Dept. of Pharmacology, Alberta Diabetes Institute, University of Alberta, 9-70 Medical Sciences Building, Edmonton, AB T6G 2H7, Canada.
Dept. of Cellular and Molecular Biology, Xenon Pharmaceuticals Inc., 3650 Gilmore Way, Burnaby, BC V5G 4W8, Canada.
Function (Oxf). 2023 May 4;4(4):zqad021. doi: 10.1093/function/zqad021. eCollection 2023.
Kv7 (KCNQ) voltage-gated potassium channels are critical regulators of neuronal excitability and are candidate targets for development of antiseizure medications. Drug discovery efforts have identified small molecules that modulate channel function and reveal mechanistic insights into Kv7 channel physiological roles. While Kv7 channel activators have therapeutic benefits, inhibitors are useful for understanding channel function and mechanistic validation of candidate drugs. In this study, we reveal the mechanism of a Kv7.2/Kv7.3 inhibitor, ML252. We used docking and electrophysiology to identify critical residues involved in ML252 sensitivity. Most notably, Kv7.2[W236F] or Kv7.3[W265F] mutations strongly attenuate ML252 sensitivity. This tryptophan residue in the pore is also required for sensitivity to certain activators, including retigabine and ML213. We used automated planar patch clamp electrophysiology to assess competitive interactions between ML252 and different Kv7 activator subtypes. A pore-targeted activator (ML213) weakens the inhibitory effects of ML252, whereas a distinct activator subtype (ICA-069673) that targets the voltage sensor does not prevent ML252 inhibition. Using transgenic zebrafish larvae expressing an optical reporter (CaMPARI) to measure neural activity in-vivo, we demonstrate that Kv7 inhibition by ML252 increases neuronal excitability. Consistent with in-vitro data, ML213 suppresses ML252 induced neuronal activity, while the voltage-sensor targeted activator ICA-069673 does not prevent ML252 actions. In summary, this study establishes a binding site and mechanism of action of ML252, classifying this poorly understood drug as a pore-targeted Kv7 channel inhibitor that binds to the same tryptophan residue as commonly used pore-targeted Kv7 activators. ML213 and ML252 likely have overlapping sites of interaction in the pore Kv7.2 and Kv7.3 channels, resulting in competitive interactions. In contrast, the VSD-targeted activator ICA-069673 does not prevent channel inhibition by ML252.
Kv7（KCNQ）电压门控钾通道是神经元兴奋性的关键调节因子，也是开发抗癫痫药物的候选靶点。药物发现工作已经确定了能够调节通道功能的小分子，并揭示了 Kv7 通道生理作用的机制见解。虽然 Kv7 通道激活剂具有治疗益处，但抑制剂对于理解通道功能和候选药物的机制验证很有用。在这项研究中，我们揭示了 Kv7.2/Kv7.3 抑制剂 ML252 的作用机制。我们使用对接和电生理学来确定与 ML252 敏感性相关的关键残基。值得注意的是，Kv7.2[W236F]或 Kv7.3[W265F]突变强烈减弱了 ML252 的敏感性。该通道中的这种色氨酸残基也需要对某些激活剂（包括瑞替加滨和 ML213）敏感。我们使用自动化平面膜片钳电生理学来评估 ML252 与不同 Kv7 激活剂亚型之间的竞争性相互作用。靶向通道的激活剂（ML213）减弱了 ML252 的抑制作用，而靶向电压传感器的不同激活剂亚型（ICA-069673）则不能阻止 ML252 的抑制作用。使用表达光学报告物（CaMPARI）的转基因斑马鱼幼虫在体内测量神经活动，我们证明了 ML252 抑制 Kv7 会增加神经元兴奋性。与体外数据一致，ML213 抑制 ML252 诱导的神经元活动，而电压传感器靶向激活剂 ICA-069673 则不能防止 ML252 的作用。总之，这项研究建立了 ML252 的结合位点和作用机制，将这种了解甚少的药物归类为靶向通道的 Kv7 通道抑制剂，它与常用的靶向通道的 Kv7 激活剂结合到相同的色氨酸残基上。ML213 和 ML252 可能在 Kv7.2 和 Kv7.3 通道的孔内有重叠的相互作用位点，导致竞争性相互作用。相比之下，VSD 靶向激活剂 ICA-069673 不能阻止 ML252 对通道的抑制作用。
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