Development of Distal Nephron Diuretics Targeting Kir4.1/5.1 Heteromeric Potassium Channels

总结 水肿性液体潴留引起的肿胀是心力衰竭（HF）常见的危及生命的症状， 慢性肾病（CKD）。袢利尿剂通常作为一线治疗处方，以快速减少 HF和CKD患者的细胞外液容量负荷。这类利尿剂通过抑制NaCl 重吸收在汉勒袢的粗升支（TAL），并增加输送NaCl和液体， 远侧肾单位由远曲小管（DCT）和集合管（CD）组成。响应于 增加NaCl和液体负荷，DCT和CD通过上调 特定离子转运蛋白和通道的表达。这种补偿机制减少了 环利尿剂的有效性，并引起环利尿剂抵抗。越来越多的共识 肾脏病学家认为，远端作用利尿剂抑制DCT中钠（Na+）重吸收（即噻嗪 应在TAL下游给予利尿剂）或CD（保钾利尿剂）， 克服袢利尿剂抵抗。然而，这两类利尿剂都有严重的局限性， 发现更有效、更安全、新机制的远端利尿剂，以规避袢利尿剂抵抗。 在本申请中，我们提出发现异聚Kir4.1/5.1的第一个有效的和选择性的抑制剂， 钾通道，最近出现的NaCl重吸收和激酶信号的关键调节 在远端肾单位在目标1中，我们将采用一种经过充分验证的基于荧光的铊通量测定法来筛选 来自范德比尔特化学生物学研究所图书馆的大约110，000种结构不同的化合物 在HEK-293细胞中异源表达的Kir4.1/5.1通道的新型抑制剂。一系列次级 然后，将使用铊通量测定以及高通量自动膜片钳电生理学， 在一个广泛的相关小组中评估Kir4.1/5.1确认抑制剂的效力和选择性 内向整流钾（Kir）通道。在目标2中，我们将根据药物组合选择最有前途的Kir4.1/5.1抑制剂。 其效力、选择性、化学结构和体外代谢稳定性，以开发类似物 图书馆使用最先进的药物化学技术，以优化药理学 用于体内给药的抑制剂的性质。在目标3中，我们将使用单通道分析来测试活动 在新鲜分离的肾小管中对天然大鼠和人Kir4.1/5.1通道的铅抑制剂。此外，本发明还提供了一种方法， 我们将检验抑制Kir4.1/5.1诱导大鼠肾脏排泄Na+、K+和水的假设。 这些目标的实现将为评估治疗潜力提供急需的工具化合物 Kir4.1/5.1通道作为HF和CKD患者袢利尿剂抵抗的利尿剂靶点。

SUMMARY Swelling caused by edematous fluid retention is a common, life-threatening symptom of heart failure (HF) and chronic kidney disease (CKD). Loop diuretics are often prescribed as a first-line therapy to quickly reduce the extracellular fluid volume burden in HF and CKD patients. This class of diuretic works by inhibiting NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop, and increases the delivery of NaCl and fluid to the distal nephron comprised of the distal convoluted tubule (DCT) and collecting duct (CD). In response to the increased NaCl and fluid load, the DCT and CD increase their NaCl reabsorbing capacity by upregulating the expression of specific ion transporters and channels. This compensatory mechanism diminishes the effectiveness of loop diuretics and gives rise to loop diuretic resistance. A growing consensus among nephrologists is that distally acting diuretics that inhibit sodium (Na+) reabsorption in the DCT (i.e. thiazide diuretics) or CD (potassium-sparing diuretics) downstream of the TAL should be administered in an effort to overcome loop diuretic resistance. However, both diuretic classes have critical limitations that highlight the need to discover more effective, safer, and novel-mechanism distal diuretics for circumventing loop diuretic resistance. In this application, we propose to discover the first potent and selective inhibitors of heteromeric Kir4.1/5.1 potassium channels, which have emerged recently as key regulators of NaCl reabsorption and kinase signaling in the distal nephron. In Aim 1, we will employ a fully validated, fluorescence-based thallium-flux assay to screen approximately 110,000 structurally diverse compounds from the Vanderbilt Institute of Chemical Biology library for novel inhibitors of Kir4.1/5.1 channels heterologously expressed in HEK-293 cells. A series of secondary thallium-flux assays, as well as high-throughput automated patch clamp electrophysiology, will then be used to evaluate the potency and selectivity of confirmed inhibitors for Kir4.1/5.1 over an extensive panel of related inward rectifier potassium (Kir) channels. In Aim 2, we will select the most promising Kir4.1/5.1 inhibitors based on their potency, selectivity, chemical structure, and in vitro metabolic stability properties to develop analog libraries using state-of-the-art medicinal chemistry techniques with the goal of optimizing the pharmacological properties of inhibitors for in vivo administration. In Aim 3, we will use single channel analysis to test the activity lead inhibitors against native rat and human Kir4.1/5.1 channels in freshly isolated kidney tubules. In addition, we will test the hypothesis that inhibition of Kir4.1/5.1 induces renal excretion of Na+, K+, and water in rats. Completion of these aims will provide critically needed tool compounds for evaluating the therapeutic potential of Kir4.1/5.1 channels as a diuretic target in the setting of loop diuretic resistance in HF and CKD patients.