(1078-C) A novel cell-based assay to identify the FLT3 kinase construct used in enzyme inhibition assays that best represents the activity of cellular FLT3
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: FMS-like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase (RTK) that plays a key role in hematopoiesis. Mutations of this protein are associated with hematologic malignancies. Our focus was to develop a FLT3 enzymatic selectivity assay for another RTK program. FLT3 consists of six domains: the extracellular domain (ED), transmembrane domain (TD), juxtamembrane domain (JMD), tyrosine kinase domain, kinase-insert domain, and C-terminal intracellular domain. Here, we describe the characterization of FLT3 enzyme assays using two FLT3 proteins, one with a partial JMD and one with full-length JMD. RTK inhibitors yielded significantly different IC50 values between the two FLT3 constructs, suggesting a role of the JMD in FLT3 inhibitor activity. To further explore the role of the JMD, we tested known type I and II kinase inhibitors. Type II inhibitors (which bind to the inactive kinase) demonstrated large potency differences between the two FLT3 constructs, being more potent towards the FLT3 protein with full-length JMD, while type I inhibitors (which bind to the inactive and active kinase) did not show significant potency differences. To identify the FLT3 kinase protein that better represents the cellular activity of the entire FLT3 protein, we developed a novel cell-based assay that measures RTK phosphorylation using a pLISA luminescence detection method. This assay demonstrated that results from the FLT3 protein containing the full-length JMD correlated better with those from full length cellularly expressed FLT3, and is therefore a better enzyme assay predictor of in vitro cellular rank order potency.