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Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow
Mark P Fereshteh1, Xin Li2, Sha Li2
1Bristol-Myers Squibb Company, Princeton, NJ, USA Mark.Fereshteh@bms.com.
Abstract:
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.
Insights
Researchers developed a high-throughput assay to screen Janus-associated kinase (JAK) inhibitors in human blood. This new method increases efficiency and enables the discovery of novel JAK/STAT signaling inhibitors.
Area of Science:
- Pharmacology and Drug Discovery
- Immunology
- Biotechnology
Background:
- Oral Janus-associated kinase (JAK) inhibitors are under clinical development for various conditions.
- Assessing JAK inhibition and its biological effects in human blood is crucial but traditionally low-throughput and expensive.
- Existing methods for evaluating JAK/STAT signaling in blood lack efficiency for large-scale screening.
Purpose of the Study:
- To develop a high-throughput, flow-based assay for screening small molecules targeting JAK/STAT signaling in human whole blood.
- To enable sensitive measurement of target engagement and biological outcomes of JAK inhibitors.
- To overcome limitations of conventional manual flow cytometry for analyzing signaling events in blood.
Main Methods:
- Development of a robust 384-well, high-throughput flow-based assay platform.
- Utilized automation technologies and process optimizations to address sample handling and data analysis challenges.
- Evaluated small molecule compounds targeting JAKs in peripheral human whole blood.
Main Results:
- The developed assay provides a highly sensitive analysis of JAK/STAT signaling events in human blood.
- Automation and optimized processes improved sample throughput significantly compared to manual methods.
- The assay platform successfully facilitated the measurement of target engagement for JAK inhibitors.
Conclusions:
- A novel, high-throughput flow-based assay for screening JAK/STAT inhibitors in human whole blood has been established.
- This platform overcomes previous throughput and cost limitations, enabling efficient drug discovery.
- The assay facilitates the development and identification of novel JAK/STAT pathway inhibitors.
