Takenaka Mai, Kodama Masami, Murayama Takashi, Ishigami-Yuasa Mari, Mori Shuichi, Ishida Ryosuke, Suzuki Junji, Kanemaru Kazunori, Sugihara Masami, Iino Masamitsu, Miura Aya, Nishio Hajime, Morimoto Sachio, Kagechika Hiroyuki, Sakurai Takashi, Kurebayashi Nagomi
Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.).
Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
Mol Pharmacol. 2023 Dec;104(6):275-286. doi: 10.1124/molpharm.123.000720. Epub 2023 Sep 7.
Type 2 ryanodine receptor (RyR2) is a Ca release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca signals from R-CEPIA1er, a genetically encoded ER Ca indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca signal. All of the three compounds suppressed spontaneous Ca oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca-dependent [H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca waves without substantial effects on the action-potential-induced Ca transients. These results confirm that ER Ca-based screening is useful for identifying modulators of ER Ca release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca wave generation without substantially affecting physiological action-potential induced Ca transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.
2型兰尼碱受体（RyR2）是内质网（ER）/肌浆网上的一种钙释放通道，在心脏兴奋-收缩偶联中起核心作用。RyR2功能亢进与儿茶酚胺能多形性室性心动过速和心力衰竭患者的室性心律失常有关，在这些患者中，通过过度激活的RyR2自发释放钙会使舒张期膜电位去极化，从而诱发触发活动。在这种情况下，抑制RyR2活性的药物有望预防心律失常，但目前尚无临床可用的RyR2抑制剂。在本研究中，我们使用最近开发的基于内质网钙的检测方法，从一个特征明确的化合物库中筛选RyR2抑制剂，通过在表达RyR2的HEK293细胞中，检测来自基因编码的内质网钙指示剂R-CEPIA1er的内质网钙信号增加来检测RyR2活性的抑制情况。通过筛选库中的1535种化合物，我们鉴定出三种化合物（氯二甲酚、甲基苔色酸酯和利鲁唑），它们能显著增加内质网钙信号。这三种化合物均抑制了表达RyR2的HEK293细胞中的自发钙振荡，并相应降低了钙依赖性[H]兰尼碱结合活性。在来自RyR2突变小鼠的心肌细胞中，这三种化合物有效抑制了异常钙波，而对动作电位诱导的钙瞬变没有实质性影响。这些结果证实基于内质网钙的筛选对于鉴定内质网钙释放通道的调节剂是有用的，并表明RyR2抑制剂有潜力被开发为一类新型抗心律失常药物。意义声明：我们使用基于内质网钙的检测方法，成功地从一个特征明确的化合物库中鉴定出三种具有RyR2抑制作用的化合物，并证明这些化合物抑制了致心律失常钙波的产生，而对心肌细胞中生理动作电位诱导的钙瞬变没有实质性影响。本研究将有助于开发RyR2特异性抑制剂，作为治疗由RyR2过度激活引起的危及生命的心律失常的潜在新型药物。
