Liu Jay, Chen Tongming, Norris Tyrell, Knappenberger Kathy, Huston Julie, Wood Michael, Bostwick Robert
HTS Center and Global Support Department, AstraZeneca Pharmaceuticals, C253H Wilmington, DE 19850, USA.
Assay Drug Dev Technol. 2008 Dec;6(6):781-6. doi: 10.1089/adt.2008.161.
The ionotropic -aminobutyric acid (GABA)A receptors are an important family of drug targets for a variety of neurological and psychiatric disorders. Selective modulation of certain subtypes of the receptor could lead to novel or improved therapies. However, the discovery of subtype-selective compounds has been hampered by the lack of a high-throughput functional assay and the difficulty in establishing stable cell lines expressing GABAA receptor in a proper subunit composition. To meet drug discovery need we developed a fluorescent imaging plate reader(FLIPR)-based membrane potential assay with sufficient robustness and reproducibility for use in a high-throughput format. Two major subtypes of GABAA receptor were used: GABAA1 and GABAA2, which are composed of (alpha1)2(beta2)2gama2 and (alpha1)2(beta3)2gama2, respectively. We expressed the receptors by transiently co-transfecting cells with the three subunit DNAs in separate constructs, and by controlling the ratio of the DNA amount for each subunit transfected we forced the cells to express GABAA receptors in a pharmacologically relevant form. A large batch of transfected human embryonic kidney 293 cells were cryopreserved and used to screen and evaluate GABAA modulators.In these cells, agonist activation of GABAA receptor resulted in Cl- efflux and membrane depolarization, which was detected by FLIPR as an increase in fluorescence signal. Based on our characterization of several known GABAA modulators and a test set of compounds known to bind to the GABAA benzodiazepine site, we have demonstrated the validity and utility of this assay for discovery of novel pharmacological agents acting at GABAA receptors.
离子型γ-氨基丁酸（GABA）A受体是多种神经和精神疾病的重要药物靶点家族。对该受体某些亚型的选择性调节可能会带来新的或改进的治疗方法。然而，由于缺乏高通量功能检测方法以及难以建立以适当亚基组成表达GABAA受体的稳定细胞系，亚型选择性化合物的发现受到了阻碍。为满足药物发现的需求，我们开发了一种基于荧光成像酶标仪（FLIPR）的膜电位检测方法，该方法具有足够的稳健性和可重复性，可用于高通量检测。我们使用了GABAA受体的两种主要亚型：GABAA1和GABAA2，它们分别由（α1）2（β2）2γ2和（α1）2（β3）2γ2组成。我们通过在单独的构建体中用三种亚基DNA瞬时共转染细胞来表达受体，并通过控制每个转染亚基的DNA量的比例，迫使细胞以药理学相关的形式表达GABAA受体。大量转染的人胚肾293细胞被冻存，并用于筛选和评估GABAA调节剂。在这些细胞中，GABAA受体的激动剂激活导致Cl-外流和膜去极化，FLIPR检测到荧光信号增加。基于我们对几种已知GABAA调节剂的表征以及一组已知与GABAA苯二氮䓬位点结合的化合物的测试集，我们证明了该检测方法在发现作用于GABAA受体的新型药理剂方面的有效性和实用性。
