Molecular pharmacology and physiology of kidney potassium transport

描述（由申请人提供）：抑制整流钾（Kir）通道是多种生理过程的关键调节剂，可能代表疾病的新型药物靶点。然而，由于缺乏靶向内向整流器的药物样化合物，它们的治疗潜力尚未被直接测试。缺乏选择性“探针”也阻碍了确定某些Kir通道的生理功能的努力。为了克服这一巨大的障碍并为研究内向整流生理学创造新的机会，研究人员对ROMK（Kir1.1）的小分子调节剂进行了20多万种化合物的高通量筛选（HTS），ROMK是一类新型利尿剂的推定靶点。一种名为VU 590的化合物在纳摩尔浓度下抑制ROMK，在低微摩尔范围内抑制Kir7.1，使其成为这两种通道的第一种小分子抑制剂。研究人员继续使用药物化学来合理设计一种称为VU 591的纳摩尔亲和探针，该探针对ROMK具有高度选择性，超过60种潜在的脱靶点，包括内向整流器和BK通道。在目标1中，研究人员将采用最先进的分子建模技术，原子结构指导的诱变和电生理学来确定ROMK和Kir7.1中的VU 590/591结合位点。VU 591对ROMK具有显著的选择性，因此代表了进一步开发用于动物研究的有希望的候选者。在目标2中，研究人员将首先通过评估VU 591在低流量和高流量条件下对分离的灌注皮质集合管中K和Na转运的影响来确定VU 591是否在天然组织中具有活性。研究人员还发现了一种G蛋白调节的内向整流器（GIRK）的纳摩尔亲和力抑制剂，这是房颤的一个假定治疗靶点。在目标3中，研究人员将使用药物化学，结构导向诱变和电生理学来定义这种称为VU 592的新型化合物的分子结合位点。这些研究将为内向整流器的原子结构提供重要的新见解，并产生急需的探针，以确定这些通道的综合生理学和治疗潜力。敷设总结：研究人员将结合联合收割机药物化学，先进的计算技术和经典的生理学方法，开发针对钾通道的药物样化合物，这些化合物可能成为高血压，水肿和心律失常的治疗靶点。 公共卫生相关性：内向整流钾通道在多种细胞功能中起着重要的生理作用，并可能成为新的药物靶点。然而，缺乏选择性药理学“探针”阻碍了探索大多数Kir通道的整合生理学和治疗潜力的努力。本文拟利用药物化学、原子结构导向突变、肾小管微灌流和电生理学等方法，开展Kir1.1、Kir7.1和Kir 3通道的小分子药理学研究。

DESCRIPTION (provided by applicant): Inwardly rectifying potassium (Kir) channels are key regulators of diverse physiological processes and may represent novel drug targets for diseases. Their therapeutic potential has not been tested directly, however, due to the lack of drug-like compounds targeting inward rectifiers. The lack of selective "probes" has also hindered efforts to define the physiological functions of some Kir channels. To overcome this formidable barrier and create new opportunities for studying inward rectifier physiology, the investigators performed a high- throughput screen (HTS) of more than 200,000 compounds for small-molecule modulators of ROMK (Kir1.1), a putative target for a novel class of diuretic. One compound, termed VU590, inhibits ROMK at nanomolar concentrations and Kir7.1 in the low micromolar range, making it the first small-molecule inhibitor of both channels. The investigators went on to use medicinal chemistry to rationally design a nanomolar-affinity probe, termed VU591, which is highly selective for ROMK over more than 60 potential off targets, including inward rectifiers and BK channels. In Aim 1, the investigators will employ state-of-the-art molecular modeling techniques, atomic structure-guided mutagenesis and electrophysiology to define the VU590/591 binding sites in ROMK and Kir7.1. VU591 is remarkably selective for ROMK and therefore represents a promising candidate for further development for use in animal studies. In Aim 2, the investigators will first determine if VU591 is active in the native tissue by assessing its effects on K and Na transport in isolated perfused cortical collecting ducts under low- and high-flow conditions. The investigators also discovered a nanomolar-affinity inhibitor of a G-protein regulated inward rectifier (GIRK), a putative therapeutic target for atrial fibrillation. In Aim 3, the investigators will use medicinal chemistry, structure-guided mutagenesis and electrophysiology to define the molecular binding sites for this novel compound termed VU592. These studies will provide important new insights into the atomic structures of inward rectifiers and generate critically needed probes with which to define the integrative physiology and therapeutic potential of these channels. Lay summary: The investigators will combine medicinal chemistry, advanced computational techniques and classical physiological methods to develop drug-like compounds targeting potassium channels that could be therapeutic targets for hypertension, edema and cardiac arrhythmia. PUBLIC HEALTH RELEVANCE: Inward rectifying potassium (Kir) channels play key physiological roles in diverse cellular functions and may represent novel drug targets. However, the lack of selective pharmacological "probes" has hindered efforts to explore the integrative physiology and therapeutic potential of most Kir channels. Here we propose to employ medicinal chemistry, atomic structure-guided mutagenesis, kidney tubule microperfusion and electrophysiology to develop the small-molecule pharmacology for Kir1.1, Kir7.1 and Kir3 channels.