CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC₅₀ = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

CLC-2 是一种电压门控氯离子通道，广泛表达于哺乳动物组织中。在中枢神经系统中，CLC-2 出现在神经元和神经胶质细胞中。定义该通道如何促进中枢神经系统正常和病理生理功能的研究提出了尚未解决的问题，部分原因是缺乏调节 CLC-2 活性的精确药理学工具。_{在此，我们描述了 AK-42 的开发和优化，AK-42 是一种具有纳摩尔效力 (IC 50} = 17 ± 1 nM)的 CLC-2 特异性小分子抑制剂。AK-42 比 CLC-1（最接近的 CLC-2 同源物）表现出前所未有的选择性（>1,000 倍），并且对脑组织中表达的一组 61 个常见通道、受体和转运蛋白没有表现出脱靶作用。通过诱变和动力学研究验证的计算对接表明 AK-42 与通道孔上方的细胞外前庭结合。在小鼠 CA1 海马锥体神经元的电生理记录中，AK-42 急剧且可逆地抑制 CLC-2 电流；从 CLC-2 敲除小鼠的脑切片中未观察到对电流的影响。这些结果使 AK-42 成为研究 CLC-2 神经生理学的强大工具。