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

摘要 水肿性液体滞留引起的肿胀是一种常见的、危及生命的心力衰竭(HF)和 慢性肾病(CKD)。环状利尿剂通常被作为一线治疗药物来开出，以迅速减少 心衰和慢性肾脏病患者的细胞外液容量负荷。这类利尿剂通过抑制氯化钠起作用 在Henle‘s环的粗大升支(TAL)重吸收，并增加氯化钠和液体的输送到 远端肾单位由远端曲管(DCT)和集合管(CD)组成。为回应这一事件 增加了氯化钠和液体负荷，DCT和Cd通过上调 特定离子转运体和通道的表达。这种补偿机制减少了 环状利尿剂的有效性和引起环状利尿剂抵抗。越来越多的人达成共识 肾病学家认为，抑制DCT中钠(Na+)重吸收的远端作用利尿剂(即噻嗪 应在TAL下游使用利尿剂)或CD(保钾利尿剂)，以努力 克服循环利尿剂抵抗。然而，这两类利尿剂都有严重的局限性，这突显了 寻找更有效、更安全、新机制的远端利尿剂，以绕过环状利尿剂抵抗。 在这个应用中，我们打算发现第一个有效的和选择性的异构体Kir4.1/5.1的抑制剂 最近出现的钾通道，它是盐重吸收和激酶信号的关键调节因子 在远端肾单位。在目标1中，我们将使用一种完全有效的、基于荧光的铊通量分析来筛选 来自范德比尔特化学生物学研究所图书馆的大约11万种结构不同的化合物 针对在HEK-293细胞中异源表达的Kir4.1/5.1通道的新型抑制剂。一系列次要的 然后，铊通量分析以及高通量自动膜片钳电生理学将被用于 通过广泛的相关小组评估已确认的Kir4.1/5.1抑制剂的效力和选择性 内向整流钾(KIR)通道。在目标2中，我们将选择最有希望的Kir4.1/5.1抑制剂 对它们的效力、选择性、化学结构和体外代谢稳定性特性进行研究以开发类似物 图书馆使用最先进的药物化学技术，目标是优化药理 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 铅抑制剂对新鲜分离的肾小管中天然大鼠和人Kir4.1/5.1通道的作用。此外, 我们将验证这样的假设，即抑制Kir4.1/5.1会诱导大鼠肾脏排泄Na+，K+和水。 这些目标的完成将为评估治疗潜力提供急需的工具化合物 在心力衰竭和慢性肾脏病患者的环状利尿剂抵抗设置中，Kir4.1/5.1通道作为利尿剂靶点。

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.