Dissecting the binding landscape of four cKIT inhibitors through an integrated multifaceted approach.

Abstract

The receptor tyrosine kinase cKIT plays a pivotal role in a variety of physiological processes and is implicated in a broad spectrum of pathological conditions. Its activity is controlled by phosphorylation-dependent conformational changes, which also influence the binding mode of small-molecule inhibitors. We employed an integrated experimental and computational strategy to characterize the conformational landscape of cKIT and to elucidate the binding mechanisms of four inhibitors, Avapritinib, Olverembatinib, Labuxtinib and Cenisertib. A combination of Surface Plasmon Resonance (SPR), enzymatic assay, Molecular Dynamics (MD) simulations, stability energy evaluation, Limited Proteolysis coupled to Mass Spectrometry (LiP-MS) and X-Ray crystallography was used to investigate both active and inactive kinase states. Avapritinib preferentially binds the active form of cKIT, as supported by SPR kinetics, LiP-MS patterns, and MD results. Structural data further confirm that this compound occupies the ATP-binding pocket, consistent with a Type I inhibitor. Olverembatinib and Labuxtinib exhibit high affinity for the inactive kinase, showing stronger binding to the inactive form by SPR and inducing extensive protection of residues spanning the ATP-binding pocket in LiP-MS experiment. MD analysis reveals the burial of key pocket residues, supporting a Type II inhibition mode. Cenisertib exhibits a more complex behavior. While SPR indicates binding to both kinase states, LiP-MS, MD and X-Ray crystallography analyses reveal distinct interaction patterns depending on phosphorylation. Overall, this work highlights how distinct inhibitors exploit different conformational states of cKIT and demonstrates the value of integrating structural analyses, biophysical measurements, calculations and molecular simulations to define the mechanism of kinase inhibition.