Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow Cytometry
Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow Cytometry
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DOI：
10.1177/1087057116645095
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发表时间：
2016-09-01
影响因子：
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通讯作者：
Zhang, Litao
中科院分区：
文献类型：
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作者：
Fereshteh, Mark P.;Li, Xin;Zhang, Litao
以Janus-associated kinase （JAKs）为靶点的口服药物是一种具有临床开发前景的新型药物。为了更好地了解JAK抑制与生物学结果之间的关系，我们在外周人全血中评估了靶向JAK的化合物。到目前为止，这些分析是低通量和昂贵的。在这里，我们开发了一种强大的384孔，高通量基于流动的检测方法来筛选人全血中JAK/STAT信号抑制的小分子。该分析平台提供了血液中信号事件的高灵敏度分析，并促进了目标接触的测量。此外，这里开发的自动化技术和过程优化克服了样品完整性、处理和多参数数据分析瓶颈，而不影响分析性能。与传统的手工流式细胞术方法相比，这些努力显著提高了样品通量，并促进了新型JAK/STAT抑制剂的开发。
Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors.