A multi-proteomics approach identifies non-catalytic effects of ATP-competitive kinase inhibition

Kinases are essential regulators of most cellular processes, and their activity is tightly controlled. Aberrant kinase function can contribute to the development and progression of various diseases. Small molecule inhibitors offer a means to correct this dysregulated kinase activity. Type I inhibitors are the largest group of kinase inhibitors and target the ATP-binding pocket of kinases in an ATP-competitive manner. While this mode of action has been well studied, it is largely unclear how these inhibitors could affect non-catalytic kinase functions by remodeling the structural dynamics and subsequent complex formation of kinases. Here we present a proteomics workflow based on affinity purification (AP) and limited proteolysis (LiP) coupled with mass spectrometry (MS) to probe changes in complex formation and kinase structure induced by these compounds. We show that inhibition with SGC-CAMKK2-1 induces CAMKK2 complex formation with known and candidate substrates, including the metabolic regulator AMPK, typically associated with activation of CAMKK2. To understand the mechanisms underlying inhibitor induced protein sequestration, we explored structural changes upon inhibition of CAMKK2 with the inhibitors SGC-CAMKK2-1 and staurosporine using AP-LiP-MS. Both inhibitors induced allosteric changes at the autoinhibitory domain, which is consistent with a relief of autoinhibition upon inhibitor binding. We propose that the inhibitor locks the kinase in a pseudoactive conformation that resembles the active, non-autoinhibited conformation, promoting stable complex formation with substrates and other proteins, potentially altering their activity. We observed similar structural changes upon inhibition of the related kinases CHEK1, DCLK1, CAMK1 and PKCα, suggesting a more general mechanism underlying Type I inhibition of kinases with autoinhibitory domains. We propose that holistic characterization of the effects of Type I inhibition can reveal crucial new insights into the molecular mechanisms that drive cellular phenotypes of existing and future kinase therapeutics beyond inhibition of kinase mediated substrate phosphorylation, to allow future expansion of the activities that could be targeted by ATP-competitive kinase inhibitors.