一的效力和选择性， VU206，了解其分子作用机制，并测试其拯救选择EAST的功能的能力 突变通道在目标1中，将采用药物化学来创建VU 206的迭代类似物文库， 评估化学支架的官能团变化与效力之间的关系， 对Kir4.1/5.1的选择性。将使用已建立的定量铊通量测定来筛选VU 206， 针对Kir4.1/5.1和十个不同Kir通道的模拟库。具有最大效力的增效剂， 选择性将使用"金标准"手动膜片钳电生理学来确认。最后，VU206和/或 作为步骤，将针对天然大鼠远曲小管Kir4.1/5.1通道测试优化的增效剂 开发一种体内活性通道增强剂。在目标2中，我们将确定分子机制 关于VU206我将使用单通道分析来检验VU206增强Kir4.1/5.1的假设 通过增加的开态概率（Po）的活动，而不是增加单位电导。VU206的影响 对Po的开放将与打开螺旋束交叉（HBC）门相一致， 通过细胞内pH值的变化调节通道。接下来，我们将系统地评估 EAST门控突变R65 P、A167 V和R175 Q与细胞内pH和VU206的变化的关系，以确定 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 目的将导致一流的增效剂Kir4.1/5.1的发展，提供重要的新见解， Kir4.1/5.1渠道如何能够被开放，并有助于不断增长的"工具包"，以评估 Kir4.1/5.1通道在肾小管调节中的整合生理和治疗潜力 功能重要的是，这种奖学金的经验将准备我作为一个独立的成功的职业生涯 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.