酸敏感离子通道(ASIC)在细胞外酸化变化的感知和响应中起关键作用。这些质子门控阳离子通道对神经元功能至关重要,比如学习和记忆,恐惧,机械感觉和内部调节,如突触可塑性。此外,它们在神经元变性中起关键作用,缺血性神经元损伤,癫痫发作终止,疼痛感应,等。功能性ASIC是与(ASIC1-ASIC3)同源亚基形成的同源或异源三聚体。ASIC1a,中枢神经系统(CNS)的主要ASIC同工型,在细胞外区域有一个酸性口袋,这是通道门控的关键调节器。越来越多的数据表明,ASIC1a通道是治疗各种神经系统疾病的潜在治疗靶点,包括中风,癫痫和疼痛。许多研究旨在识别ASIC通道的变构调节剂。然而,对ASIC的监管仍然知之甚少。使用所有可用的晶体结构,对应于ASIC1的不同功能状态和分子动力学模拟(MD)协议,我们分析了通道失活的过程。然后,我们应用分子对接程序来预测适合阿米洛利结合的蛋白质构象。为了确认其唯一的活性阻断剂对ASIC1状态过渡路线的影响,我们通过另一次MD模拟运行研究了该复合物。进一步的实验评估了烯胺文库中出现的具有可检测的ASIC抑制活性的各种化合物。我们对阿米洛利抑制ASIC1a的结构基础进行了详细分析,使用计算机模拟方法的组合来可视化其与开放状态下的离子孔的相互作用。人工激活(否则,中心孔的膨胀)导致通道结构的复杂改变,即它的跨膜结构域。输出的蛋白质构象被用作一组对接模型,适用于Enamine化学文库的高通量虚拟筛选。通过电生理学测定证实了虚拟筛选的结果,对于三种命中化合物显示出最佳结果。
Acid-sensing ion channels (ASICs) play a key role in the perception and response to extracellular acidification changes. These proton-gated cation channels are critical for neuronal functions, like learning and memory, fear, mechanosensation and internal adjustments like synaptic plasticity. Moreover, they play a key role in neuronal degeneration, ischemic neuronal injury, seizure termination, pain-sensing, etc. Functional ASICs are homo or heterotrimers formed with (ASIC1-ASIC3) homologous subunits. ASIC1a, a major ASIC isoform in the central nervous system (CNS), possesses an acidic pocket in the extracellular region, which is a key regulator of channel gating. Growing data suggest that ASIC1a channels are a potential therapeutic target for treating a variety of neurological disorders, including stroke, epilepsy and pain. Many studies were aimed at identifying allosteric modulators of ASIC channels. However, the regulation of ASICs remains poorly understood. Using all available crystal structures, which correspond to different functional states of ASIC1, and a molecular dynamics simulation (MD) protocol, we analyzed the process of channel inactivation. Then we applied a molecular docking procedure to predict the protein conformation suitable for the amiloride binding. To confirm the effect of its sole active blocker against the ASIC1 state transition route we studied the complex with another MD simulation run. Further experiments evaluated various compounds in the Enamine library that emerge with a detectable ASIC inhibitory activity. We performed a detailed analysis of the structural basis of ASIC1a inhibition by amiloride, using a combination of in silico approaches to visualize its interaction with the ion pore in the open state. An artificial activation (otherwise, expansion of the central pore) causes a complex modification of the channel structure, namely its transmembrane domain. The output protein conformations were used as a set of docking models, suitable for a high-throughput virtual screening of the Enamine chemical library. The outcome of the virtual screening was confirmed by electrophysiological assays with the best results shown for three hit compounds.