Gill Sikander, Gill Rajwant, Wen Yang, Enderle Thilo, Roth Doris, Liang Dong
1 Aurora Biomed Inc. , Vancouver, Canada .
2 Roche Innovation Center Basel , Hoffmann-La Roche, Basel, Switzerland .
Assay Drug Dev Technol. 2017 May/Jun;15(4):167-177. doi: 10.1089/adt.2017.787.
A high-throughput screening (HTS) assay was developed for cotransporter, NKCC1, which is a potential target for the treatment of diverse disorders. This nonradioactive rubidium flux assay coupled with ion channel reader series provides a working screen for this target expressed in human embryonic kidney (HEK) cell line. An eightfold window of detection was achieved with the optimized assay. This new functional assay offered a robust working model for NKCC1 in determining reliable and concordant rank orders of the test compounds supporting its sensitivity and specificity. The robustness of manual assay indicated by Z' of 0.9 qualified its amenability to automation. The Z' of 0.7 was displayed by automated assay employed in high-throughput screening of compound libraries against this target. Being electrically neutral, the NKCC1 screening is difficult to achieve by both manual and automated electrophysiological techniques. These techniques, although considered gold standard, suffer from their inherent problems of being too slow to be in high-throughput format and with high running costs. In addition to being a functional assay for NKCC1, it is nontoxic as compared with thallium flux assay, which is prone to generate high number of false-positive/false-negative rates because of its innate fluorescence issues.
针对共转运体NKCC1开发了一种高通量筛选（HTS）检测方法，NKCC1是治疗多种疾病的潜在靶点。这种结合离子通道读数器系列的非放射性铷通量检测方法为在人胚肾（HEK）细胞系中表达的该靶点提供了一个有效的筛选方法。通过优化检测方法实现了八倍的检测窗口。这种新的功能检测方法为NKCC1提供了一个强大的工作模型，用于确定测试化合物可靠且一致的排名顺序，证明了其灵敏度和特异性。手动检测的稳健性通过Z'值为0.9表明其适合自动化。在针对该靶点的化合物库高通量筛选中采用的自动化检测显示Z'值为0.7。由于NKCC1呈电中性，通过手动和自动化电生理技术都难以实现对其的筛选。这些技术虽然被视为金标准，但存在固有的问题，即速度太慢无法采用高通量形式，且运行成本高。除了作为NKCC1的功能检测方法外，与铊通量检测相比，它无毒，铊通量检测由于其固有的荧光问题容易产生大量假阳性/假阴性率。
