Urea transport inhibitors as a new class of diuretics

描述（由申请人提供）：肾脏产生浓缩尿液涉及一种逆流增殖机制，该机制由肾小管上皮细胞中的尿素转运蛋白（UT）-A型尿素转运蛋白促进，以及一种逆流交换机制，该机制由微血管（直血管）内皮中的UT-B促进。预计UT功能的丧失会破坏尿浓缩能力。我们建议将UT作为开发新型利尿剂的新靶点，我们称之为“利尿剂”，具有新的作用机制和独特的临床适应症。该提案的主要目标是提供 用于临床开发的药物样、经验证的UT抑制剂。其他可交付成果包括生成有效的UT选择性抑制剂作为研究工具，并使用这些工具，通过化学敲除在啮齿动物模型中生成关于肾UT生理学的新数据，认识到其优于基因敲除。在目标1中，一种新的高通量筛选将用于鉴定具有不同UT亚型选择性特征的UT抑制剂。这一目标来自于UT-B和UT-A1抑制剂检测开发和鉴定的大量初步数据。将针对每种主要的肾脏UT亚型UT-A1、UT-A2和UT-A3进行筛选，以鉴定用于研究结构-活性关系和选择性特征的活性化合物。在目标2中，将确定目标1中确定的化合物的UT抑制机制和药理学，以建立用于动物试验的化合物优先列表。目标化合物特性包括高UT抑制效力（低nM IC50）和良好的药理学特征。研究将包括：（a）使用细胞培养模型-抑制可逆性、动力学、侧性和尿素竞争;（B）通过计算化学-抑制效力和选择性的分子基础;和（c）使用大鼠-药代动力学、肾/尿蓄积和毒性。在目标3中，啮齿动物模型和UT抑制剂将用于表征UT在尿浓缩功能中的作用，并获得UT抑制剂治疗水肿的概念验证。UT抑制剂在体内的靶向作用包括增加尿排出量和降低尿浓缩能力，以及在临床相关的液体积聚状态下减轻水肿。在大鼠中进行的研究将包括在正常水合和脱水DDAVP期间测量化合物对尿量、渗透压摩尔浓度和尿素浓度以及血清尿素浓度的影响。还将在充血性心力衰竭水肿的大鼠模型中测试化合物。该提案的结果将包括药物样的，经验证的UT抑制剂，用作研究工具和临床开发，以及关于UT在尿浓缩机制中作用的新信息。

DESCRIPTION (provided by applicant): The generation of a concentrated urine by the kidney involves a countercurrent multiplication mechanism, which is facilitated by urea transporter (UT)-A-type urea transporters in tubule epithelial cells, and a countercurrent exchange mechanism, which is facilitated by UT-B in microvascular (vasa recta) endothelia. Loss of UT function is predicted to disrupt urinary concentrating ability. We propose UTs as novel targets for the development of a new type of diuretic, which we call 'urearetic', with a novel mechanism of action and a unique clinical indication profile. The primary goal of this proposal is to deliver drug-like, validated UT inhibitors for clinical development. Additional deliverables include the generation of potent UT-selective inhibitors as research tools, and, using these tools, to generate new data on renal UT physiology in rodent models by chemical knockout, recognizing its advantages over gene knockout. In Aim 1, a novel high-throughput screen will be used to identify UT inhibitors with different UT isoform selectivity profiles. This aim follows from extensive preliminary data on assay development and identification of UT-B and UT-A1 inhibitors. Screening against each of the major renal UT isoforms, UT-A1, UT-A2 and UT-A3 to identify active compounds for study structure-activity relationships and selectivity profiles will e conducted. In Aim 2, UT inhibition mechanisms and pharmacology of compounds identified in Aim 1 will be determined in order to establish a prioritized list of compounds for animal testing. Target compound properties include high UT inhibition potency (low nM IC50) and good pharmacological profile. Studies will include: (a) using cell culture models - inhibition reversibility, kinetics, sidedness and urea competition; (b) by computational chemistry - the molecular basis of inhibition potency and selectivity; and (c) using rats - pharmacokinetics, renal/urine accumulation and toxicity. In Aim 3, rodent models and UT inhibitors will be used to characterize the role of UTs in urinary concentrating function and to obtain proof-of-concept for UT inhibitor therapy of edema. Target effects of UT inhibitors in vivo include increasing urine output and reducing urinary concentrating ability, and reducing edema in clinically relevant states of fluid accumulation. Studies in rats will include measurements of compound effects on urine output, osmolality and urea concentration, and serum urea concentration, during normal hydration and with dehydration DDAVP. Compound(s) will also be tested in a rat model of edema in congestive heart failure. The outcomes of this proposal will include drug-like, validated UT inhibitors for use as research tools and for clinical development, and new information on the role of UTs in the urinary concentrating mechanism.