Development of an In Vivo, Brain-penetrant GIRK1/2 Potassium Channel Activator

 描述(申请人提供)：G蛋白门控内向整流钾(GIRK)通道是大脑和心脏细胞兴奋性的关键介质。GIRK通道是由四个亚基(GIRK1-4)形成的同四异四聚体复合体。虽然来自小鼠基因消融研究的证据表明，特定的GIRK通道亚型对器官生理学和行为有不同的贡献，但缺乏亚型选择性的GIRK通道探针阻碍了对与GIRK通道激活相关的后果和治疗潜力的严格评估。最近，我们使用高通量筛选方法确定了一种有前景的GIRK通道激活剂支架。这种支架被用来开发第一个亚型选择性的小分子GIRK通道激活剂ML297。相对于缺乏GIRK1的通道，ML297对含有GIRK1的通道具有很强的选择性。ML297以GIRK1依赖的方式减少小鼠的焦虑相关行为，并抑制大鼠癫痫模型的发作。然而，ML297的水溶解性差，全身给药后脑组织渗透率低，清除速度快。此外，与包括心脏GIRK通道(GIRK1/4)在内的其他GIRK1亚型相比，ML297对主要的神经元GIRK通道(GIRK1/2)只表现出适度的选择性。ML297的这些特征限制了其作为体内探针用于阐明与GIRK1/2通道激活相关的相关性和治疗潜力。因此，该项目的目标是使用ML297支架开发新的GIRK1/2激活剂，具有改进的通道亚型选择性分布和体内脑穿透剂探针所需的药代动力学特性。该项目结合了药物化学和化合物表征方面的最先进方法，并将由一个在GIRK通道生物学和体内探针开发方面具有互补专业知识的调查团队进行。这项工作围绕三个具体目标展开：1)开发有效和选择性的GIRK1/2通道激活剂，2)开发具有优化药代动力学特性的GIRK1/2通道激活剂，3)表征GIRK1/2通道激活剂在体内的药效学特性。目标1和目标2的工作将涉及迭代并行药物化学合成和测试战略， 并将生产具有优于ML297的药效学和药代动力学特性的GIRK1/2激动剂。在AIM 3中，这些化合物将在应激诱导热疗(SIH)测试中表征体内有效性、选择性和效力，SIH测试是一种具有很强表面有效性的抗焦虑化合物。在这项测试中，一小组显示GIRK1/2依赖效应的优化探针将在另一个焦虑模型和探索GIRK1/2探针对运动活动和协调性的影响的测试中进行进一步评估。该研究项目的完成将为GIRK1/2通道产生选择性和有效的体内探针。这些探测将有助于未来对GIRK1/2通道的相关性和治疗潜力的研究，包括更严格地评估它们在治疗糖尿病中的作用。 焦虑症相关障碍。

 DESCRIPTION (provided by applicant): G protein-gated Inwardly-Rectifying K+ (GIRK) channels are critical mediators of cell excitability in the brain and heart. GIRK channels are homo- and heterotetrameric complexes formed by four subunits (GIRK1-4). While evidence from gene ablation studies in mice suggests that specific GIRK channel subtypes make discrete contributions to organ physiology and behavior, the lack of subtype-selective GIRK channel probes has prevented rigorous evaluation of the consequences, and therapeutic potential, associated with GIRK channel activation. Recently, we identified a promising GIRK channel activator scaffold using a high-throughput screening approach. This scaffold was used to develop ML297, the first subtype-selective, small-molecule GIRK channel activator. ML297 is potent and strongly-selective for GIRK1-containing relative to GIRK1-lacking channels. ML297 reduces anxiety-related behavior in a GIRK1-dependent manner in mice, and suppresses seizures in rat epilepsy models. However, ML297 suffers from poor aqueous solubility, as well as low brain penetration and rapid clearance following systemic administration. Moreover, ML297 exhibits only modest selectivity for the predominant neuronal GIRK channel (GIRK1/2) relative to other GIRK1-containing subtypes, including the cardiac GIRK channel (GIRK1/4). These features of ML297 limit its utility as an in vivo probe for elucidating the relevance and therapeutic potential associated with GIRK1/2 channel activation. Accordingly, the goal of this project is to use the ML297 scaffold to develop new GIRK1/2 activators with an improved channel subtype selectivity profile and pharmacokinetic properties required of a brain-penetrant, in vivo probe. The project combines state-of-the-art approaches in medicinal chemistry and compound characterization, and will be conducted by an investigative team with complementary expertise in GIRK channel biology and in vivo probe development. The work is framed around three Specific AIMs: 1) To develop potent and selective GIRK1/2 channel activators, 2) To develop GIRK1/2 channel activators with optimized pharmacokinetic properties, and 3) To characterize the pharmacodynamics properties of GIRK1/2 channel activators in vivo. Work in AIMs 1 and 2 will involve an iterative parallel medicinal chemistry synthesis and testing strategy, and will yield GIRK1/2 activators with pharmacodynamic and pharmacokinetic properties superior to those of ML297. In AIM 3, these compounds will be characterized for in vivo efficacy, selectivity, and potency in the stress-induced hyperthermia (SIH) test, a paradigm with strong face validity for anxiolytic compounds. A small set of optimized probes exhibiting GIRK1/2-dependent efficacy in this test will undergo further evaluation in another anxiety model, and tests that explore the effects of the GIRK1/2 probes on motor activity and coordination. Completion of this research project will yield selective and effective in vivo probes for GIRK1/2 channels. These probes will facilitate future research into the relevance and therapeutic potential of GIRK1/2 channels, including more rigorous evaluation of their utility for treatment of anxiety-related disorders.