title: Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow Cytometry.
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.
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA Mark.Fereshteh@bms.com.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Fereshteh
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Sha
authors.initials: S
authors.lastname: Li
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Fan
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Rosemary
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Glen A
authors.initials: GA
authors.lastname: Farr
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Garrett
authors.initials: G
authors.lastname: Kolodin
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Lippy
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Naglich
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Schieven
authors.affiliation: Sanofi-Aventis, Boston, MA, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Schweizer
authors.affiliation: Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Litao
authors.initials: L
authors.lastname: Zhang
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D053612:Janus Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D050791:STAT Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects*; D054852:Small Molecule Libraries / Q000737:chemistry
chemical_list: D047428:Protein Kinase Inhibitors; D050791:STAT Transcription Factors; D054852:Small Molecule Libraries; D053612:Janus Kinases
keywords: JAK/STAT signaling; automation; flow cytometry; high throughput; human whole blood; miniaturization