A novel cell-based duplex high-throughput screening assay combining fluorescent Ca²⁺ measurement with homogeneous time-resolved fluorescence technology

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• 作者：Lá ; szló ; Kiss ; Attila Cselenyá ; k ; Á ; gnes Varga ; Andrá ; s Visegrá ; dy ; ^{visegrady@richter.hu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Cell-based assays ; High-throughput screening ; Multiplex assays and technology ; Fluorescent Ca2+ measurement ; HTRF technology
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：15 August 2016
• 年：2016
• 卷：507
• 期：Complete
• 页码：33-39
• 全文大小：706 K

文摘

Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca²⁺] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm.