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

针对 Kir4.1/5.1 异聚钾通道的远端肾单位利尿剂的开发

基本信息

批准号：
10179370
负责人：
Jerod S. Denton
金额：
$ 37.49万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-15 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10179370
关键词：
Abdomen Alanine American Biological Assay Biology Cells Chemical Structure Chemicals Chronic Kidney Failure Consensus Development Distal Distal convoluted renal tubule structure Diuretics Duct (organ) structure Effectiveness Electrophysiology (science)Excretory function Exhibits Extracellular Fluid Female Fluorescence Genes Genetic Goals Heart failure Human In Vitro Institutes Ions Kidney Knockout Mice Lead Libraries Life Limb structure Liquid substance Literature Lung Lysine Metabolic Molecular Nephrons Oral Patients Pharmaceutical Chemistry Pharmacology Phenotype Phosphotransferases Physiology Play Potassium Potassium Channel Prevalence Proline Property Rattus Renal tubule structure Resistance Role Series Sesame - dietary Signal Pathway Signal Transduction Sodium Sodium Chloride Structure Structure of ascending limb of Henle&apos s loop Swelling Symptoms Techniques Testing Thallium Therapeutic Thiazide Diuretics Thick Validation Water Work analog base drug discovery high throughput screening in vivo inhibitor/antagonist loss of function mutation male novel patch clamp patient population response small molecule thiazide tool virtual wasting

项目摘要

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.

概括水肿性液体潴留引起的肿胀是心力衰竭 (HF) 和心力衰竭 (HF) 的常见且危及生命的症状慢性肾脏病（CKD）。袢利尿剂通常被用作一线治疗，以快速减少 HF 和 CKD 患者的细胞外液容量负担。此类利尿剂通过抑制 NaCl 起作用亨利氏袢粗升肢 (TAL) 的重吸收，并增加 NaCl 和液体的输送远端肾单位由远端曲管（DCT）和集合管（CD）组成。响应增加 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。这种补偿机制会削弱袢利尿剂的有效性并引起袢利尿剂抵抗。越来越多的共识肾病专家认为，远端作用的利尿剂可抑制 DCT 中钠 (Na+) 的重吸收（即噻嗪类药物）应在 TAL 下游施用利尿剂）或 CD（保钾利尿剂），以努力克服袢利尿阻力。然而，这两种利尿剂都有严重的局限性，凸显了需要发现更有效、更安全、机制新颖的远端利尿剂来规避袢利尿剂抵抗。在此应用中，我们计划发现第一个有效且选择性的异聚 Kir4.1/5.1 抑制剂钾通道，最近已成为 NaCl 重吸收和激酶信号传导的关键调节因子在远端肾单位。在目标 1 中，我们将采用经过充分验证的基于荧光的铊通量测定来筛选来自范德比尔特化学生物学研究所图书馆的约 110,000 种结构不同的化合物用于 HEK-293 细胞中异源表达的 Kir4.1/5.1 通道的新型抑制剂。二次系列然后，铊通量测定以及高通量自动膜片钳电生理学将用于评估已确认的 Kir4.1/5.1 抑制剂在广泛的相关组合中的效力和选择性内向整流钾 (Kir) 通道。在目标 2 中，我们将选择最有前途的 Kir4.1/5.1 抑制剂根据其效力、选择性、化学结构和体外代谢稳定性特性来开发类似物使用最先进的药物化学技术的图书馆，其目标是优化药理学用于体内给药的抑制剂的特性。在目标 3 中，我们将使用单通道分析来测试活动针对新鲜分离的肾小管中的天然大鼠和人 Kir4.1/5.1 通道的先导抑制剂。此外，我们将检验抑制 Kir4.1/5.1 会诱导大鼠肾排泄 Na+、K+ 和水的假设。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 Kir4.1/5.1 通道作为 HF 和 CKD 患者袢利尿抵抗的利尿靶点。