Preclinical validation of Kir4.1/5.1 inhibitors for overcoming diuretic resistance

摘要 水肿是充血性心力衰竭(HF)和慢性肾脏疾病的一种常见的、危及生命的后果 慢性肾脏病(CKD)和慢性肝病。环状利尿剂经常被用作快速减少液体的一线治疗方法。 心衰、慢性肾脏病和肝功能衰竭患者的容量负荷。这类利尿剂通过抑制氯化钠重吸收发挥作用 在Henle‘s环的粗大升支(TAL)和增加氯化钠向远曲的输送 肾小管(DCT)和皮质集合管(CCD)组成远端肾单位。为了应对不断增加的氯化钠 负荷后，DCT和Ccd通过远端小管重塑增加其对氯化钠的重吸收能力 细胞/组织肥大和上调涉及盐的离子转运体和通道的表达 重吸收。这种代偿机制降低了环状利尿剂的有效性，并导致环状 利尿剂抵抗，这是治疗心力衰竭、慢性肾脏病和肝功能衰竭的常见临床问题。一个不断增长的 普遍认为，抑制DCT中钠(Na+)重吸收的远端作用利尿剂(即噻嗪利尿剂) 或在TAL下游给予阿米洛利，以克服环状利尿剂抵抗。 然而，这两类利尿剂都有严重的责任，这突显了需要更有效、更安全和更新颖的- 机制远端肾单位靶向利尿剂。异构体Kir4.1/5.1内向整流钾(KIR)通道 在过去的十年中已经成为潜在的远端肾单位利尿剂靶点，主要有两个原因。首先，这些 基底侧膜通道在DCT和Ccd中表达，对Na+的重吸收是必不可少的。 两个肾单位节段。其次，也是重要的是，慢性阻塞性肺疾病患者Kir4.1/5.1功能的丧失 芝麻/EAST综合征导致肾脏盐耗和低血压，提供强大的遗传 确认Kir4.1/5.1为人类利尿剂靶点。我们最近做了一个由美国国立卫生研究院资助的 (R01DK120821；Denton Pi)高通量筛选(HTS)来自Vanderbilt Institute of 化学生物学发现收集并鉴定了数百种结构多样的小分子抑制剂 Kir4.1/5.1。我们使用迭代药物化学和功能分析来推动 一流的，中等效力(IC50=0.24微米)，高选择性(9个相关KIR通道的30倍)，体外 Kir4.1/5.1的抑制剂，命名为VU6036720(PMID 35246480)。然而，不幸的是，VU6036720未能 由于高血浆蛋白结合率和快速代谢清除，在小鼠中诱导利尿反应。在目标1中 对于这一后续应用，我们建议使用药物化学和已建立的功能分析来 进一步优化药物的效力、选择性以及药物代谢和药代动力学(DMPK)特性 VU6036720和我们HTS活动中确定的其他备用脚手架。在目标2中，我们将在体内评估 优化抑制剂在利尿剂抵抗和慢性肾脏病小鼠模型中的活性。完成这项计划将 一般情况下，提供Kir4.1/5.1作为可行利尿剂靶点的临床前验证，并用于规避 尤其是与利尿剂抵抗相关的并发症。

SUMMARY Edema is a common, life-threatening consequence of congestive heart failure (HF), chronic kidney disease (CKD), and chronic liver disease. Loop diuretics are often used as a first-line therapy to quickly reduce the fluid volume burden in HF, CKD, and liver failure patients. This class of diuretic works by inhibiting NaCl reabsorption in the thick ascending limb (TAL) of Henle’s loop and increasing the delivery of NaCl to the distal convoluted tubule (DCT) and cortical collecting duct (CCD) comprising the distal nephron. In response to the increased NaCl load, the DCT and CCD increase their NaCl reabsorption capacity through distal tubule remodeling involving cell/tissue hypertrophy and by upregulating the expression of ion transporters and channels involved in NaCl reabsorption. This compensatory mechanism diminishes the effectiveness of loop diuretics and gives rise to loop diuretic resistance, which is a common clinical problem in the treatment of HF, CKD, and liver failure. A growing consensus is that distally acting diuretics that inhibit sodium (Na+) reabsorption in the DCT (i.e., thiazide diuretics) or CCD (i.e., amiloride) downstream of the TAL should be administered to overcome loop diuretic resistance. However, both diuretic classes have serious liabilities that highlight the need for more effective, safer, and novel- mechanism distal nephron-targeted diuretics. Heteromeric Kir4.1/5.1 inward rectifier potassium (Kir) channels have emerged over the last decade as potential distal nephron diuretic targets for two main reasons. First, these basolateral membrane channels are expressed in the DCT and CCD and are essential for Na+ reabsorption in both nephron segments. Secondly, and importantly, the loss of Kir4.1/5.1 function in patients with SeSAME/EAST syndrome leads to renal salt wasting and low blood pressure, providing strong genetic validation for Kir4.1/5.1 as a diuretic target in humans. We recently performed an NIH-funded (R01DK120821; Denton PI) high-throughput screen (HTS) of 80,475 compounds from the Vanderbilt Institute of Chemical Biology Discovery Collection and identified hundreds of structurally diverse small-molecule inhibitors of Kir4.1/5.1. We employed iterative medicinal chemistry and functional analysis to drive the development of the first-in-class, moderately potent (IC50 = 0.24 µM), highly selective (>30 fold over 9 related Kir channels), in vitro inhibitor of Kir4.1/5.1, named VU6036720 (PMID 35246480). Unfortunately, however, VU6036720 failed to induce a diuretic response in mice due to high plasma protein binding and rapid metabolic clearance. In Aim 1 of this follow-up application, we propose to employ medicinal chemistry and established functional assays to further optimize the potency, selectivity, and drug metabolism and pharmacokinetic (DMPK) properties of VU6036720 and other backup scaffolds identified in our HTS campaign. In Aim 2, we will evaluate the in vivo activity of optimized inhibitors in mouse models of diuretic resistance and CKD. Completion of this program will provide pre-clinical validation of Kir4.1/5.1 as a viable diuretic target, generally, and for circumventing complications associated with diuretic resistance, specifically.