Supplementary Material for: Molecular Insights into hERG Potassium Channel Blockade by Lubeluzole

Background/Aims: Lubeluzole is a benzothiazole derivative that has shown neuroprotective properties in preclinical models of ischemic stroke. However, clinical research on lubeluzole is now at a standstill, since lubeluzole seems to be associated with the acquired long QT syndrome and ventricular arrhythmias. Since the cardiac cellular effects of lubeluzole have not been described thus far, an explanation for the lubeluzole-induced QT interval prolongation is lacking. Methods: We tested the affinity of lubeluzole, its enantiomer, and the racemate for hERG channel using the patch-clamp technique. We synthesized and tested two simplified model compounds corresponding to two moieties included in the lubeluzole structure. The obtained experimental results were rationalized by docking simulation on the recently reported cryo-electron microscopy (cryo-EM) structure of hERG. Group efficiency analysis was performed in order to individuate the fragment most contributing to binding. Results: We found that lubeluzole and its R enantiomer are highly potent inhibitors of human ether-ago-go-related gene (hERG) channel with an IC50 value of 12.9 ± 0.7 nM and 11.3 ± 0.8 nM, respectively. In the presence of lubeluzole, steady-state activation and inactivation of hERG channel were shifted to more negative potentials and inactivation kinetics was accelerated. Mutations of aromatic residues (Y652A and F656A) in the channel inner cavity significantly reduced the inhibitory effect of lubeluzole. Molecular docking simulations performed on the near atomic resolution cryo-electron microscopy structures of hERG supported the role of Y652 and F656 as the main contributors to high affinity binding. Group efficiency analysis indicated that both 1,3-benzothiazol-2-amine and 3-aryloxy-2-propanolamine moieties contribute to drug binding with the former giving higher contribution. Conclusions: This study suggests the possibility to modulate lubeluzole hERG blockade by introducing suitable substituents onto one or both constituting portions of the parent compound in order to either reduce potency (i. e. torsadogenic potential) or potentiate affinity (useful for class III antiarrhythmic and anticancer agent development).

研究背景与目的：卢贝洛尔（Lubeluzole）是一种苯并噻唑类衍生物，在缺血性脑卒中的临床前模型中已展现出神经保护特性。然而，当前卢贝洛尔的临床研究已陷入停滞，因其被认为与获得性长QT综合征及室性心律失常相关。截至目前，卢贝洛尔的心肌细胞效应尚未被阐明，因此尚无针对其诱导QT间期延长的合理解释。
研究方法：本研究采用膜片钳技术（patch-clamp technique），检测了卢贝洛尔、其对映异构体（enantiomer）及外消旋体（racemate）对人类ether-ago-go相关基因（human ether-ago-go-related gene，hERG）通道的亲和力。我们合成并测试了两种简化模型化合物，分别对应卢贝洛尔结构中的两个药效团片段。通过对接模拟（docking simulation）分析近期解析的hERG冷冻电子显微镜（cryo-electron microscopy, cryo-EM）近原子分辨率结构，对实验结果进行了合理化阐释。此外，本研究开展了基团效率分析（group efficiency analysis），以明确对结合作用贡献最大的片段。
研究结果：本研究发现，卢贝洛尔及其R型对映异构体均为强效的hERG通道抑制剂，半数抑制浓度（IC50）分别为12.9±0.7 nM及11.3±0.8 nM。卢贝洛尔可使hERG通道的稳态激活与失活曲线向更负电位方向偏移，并加速其失活动力学过程。通道内腔的芳香族残基突变（Y652A与F656A）可显著削弱卢贝洛尔的抑制作用。基于hERG近原子分辨率冷冻电镜结构的分子对接模拟结果证实，Y652与F656是介导高亲和力结合的关键残基。基团效率分析结果显示，1,3-苯并噻唑-2-胺与3-芳氧基-2-丙醇胺两个片段均对药物结合存在贡献，其中前者的贡献更为显著。
研究结论：本研究表明，可通过在母药的两个构成片段（或其中之一）上引入合适的取代基，对卢贝洛尔的hERG通道阻断作用进行调控：既可降低其致扭转性室速潜能（torsadogenic potential），也可增强其结合亲和力，这一发现可为III类抗心律失常药及抗癌药物的开发提供参考。