Molecular pharmacology and physiology of kidney potassium transport

描述(由申请人提供)：内向整流钾(KIR)通道是多种生理过程的关键调节器，可能是治疗疾病的新药物靶点。然而，由于缺乏针对内向整流子的类药物化合物，它们的治疗潜力尚未直接测试。缺乏选择性的“探针”也阻碍了确定一些KIR通道的生理功能的努力。为了克服这一巨大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(Kir1.1)是一种新型利尿剂的假定靶点。一种名为VU590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，使其成为这两个通道的第一个小分子抑制剂。研究人员继续利用药物化学合理地设计了一种名为VU591的纳摩尔亲和探针，它对ROMK具有高度的选择性，超过60多个潜在的OFF靶点，包括内向整流器和BK通道。在目标1中，研究人员将使用最先进的分子建模技术、原子结构引导的突变和电生理学来确定ROMK和Kir7.1中的VU590/591结合位点。VU591对ROMK具有显著的选择性，因此是进一步开发用于动物研究的有前途的候选者。在目标2中，研究人员将首先通过评估VU591在低流量和高流量条件下对分离的灌流皮质集合管中K和Na运输的影响来确定VU591在自然组织中是否活跃。研究人员还发现了G蛋白调节的内向整流子(GIRK)的纳摩尔亲和力抑制剂，GIRK可能是房颤的治疗靶点。在目标3中，研究人员将使用药物化学、结构导向突变和电生理学来确定这种名为VU592的新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，用来定义这些通道的综合生理和治疗潜力。综述：研究人员将结合药物化学、先进的计算技术和经典的生理学方法，开发针对钾通道的类药物化合物，这些化合物可能成为高血压、浮肿和心律失常的治疗靶点。 与公共健康相关：内向整流钾(KIR)通道在不同的细胞功能中起着关键的生理作用，可能是新的药物靶点。然而，缺乏选择性的药理“探针”阻碍了探索大多数KIR通道的综合生理学和治疗潜力的努力。在这里，我们建议利用药物化学、原子结构引导的诱变、肾小管微灌流和电生理学来开发针对Kir1.1、Kir7.1和Kir3通道的小分子药理学。

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.