Allosteric Gain-of-Function and Off-Mechanism Effects of ATP-Competitive Kinase Inhibitors Revealed by Multimodal Proteomics

Protein kinases are critical regulators of cellular processes, and their dysregulation is a common hallmark of human disorders. While ATP-competitive inhibitors are the primary therapeutic strategy to block aberrant kinase activity, it is largely unknown how their binding could affect non-catalytic kinase functions via changes in kinase conformation and protein-protein interactions (PPIs). Here, we introduce a multimodal proteomics approach to systematically characterize these overlooked effects, providing a crucial framework for comprehensive target engagement analysis. Our integrated platform, which combines affinity purification coupled with limited proteolysis mass spectrometry (AP-LiP-MS), AP-MS and in vivo proximity labelling, enables comprehensive exploration of inhibitor-induced allosteric and non-catalytic functional changes. Using this pipeline, we discovered that all four kinases analyzed (DCLK1, CAMKK2, CHEK1, and PRKCA) undergo structural rearrangements upon inhibitor binding in the autoinhibitory domain (AID) and other regions. These structural shifts underlie changes in complex formation, membrane association and other non-catalytic functions. Our work suggests that these on-target, off-mechanism effects are highly prevalent and provides the analytical tools to systematically characterize a frequently overlooked phenomenon highly relevant for understanding drug side effects to guide the development of novel therapeutics.