Discovery of small molecule activators of the Slack potassium channel for use as cell-based probes for the study of fragile X syndrome

摘要 离子通道是充满水的小孔，它调节离子通过细胞膜，从而有助于 一系列基本的细胞功能。尽管许多目前可用的药物是通过与离子相互作用而起作用的 通道，绝大多数离子通道缺乏有效和选择性的小分子工具化合物 对于正确研究它们的治疗潜力是必要的。由KCNT1编码的Slack信道是 Slo钾通道家族；这个通道家族在整个身体中扮演着不同的角色，特别是 包括对神经元活动的调节。最近，人们发现一种被称为RNA结合蛋白的 脆性X智力低下蛋白(FMRP)结合并激活Slack通道。脆性X的基因沉默 1型精神发育迟滞(FMR1)导致FMRP缺失，并导致脆性X综合征(FXS)，一种X- 连锁障碍是遗传性智力残疾(ID)和单基因最常见的已知原因 自闭症谱系障碍(ASD)。对动物的研究表明，松弛活动对某些类型的 较高的认知功能。因此，我们假设FXS患者的松弛活动因缺乏而减少 FMRP有助于与疾病相关的ID，并通过以下方式使松弛活动正常化 药物治疗代表着一种潜在的治疗方法。不幸的是，目前还没有已知的有效药物 和选择性小分子松弛激活剂探针可用于研究这一假说。对这样的研究 探针是帮助验证FXS治疗的这一新方法的关键的早期步骤。建议数 研究将使用药物化学HIT优化方法来开发结构活性关系 (SAR)在我们通过高通量筛选(HTS)发现的两种不同的化学支架中 接近。我们将评估我们新合成的化合物的Slack激活剂效力和功效 一种与HTS兼容的Tl+通量分析和测量离子通道活度、电压和电流的“金标准”方法。 钳夹电生理学。我们将测试我们的化合物增加Slack通道活性的能力 在FMRP表达已被敲除的表达Slack的HEK-293细胞和分离的神经元中 来自Fmr1-/y小鼠。我们将评估化合物与Slo家族成员、其他离子通道、 最终形成一大批分子靶标。我们还将监测我们的化合物的溶解性， 膜通透性和细胞外流潜力，以确保我们的探头不会受到不良影响 这些区域中的属性。为了高效地实现我们的目标，我们设计了一条周到的关键路径，可以为 它可以为我们提供复合进度决策，并确保对复合化合物进行全面评估。完成我们的目标 将使我们能够实现我们的最终目标，即开发高质量的基于松弛激活因子细胞的探针，该探针可能是 被社区用来研究激活这个靶点作为FXS的一种新治疗方法。

ABSTRACT Ion channels are water-filled pores that mediate the passage of ions across cell membranes, thereby contributing to a multitude of essential cellular functions. Though many currently available drugs work via interaction with ion channels, the vast majority of ion channels lack the potent and selective small molecule tool compounds necessary to properly study their therapeutic potential. The Slack channel, encoded by KCNT1, is a member of the Slo family of potassium channels; this family of channels has a diversity of roles throughout the body, notably including the regulation of neuronal activity. Recently, it was discovered that an RNA-binding protein known as Fragile X mental retardation protein (FMRP) binds to and activates Slack channels. Gene silencing of fragile X mental retardation type 1 (FMR1) leads to an absence of FMRP and causes fragile X syndrome (FXS), an X- linked disorder that is the most common known cause of inherited intellectual disability (ID) and monogenic autism spectrum disorder (ASD). Studies in animals have shown that Slack activity is critical for certain types of higher cognitive function. Thus, we hypothesize that diminished Slack activity in FXS patients due to the absence of FMRP contributes to the ID associated with the disease, and normalization of Slack activity via pharmacotherapy represents a potential treatment approach. Unfortunately, there are presently no known potent and selective small molecule Slack activator probes available to investigate this hypothesis. Studies with such probes represent an essential, early step in helping to validate this novel approach to FXS therapy. The proposed research will employ a medicinal chemistry hit optimization approach to develop structure activity relationships (SAR) in two distinct chemical scaffolds that we discovered through a high-throughput screening (HTS) approach. We will assess the Slack activator potency and efficacy of our newly synthesized compounds using an HTS-compatible Tl+ flux assay and the “gold standard” method for measuring ion channel activity, voltage- clamp electrophysiology. We will test the capacity of our compounds to increase the activity of Slack channels in Slack-expressing HEK-293 cells where FMRP expression has been knocked out as well as in neurons isolated from Fmr1-/y mice. We will evaluate the selectivity of compounds versus Slo family members, other ion channels, and ultimately a broad panel of molecular targets. We will also monitor our compounds with regard to solubility, membrane permeability, and potential for cellular efflux to ensure that our probes will not be limited by poor attributes in these areas. To reach our goal efficiently, we have designed a thoughtful critical path that can serve us with compound progression decisions and ensure a holistic evaluation of compounds. Completion of our aims will enable us to reach our end goal of developing high quality Slack activator cell-based probes that may be used by the community to study activation of this target as a novel treatment for FXS.