Mapping the ATP-Binding Pockets of FLT3 and TAK1: A Structural Basis for Dual Inhibition by FLT3-Targeted Therapies

Cutting edge protein kinase inhibitors are often plagued by unpredicted off target binding that can limit their therapeutic window, impacting clinical outcomes. However, such off-target binding may provide in vivo clinical evidence supporting therapeutic avenues in nonrelated diseases. We have recently identified off target transforming growth activated kinase beta-1 (TAK1) affinity in FDA approved FLT3 inhibitors. The receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) serves as a key regulator of hematopoietic stem cell survival and proliferation. Unlike FLT3, TAK1 is a serine threonine kinase involved in inflammatory signaling in the MAPK pathway. Despite very little sequence homology between the TAK1 and FLT3, through targeted inhibitor development and kinome wide screening we identified a strong structural homology between the active sites of these two kinases that leads to cross reactivity of several FLT3 inhibitors in clinical use with TAK1. Enzymatic and in silico modeling confirmed binding and affinity of many common FLT3 inhibitors against TAK1. Furthermore, we explored the in vitro implications of off target TAK1 inhibition by FLT3 inhibitors in human PBMCs stimulated with lipopolysaccharides (LPS) on downstream cytokine production. Our results confirm intracellular inhibition of TAK1 by several FLT3 inhibitors in clinical use. Given that FLT3 inhibitors are administered chronically to treat AML, the nonharmful unintended off target inhibition of TAK1 in vivo by current FLT3 inhibitors provides some insight into the safety and tolerability of chronic TAK1 inhibition in patients.