Kinase activation is often driven by the phosphorylation of residues in key regulatory elements, including, among others, the activation loop (A-loop) and receptor tyrosine kinase (RTK) autoinhibitory juxtamembrane (JM) domains. A-loop phosphorylation stabilizes a catalytically active kinase conformation characterized by a “DFG-in” structure at the loop’s N-terminus, whereas JM domain phosphorylation relieves autoinhibition by dissociating the JM domain from the catalytic domain, where it locks down an inactive “DFG-out” A-loop conformation and sterically occludes the active site. For both of these mechanisms, inhibitor binding affinity can be highly activation state-dependent. For example, the Type II ABL/KIT inhibitor imatinib, which recognizes an inactive “DFG-out” kinase conformation, binds with high affinity to nonphosphorylated ABL, but with reduced affinity (30-fold) to activated ABL, phosphorylated on the A-loop. Imatinib also has a dramatic affinity preference (1,000-fold) for non-autoinhibited KIT (JM domain phosphorylated and dissociated) over the autoinhibited state (JM domain nonphosphorylated and docked) that is explained by steric clashes between the inhibitor and the docked JM domain. The activation state dependence of binding reflects an inhibitor’s binding mode and therefore provides a rapid biochemical method to gain structural insights in the absence of cocrystal data.
Panel Highlights and Benefits
- Classify inhibitors as having Type I or Type II binding modes without a requirement for cocrystal structures
- Further differentiate the detailed binding modes of inhibitors within the Type I and Type II classes
- Collect activation state-specific biochemical PDGFR family RTK inhibition data that are required to predict & interpret potency in cellular assays
