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

 描述（由申请人提供）：G蛋白门控内向整流K+（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（GIRK 1 -4）形成的同源和异源四聚体复合物。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独立的贡献，但缺乏亚型选择性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，我们确定了一个有前途的GIRK通道激活剂支架使用高通量筛选方法。这种支架被用于开发ML 297，这是第一种亚型选择性小分子GIRK通道激活剂。相对于GIRK 1缺乏通道，ML 297对含GIRK 1通道具有强效和强选择性。ML 297在小鼠中以GIRK 1依赖性方式减少焦虑相关行为，并在大鼠癫痫模型中抑制癫痫发作。然而，ML 297的水溶性差，以及全身给药后的脑渗透性低和清除迅速。此外，ML 297对主要神经元GIRK通道（GIRK 1/2）仅表现出相对于其他含GIRK 1的亚型（包括心脏GIRK通道（GIRK 1/4））的适度选择性。ML 297的这些特征限制了其作为阐明与GIRK 1/2通道激活相关的相关性和治疗潜力的体内探针的实用性。因此，该项目的目标是使用ML 297支架开发新的GIRK 1/2激活剂，其具有脑渗透剂体内探针所需的改善的通道亚型选择性特征和药代动力学特性。该项目结合了药物化学和化合物表征的最先进方法，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。本工作围绕三个具体目标：1）开发有效和选择性的GIRK 1/2通道激活剂，2）开发具有优化药代动力学特性的GIRK 1/2通道激活剂，3）表征GIRK 1/2通道激活剂的体内药效学特性。AIM 1和2的工作将涉及迭代的平行药物化学合成和测试策略， 并将产生药效学和药代动力学性质上级ML 297的GIRK 1/2活化剂。在AIM 3中，这些化合物将在应激诱导的高热（SIH）试验中表征体内功效、选择性和效力，SIH试验是抗焦虑化合物具有强表面有效性的范例。在该测试中表现出GIRK 1/2依赖性功效的一小组优化探针将在另一个焦虑模型中进行进一步评估，并进行探索GIRK 1/2探针对运动活动和协调的影响的测试。该研究项目的完成将产生选择性和有效的GIRK 1/2通道的体内探针。这些探针将促进未来对GIRK 1/2通道的相关性和治疗潜力的研究，包括更严格地评估其治疗糖尿病的效用。 焦虑相关的疾病

 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.