Drug Discovery Platform Technology Research Group, Korea Research Institute of Chemical Technology, Daejeon, South Korea.
Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Daejeon, South Korea.
Toxicol Appl Pharmacol. 2014 Feb 1;274(3):361-71. doi: 10.1016/j.taap.2013.12.012. Epub 2013 Dec 26.
Nefazodone was used widely as an antidepressant until it was withdrawn from the U.S. market in 2004 due to hepatotoxicity. We have investigated methods to predict various toxic effects of drug candidates to reduce the failure rate of drug discovery. An electrophysiological method was used to assess the cardiotoxicity of drug candidates. Small molecules, including withdrawn drugs, were evaluated using a patch-clamp method to establish a database of hERG inhibition. Nefazodone inhibited hERG channel activity in our system. However, nefazodone-induced hERG inhibition indicated only a theoretical risk of cardiotoxicity. Nefazodone inhibited the hERG channel in a concentration-dependent manner with an IC50 of 45.3nM in HEK-293 cells. Nefazodone accelerated both the recovery from inactivation and its onset. Nefazodone also accelerated steady-state inactivation, although it did not modify the voltage-dependent character. Alanine mutants of hERG S6 and pore region residues were used to identify the nefazodone-binding site on hERG. The hERG S6 point mutants Y652A and F656A largely abolished the inhibition by nefazodone. The pore region mutant S624A mildly reduced the inhibition by nefazodone but T623A had little effect. A docking study showed that the aromatic rings of nefazodone interact with Y652 and F656 via π-π interactions, while an amine interacted with the S624 residue in the pore region. In conclusion, Y652 and F656 in the S6 domain play critical roles in nefazodone binding.
奈法唑酮曾被广泛用作抗抑郁药，但由于其肝毒性，于 2004 年从美国市场撤出。我们一直在研究预测候选药物各种毒性作用的方法，以降低药物发现的失败率。本研究采用电生理学方法评估候选药物的心脏毒性。采用膜片钳法评估小分子（包括已撤出药物），建立 hERG 抑制数据库。奈法唑酮在我们的系统中抑制 hERG 通道活性。然而，奈法唑酮诱导的 hERG 抑制仅表明存在心脏毒性的理论风险。奈法唑酮以浓度依赖的方式抑制 hERG 通道，在 HEK-293 细胞中的 IC50 为 45.3nM。奈法唑酮加速了 hERG 通道的失活恢复和起始。奈法唑酮还加速了 hERG 通道的稳态失活，尽管它没有改变电压依赖性特征。使用 hERG S6 和孔区残基的丙氨酸突变体来鉴定 hERG 上的奈法唑酮结合位点。hERG S6 点突变 Y652A 和 F656A 大大消除了奈法唑酮的抑制作用。孔区突变体 S624A 轻度降低了奈法唑酮的抑制作用，但 T623A 几乎没有影响。对接研究表明，奈法唑酮的芳环通过 π-π 相互作用与 Y652 和 F656 相互作用，而一个胺与孔区的 S624 残基相互作用。总之，S6 结构域中的 Y652 和 F656 在奈法唑酮结合中起关键作用。
