Small-molecule probes for study of CLC-2 chloride-channel function in the central nervous system

项目总结 ClC氯通道家族是一类膜蛋白，它控制着 氯离子穿过细胞膜。9个独特的CLC同源基因在 哺乳动物的组织和功能在不同的生理作用中，从电刺激 肌肉和神经元对电解质平衡的调节。其中一个亚型CLC-2是一种电压- 依赖通道在大脑中广泛表达。尽管ClC-2在大脑中的存在 已经知道几十年了，这种CLC同系物在神经元信号转导和适当的 大脑功能仍然知之甚少，部分原因是缺乏强大的和选择性的 能够研究这一通道的分子生理学的小分子工具。最近 我们实验室的突破现在为开发专门的小分子工具打开了大门 致《中图法》-2。通过化合物文库筛选，我们发现了抑制ClC-2的化合物 活动。我们将其中的一种开发成了一种有效的选择性ClC-2抑制剂FA44，它 CLC-2的IC50为18 NM，对最接近的CLC同源基因或 由65个中枢神经系统通道、受体和转运体组成的小组。FA44的药效和选择性 我们对野生型脑片的电生理记录进一步支持ClC-2 对照ClC-2基因敲除小鼠。在这个项目中，我们将继续合作努力，以 开发、表征和使用化学工具化合物来研究ClC-2。在目标1中，我们将 确定抑制ClC-2的作用机制和分子决定因素。在目标2中， 我们将开发新的探针，包括小分子激活剂和荧光成像 用于本地化通道表达式的探测。在目标3中，我们将利用我们的工具化合物来 CLC-2在兴奋性突触传递和网络兴奋性中的作用 并评估ClC-2功能障碍与癫痫之间潜在的致病联系。 我们团队在合成化学(杜波依斯)、离子通道结构和功能方面的综合专业知识 (Maduke)、计算(Dror)和细胞神经科学/癫痫(Huguenard)是理想的位置 美国推进这一研究计划。

PROJECT SUMMARY The CLC chloride channel family is a class of membrane proteins that controls the flux of chloride ions across cell membranes. Nine unique CLC homologs are differentially expressed in mammalian tissue and function in diverse physiological roles, ranging from electrical excitation of muscles and neurons to regulation of electrolyte balance. One subtype, CLC-2, is a voltage- dependent channel expressed broadly in the brain. Although the presence of CLC-2 in the brain has been known for decades, the role of this CLC homolog in neuronal signaling and proper brain function remains poorly understood, in part due to the absence of potent and selective small-molecule tools that enable studies of the molecular physiology of this channel. A recent breakthrough in our laboratories now opens the door to developing small molecule tools specific to CLC-2. Through a compound-library screen, we identified ‘hit’ compounds that inhibit CLC-2 activity. We developed one of these into a potent and selective CLC-2 inhibitor, FA44, which has an IC50 of 18 nM for CLC-2 and no off-target effects on the closest CLC homolog or on a panel of 65 CNS channels, receptors, and transporters. The efficacy and selectivity of FA44 for CLC-2 is further supported by our electrophysiological recordings of brain slices from wild-type versus CLC-2 knock-out mice. In this project, we will continue our collaborative efforts to develop, characterize, and use chemical tool compounds for studying CLC-2. In Aim 1, we will identify the mechanism of action and molecular determinants for inhibition of CLC-2. In Aim 2, we will develop novel probes, including small-molecule activators and fluorescent imaging probes for localizing channel expression. In Aim 3, we will leverage our tool compounds to query the role of CLC-2 in excitatory synaptic transmission and network excitability in the thalamus and to evaluate the potential causative link between CLC-2 malfunction and epilepsy. Our team’s combined expertise in synthetic chemistry (Du Bois), ion-channel structure-function (Maduke), computation (Dror), and cellular neuroscience/epilepsy (Huguenard) ideally positions us to advance this research program.