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

• 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
• 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' 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

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 由水肿液保留引起的肿胀是一种常见的，威胁生命的心力衰竭症状（HF）和 慢性肾脏病（CKD）。循环利尿剂通常被处方为一线疗法，以快速减少 HF和CKD患者的细胞外流体体积负担。这类利尿作品通过抑制NaCl 在亨尔环的厚肢（tal）中重吸收，并增加NaCl和流体的递送 由远端曲折小管（DCT）和收集管道（CD）组成的远端肾单位。回应 NaCl和流体负荷增加，DCT和CD通过上调增加了NaCl的重吸收能力 特定离子转运蛋白和通道的表达。这种补偿机制减少了 循环利尿剂的有效性，并引起循环利尿剂抗性。越来越多的共识 肾病学家是抑制DCT中钠（Na+）重吸收的远端作用利尿剂（即噻嗪类 利尿剂）或cd（含钾的利尿剂）应在努力下进行。 克服循环利尿剂。但是，两个利尿类都有关键的局限性，突出了需求 发现更有效，更安全和新型的机制远端利尿剂，以绕过循环利尿剂耐药性。 在此应用中，我们建议发现异元Kir4.1/5.1的第一个有效和选择性抑制剂 钾通道最近出现为NaCl重吸收和激酶信号传导的关键调节剂 在远端肾单位中。在AIM 1中，我们将采用完全验证的，基于荧光的thallium-flux测定法 范德比尔特化学生物学图书馆的结构上大约有110,000种结构上的化合物 对于在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 然后将使用thallium-flux分析以及高通量自动化贴片夹电生理学，然后将用于 评估KIR4.1/5.1确认的抑制剂的效力和选择性，相关面板 内向整流钾（KIR）通道。在AIM 2中，我们将选择最有希望的Kir4.1/5.1抑制剂 关于它们的效力，选择性，化学结构和体外代谢稳定性，以发展模拟 使用最先进的药物化学技术的图书馆，目的是优化药理 体内给药抑制剂的特性。在AIM 3中，我们将使用单个通道分析测试活动 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， 我们将检验以下假设：抑制Kir4.1/5.1会诱导大鼠Na+，K+和水的肾脏排泄。 这些目标的完成将提供急需的工具化合物，以评估治疗潜力 在HF和CKD患者中，Kir4.1/5.1通道作为利尿剂利尿剂的利尿剂。