A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors
A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors
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DOI：
10.1016/s0003-2697(03)00425-1
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发表时间：
2003-10-15
影响因子：
2.9
通讯作者：
Warrior, U
中科院分区：
文献类型：
--
作者：
Gopalakrishnan, SM;Moreland, RB;Warrior, U
G蛋白偶联受体（GPCR）激动剂和拮抗剂先导物的鉴定对制药和生物技术行业至关重要。我们报告利用一种新的，高密度，以及较少的筛选平台称为微阵列化合物筛选（muARCS），测试8640化合物的足迹的标准微升板的识别新的激动剂的特定G-蛋白偶联受体。虽然与Ga（q）蛋白偶联的受体可以通过基于荧光的Ca 2+释放测量容易地评估，但是与Ga（s）或Ga（i/o）蛋白偶联的许多GPCR不适合以这种高通量方式进行功能评价。在这项研究中，人多巴胺D-4.4受体，其通常通过Ga（i/o）蛋白偶联以抑制腺苷酸环化酶并降低细胞内cAMP的水平，通过在HEK-293细胞中稳定共表达该受体与嵌合G（alphaqo 5）蛋白而偶联至细胞内Ca 2+释放。在muARCS格式中，表达D-4.4受体和Galphaq（α 5）蛋白的细胞用fluo-4预加载，浇铸到1%琼脂糖凝胶中，放置在化合物片上，并使用ViewLux电荷耦合器件成像系统连续成像。多巴胺和其他激动剂引起的荧光反应，出现在一个时间和浓度依赖性的方式亮点增加。利用这项技术，快速筛选了260，000种化合物的库，并鉴定了几种新型激动剂。这些激动剂使用荧光成像读板仪测定进一步表征。优异的确认率加上增强的效率和通量使muARCS能够作为筛选和鉴定新型GPCR激动剂的替代平台。(C)2003年爱思唯尔公司All rights reserved.
The identification of agonist and antagonist leads for G-protein-coupled receptors (GPCRs) is of critical importance to the pharmaceutical and biotechnology industries. We report on the utilization of a novel, high-density, well-less screening platform known as microarrayed compound screening (muARCS) that tests 8640 compounds in the footprint of a standard microliter plate for the identification of novel agonists for a specific G-protein-coupled receptor. Although receptors coupled to the Galpha(q) protein can readily be assessed by fluorescence-based Ca2+ release measurements, many GPCRs that are coupled to Galpha(s) or Galpha(i/o) proteins are not amenable to functional evaluation in such a high-throughput manner. In this study, the human dopamine D-4.4 receptor, which normally couples through the Galpha(i/o) protein to inhibit adenylate cyclase and to reduce levels of intracellular cAMP, was coupled to intracellular Ca2+ release by stably coexpressing this receptor with a chimeric G(alphaqo5) protein in HEK-293 cells. In muARCS format, the cells expressing D-4.4 receptor and Galphaq(o5) protein were preloaded with fluo-4, cast into a 1% agarose gel, placed above the compound sheets, and imaged successively using a ViewLux charge-coupled device imaging system. Dopamine and other agonists evoked an increase in fluorescence response that appeared as bright spots in a time- and concentration-dependent manner. Utilizing this technology, a library of 260,000 compounds was rapidly screened and led to the identification of several novel agonists. These agonists were further characterized using a fluorometric imaging plate reader assay. Excellent confirmation rates coupled with enhanced efficiency and throughput enable muARCS to serve as an alternative platform for the screening and identification of novel GPCR agonists. (C) 2003 Elsevier Inc. All rights reserved.