Chaudhary Khuram W, O'Neal Janet M, Mo Zun-Li, Fermini Bernard, Gallavan Robert H, Bahinski Anthony
Pfizer Inc., Chesterfield, MO 63017, USA.
Assay Drug Dev Technol. 2006 Feb;4(1):73-82. doi: 10.1089/adt.2006.4.73.
Inhibition of the delayed-rectifier potassium channel current, human ether-a-go-go (hERG), by pharmaceutical agents can lead to acquired long QT syndrome and the generation of potentially lethal arrhythmias and sudden death. There remains an unmet need for higher-throughput assays to screen compounds in preclinical development for the potential to block hERG and cause QT prolongation. We evaluated the rubidium efflux assay for its ability to determine block of the hERG potassium channel. hERG-transfected human embryonic kidney-293 cells were cultured on 96-well assay plates and loaded with rubidium ion by incubating in media in which potassium was replaced by 5.4 mM Rb+. Cells were exposed to test compounds and then depolarized with a K+ channel opening buffer containing 50 mM K+. The supernatant was removed, and cells were lysed using 0.1% Triton X-100. Concentration-response curves were generated for test agents by determining the Rb+ efflux using a flame atomic absorption spectrometer. Multiple trials with cisapride yielded 50% inhibitory concentration values between 308.1 +/- 11 nM to 456.3 +/- 24 nM for inhibition of Rb+ efflux and a Z factor of 0.80 +/- 0.07 (n = 5 plates, 12 wells per plate). The values for inhibition of the hERG channel exhibited a rightward shift in potency as compared to those measured using electrophysiological techniques. In addition, we evaluated 19 blinded compounds at 10 microM in the Rb+ efflux assay, and compared results to those using patch clamp electrophysiology and the dofetilide displacement binding assay. The dofetilide displacement binding assay yielded a good correlation with electrophysiological measurements of hERG block. The rubidium efflux assay lacked sensitivity to consistently identify significant channel blockade. In conclusion, the rubidium efflux assay provides a higher-throughput means to identify potent hERG channel blocking agents, but lacks the sensitivity required to accurately determine the potency of blockade.
药物制剂对延迟整流钾通道电流——人类醚 - 去极化相关基因（hERG）的抑制作用，可导致获得性长QT综合征，并引发潜在致命性心律失常和猝死。在临床前研发阶段，仍迫切需要更高通量的检测方法来筛选化合物，以评估其阻断hERG并导致QT间期延长的可能性。我们评估了铷外流检测法测定hERG钾通道阻断的能力。将转染hERG的人胚肾 - 293细胞培养在96孔检测板上，并通过在钾离子被5.4 mM Rb⁺替代的培养基中孵育来加载铷离子。将细胞暴露于测试化合物，然后用含有50 mM K⁺的钾通道开放缓冲液使其去极化。去除上清液，并用0.1% Triton X - 100裂解细胞。通过使用火焰原子吸收光谱仪测定Rb⁺外流，生成测试药物的浓度 - 反应曲线。对西沙必利进行的多次试验得出，抑制Rb⁺外流的50%抑制浓度值在308.1±11 nM至456.3±24 nM之间，Z因子为0.80±0.07（n = 5个板，每个板12孔）。与使用电生理技术测量的值相比，hERG通道抑制值在效价上表现出向右偏移。此外，我们在铷外流检测中评估了19种10 μM的盲法化合物，并将结果与使用膜片钳电生理学和多非利特置换结合检测的结果进行比较。多非利特置换结合检测与hERG阻断的电生理测量结果具有良好的相关性。铷外流检测缺乏持续识别显著通道阻断的敏感性。总之，铷外流检测提供了一种更高通量的方法来鉴定强效hERG通道阻断剂，但缺乏准确测定阻断效价所需的敏感性。
