hERG钾离子通道变构调节剂的发现、优化及药理活性研究

The hERG K+ channel mediates the rapid component of the delayed rectifier potassium current, and plays a vital role in regulating cardiac phase III repolarization of the action potential of human ventricular myocytes. Genetic dysfunction (loss/gain-of-function) or pharmacological inhibition of the hERG channel leads to alterations of the action potential duration, QT interval lengthening or shortening, and the development of serious cardiac side effects. In the previous study, we have designed and synthesized a series of benzophenone analogues, which demonstrate negative and positive allosteric modulations on the binding of hERG blockers to the channel. Based upon the results from in vitro and ex-vivo pharmacological assays, we have found that negative allosteric modulators (NAMs) of the hERG channel significantly alleviated drug-induced cardiotoxicity via combination therapy while not affecting the on-target potency of test drugs. Furthermore, these NAMs have also been unraveled to rescue the congenital long QT syndromes (LQTSs) in a human pluripotent stem cell-derived cardiomyocyte model. Apart from NAMs, we have obtained several positive allosteric modulators (PAMs) of the hERG channel. Unlike the pharmacological activities of NAMs, we postulate that PAMs exert the potential to mitigate acquired or inherited short QT syndromes (SQTSs). However, NAMs and PAMs of the hERG channel have rarely been reported to date, and even worse, most of the disclosed NAMs and PAMs have shown inferior modulations on the channel. In this research project, we will first continue optimizing benzophenone compounds and characterizing their allosteric activities in order to obtain more potent NAMs and PAMs of the hERG channel. Photoaffinity assays coupled with proteomics and computational docking techniques will be then used to validate the specific binding sites of benzophenone NAMs and PAMs at the hERG channel. Thereafter, structure-based virtual screening, chemical synthesis and optimization, and structure-activity relationship will be comprehensively applied in the search of new ligands with high allosteric potencies at the hERG channel. Finally, a plethora of in-vitro, ex-vivo, and in-vivo pharmacological experiments will be performed to evaluate the influences of NAMs and PAMs on drug-induced hERG cadiotoxicity, inherited LQTSs and SQTSs, as well as other relevant arrhythmias. This study will pave the way for hERG-targeted drug discovery and also facilitate the translational investigations of allosteric modulators of the hERG channel on clinical trials in the future.

hERG钾离子通道因介导钾离子外流在心肌细胞动作电位的三期复极化过程中发挥着至关重要的作用。hERG基因功能获得或缺失性突变以及药物阻断钾离子通道，均可引起严重的心脏毒性进而危及患者生命。在前期研究工作中，我们设计合成了一系列二苯甲酮类hERG钾离子通道变构调节剂，并通过体外和离体实验证实部分负向变构调节剂经联合用药能够有效降低常见药物的心脏毒副作用且不影响药物本身的治疗功效，同时其单独使用亦可显著缓解遗传性QT间期延长综合症。此外，我们也发现数个hERG钾离子通道的正向变构调节剂；与上述负向变构调节功能相反，我们推测这些正向变构调节剂在治疗先天性及获得性QT间期缩短综合症方面具有极高的应用价值。截至目前，这些变构调节剂的数量依旧较少，且其变构调节活性普遍不高。因此，在该研究项目中，我们首先将会利用经典药物化学理论针对已发现的二苯甲酮类化合物进行广泛深入的结构修饰和优化，并测定其变构调节活性；然后利用光亲和标记技术、蛋白质组学以及计算机模拟分子对接手段鉴定这些变构调节剂在hERG通道蛋白内部的特异性结合位点；再通过基于计算机模拟药物设计的虚拟筛选方案寻找新型hERG钾离子通道负向及正向变构调节剂，并对其进行结构的优化改造同时测定其变构调剂活性；最终通过体外、离体以及体内药理活性实验综合评价hERG钾离子通道变构调节剂在降低药物心脏毒性、缓解QT间期延长或缩短综合症、改善心律失常等方面的临床应用功效，为以hERG钾离子通道为作用靶点的创新药物研发及其临床转化应用研究奠定坚实的基础。

近年来，基于hERG通道的研究工作主要体现在药物的安全性评价方面，而以hERG为治疗靶标的新药开发工作鲜有提及。鉴于此，我们以二苯甲酮类hERG通道调节剂为先导化合物，利用经典的药物化学改造方案以及基于结构的合理药物设计策略，设计合成了53个结构新颖的hERG通道调节剂，并建立了全细胞膜片钳实验筛选和测定目标化合物对hERG通道介导钾电流的激活或抑制活性，发现一系列活性高于先导化合物的hERG通道激活剂以及少量的hERG通道抑制剂，并归纳总结了化合物的构效关系。基于上述结果，我们利用DUD.E在线服务器以及LigandScout软件建立和验证了药效团模型，以此虚拟筛选了270万个化合物，得到99个可能具有hERG通道激活活性的化合物。然后，我们购买或合成了这些化合物，并利用全细胞膜片钳实验测定了其生物活性，发现9个具有全新结构的hERG通道激活剂。接着，我们归纳总结了这些化合物的结构通式，对其展开深入的结构修饰和优化，现已合成目标化合物15个。同时，我们基于高活性的二苯甲酮类hERG激活剂设计合成了5个分子探针，利用微量热泳动实验（MST）以及全细胞膜片钳实验发现2个能够直接与hERG蛋白结合且显著增加hERG通道介导钾电流的探针分子。同时，我们建立了光亲和标记实验，目前正在利用串联质谱技术鉴定该类化合物在hERG通道蛋白内部的特异性结合位点。最后，我们利用体外和离体实验模型以及病人源多能干细胞诱导分化的心肌细胞模型评估了活性较高的hERG通道激活剂的药理活性，发现2个候选药物能够显著缓解获得性或先天性长QT综合征，通过联合用药能够降低药物的hERG心脏毒性。进一步的机制研究表明，候选药物通过影响hERG通道的门控特性，即：加速通道激活同时减缓通道失活，从而发挥增强hERG通道介导钾电流的作用。